WHOLE MODULE Flashcards
1
Q
What are the two opposing theories of early development
A
Epigenesis - Aristotle belief that development is a gradual process, stage by stage, whereby new functions and characteristics are added on top of others. The other theory is that everything is already present and pre-established in the fertilised egg and all is required is amplification
2
Q
What is meant by preformationism
A
The idea that organs develop from miniature versions of themselves as a homunculus (little human)
3
Q
Explain Wissman’s theory of how cells become different
A
Two types of cells exist, somatic and germ cells. Germ cells contain determinants (germ plasm) whereas somatic cells only contain a subset of these determinants which define its particular set of characteristics
4
Q
Recall the experiments carried out by Wilhelm Roux on frog embryos
A
Roux killed half of the cells at the 2 and 4-cell stage and looked at this effect on the developing embryo. It was noticed that ablation of these cells lead to the production of half embryos. This lead him to believe that at that stage each cell contributes uniquely to the development of the embryo and that the cell that wasn’t killed is only capable of adopting its primary fate.
5
Q
How do the experiments carried out by Hans Driesch differ to those of Roux
A
Hans Driesch carried out similar experiments on sea urchin embryos. He divided the embryos up at the 2 and 4 cell stage and allowed them to develop, each cell in isolation from the others. He believed that these cells would give rise to the relevant part of the body plan already decided on. However each of the separated cells gave rise to a separate sea urchin proving that these cells were totipotent and could give rise to any of the cells of the body.
6
Q
How does cell shape influence behaviour
A
Epithelial cells are fixed and immobile whereas mesenchymal cells allow migration. Thus epithelial to mesenchymal transitions (EMTs) are essential during development particularly in gastrulation.
7
Q
What are the four types of cell-cell communication
A
Paracrine, autocrine, Juxtacrine and endocrine
8
Q
Which types of cell signalling occur in development
A
Paracrine, autocrine and Juxtacrine
9
Q
Which type of cell-cell communication involves a factor acting on receptors on the same cell that produced it
A
Autocrine
10
Q
Which type of cell-cell communication involves two cells communicating with each other by direct contact
A
Juxtacrine
11
Q
Which type of cell-cell communication involves a factor acting on a receptor on a neighbouring cell
A
Paracrine
12
Q
Give an example of a juxtacrine signalling mechanism seen in development
A
Delta-notch
13
Q
Give example(s) of paracrine/autocrine signalling mechanisms seen in development
A
Shh, Wnt, TGF?, BMP and FGF signalling
14
Q
What are the two types of cell division seen during development and how do they differ
A
Symmetric cell division - progeny divide and give rise to two identical daughter cells. Asymmetric cell division – progeny divide and give rise to two daughters with different characteristics
15
Q
How is asymmetric cell division achieved
A
Cytoplasmic, extracellular or membranal determinants are segregated/distributed unevenly in the precursor cells so that when it divides the daughters contain different levels
16
Q
Every cell has the potential to communicate with other cells, T or F
A
F - some cells dont
17
Q
Cells need to be competent to respond to a signal by receiving and transmitting a signal to the nucleus, T or F
A
T
18
Q
What type of signal initiates a new program
A
Instructive signals
19
Q
What type of signal provides a favourable environment for a specific program
A
Permissive signals
20
Q
What is meant by a morphogen
A
A morphogen is a chemical (usually a diffusible signalling molecule) whose concentration varies forming a concentration gradient
21
Q
What are the three requirements of a molecule for it to be a morphogen
A
Produced from a localised source, usually sensed through cell surface receptors and cells respond differently to difference concentrations of the morphogen
22
Q
What are the common features of signal transduction pathways
A
Ligand binding to a receptor causes its activation which leads to the transduction of the signal to the nucleus via a cascade if secondary messenger activation. This in-turn leads to the activation of a transcription factor which induces the transcription of specific target genes
23
Q
Gene content is identical in most cells and transcription/translation dictates the protein content of a cell, T or F
A
T
24
Q
At what levels does the cell operate control on a gene program
A
Production of mRNA, processing and stability of mRNA, production of proteins and the activity of proteins
25
What are the two ways in which the cell controls the genetic program at the level of gene transcription
Differential gene expression, enhancer-mediated control
26
What other two methods are the of controlling the genetic program
miRNA, controls at the level of chromatin
27
What feature of cells is essential for the normal development, growth and repair of organs and how is this achieved
Tissue homeostasis - achieved by a balance of new cell formation and knowledge of how many cells are required by a tissue and when it needs to build more
28
What are the effects of disruption of tissue homeostasis
Contributes to ageing and degeneration
29
What are the four processes underlying development
Pattern formation, morphogenesis, cell differentiation and growth
30
What is meant by pattern formation
The organisation of cells within the body and how they occupy particular points in the xyz axis
31
What is meant by the term morphogenesis
The creation of shape
32
Outline the process of morphogenesis
How a cell/organ/tissue adopts a particular 3D shape and how cells move and reorganise during development to generate functional organisms
33
What four attributes of cells determines morphogenesis
Adhesion, migration, death and shape
34
Define cell differentiation
The process by which cells become different from one another and acquire specialised properties
35
What governs differentiation
Changes in gene expression which dictate the repertoire of proteins synthesised by a cell
36
Differentiation is an increase in cell fate and specialisation over time that corresponds to a decrease in pluripotency, T or F
F - differentiation is a gradual restriction in cell fate over time whereby specialisation increases and pluripotency decreases
37
What attributes of cells in development contributes to growth or a tissue/organ
Cell proliferation, enlargement and accretion
38
What is meant by cell accretion
Growth of a tissue/organ that occurs by a gradual accumulation of additional layers of cells
39
Describe Haeckels funnel model of development
Embryos start off developing similarly but become progressively different as time goes on
40
How does the Von Baer Hourglass model differ to the Haeckel funnel model
Embryos start off developing differently, but then converge at gastrulation, showing substantially conserved phenotypes. After this point they then diverge again
41
How can the spatial and temporal expression of a gene of interest be studied in development, what can you actually measure
You can investigate where and when the mRNA of this gene is transcribed
42
List the 5 techniques that can be used to determine where/when a gene is expressed in the embryo
In situ hybridisation, northern blotting, reverse transcriptase PCR, micro-arrays and reporter lines
43
Outline the process of in situ hybridisation
A probe with complementary base pairs to the target gene is synthesised and labelled with a digoxigenin (DIG) immune-tag. This will bind to the target sequence of the gene of interest and then a secondary antibody binds to the DIG label. Binding of the secondary antibody leads to an enzymatic reaction that produces a reaction product that indirectly marks all the cells that produce that specific mRNA.
44
What is the major disadvantage of in situ hybridisation
You have to kill the embryo in order to carry out the technique
45
Explain the process of reporter line transgenics
Introduce a coding sequence for a reporter gene i.e. GFP, RFP or ?-gal into the coding sequence of the gene of interest. This will result in the reporter gene being under control of the target genes promoter sequence. Hence, wherever the gene of interest is expressed the reporter will be too. This can be easily visualised or investigated
46
What methods can be used to determine if a protein is actually expressed at the same time as a gene by investigating if the mRNA produced is actually transcribed
Western blotting, immunohistochemistry
47
Explain how immunofluorescence can be used to visualise where a protein is expressed during development
Introducing labelled tags to antibodies for various epitopes of a protein of interest allow you to visualise where these epitopes and proteins are expressed
48
Gain and loss of function experiments are used to investigate if target genes are essential in development, T or F
T
49
How can loss of function mutations be used to test for essential genes in development
By introducing a premature STOP codon into the target sequence you can investigate how essential a gene is in development. Loss of function mutations results in a disruption in the expression or function of the mutated genes
50
How can gain of functions mutations be used to investigate essential developmental genes
Mutations in regulatory regions of a DNA sequence can lead to increased levels of transcription. This confers a gain in the activity of the mutated protein
51
What is the difference between forwards and reverse genetics
Forward genetics investigates the identity of genetic mutations responsible for a specific observed phenotype. In contrast, reverse genetics seeks to characterise the phenotype produced from a particular gene mutation
52
How can tissue ablation be used in the study of gene regulation during development
Tissue ablation and ectopic grafting experiments can be correlated with levels of transcription factors. You can also investigate is a transcription factor soaked bead is sufficient to direct gene expression
53
How can the tissues/organs that are derived from cells that express a certain gene be elucidated
This is investigated in order to determine if every single tissue expressing a factor originated from the early structure expressing it. This can be investigated through fate mapping and determination experiments.
54
Chick-quail chimera labelling with dyes has been used to investigate cell fate and determination through dye labelling of progenitor cells, T or F
T
55
Define a morphogen
A soluble secreted molecule that acts at a distance to specify the fates of cells
56
What are the two main attributes of a morphogen
Morphogens act at a distance to induce different output or cell fates at different concentrations by forming a gradient in the embryo.
57
How is higher information encoding achieved by morphogens
More information is encoded by the higher morphogen concentrations closer to the source
58
What two mechanisms can account for the different cell fates achieved by morphogens
Temporal diffusion of the secreted signals from the source to where it is destroyed/sunk. Similarly, the magnitude of the morphogen concentration also causes the acquisition of different cell fates
59
Why are morphogen gradients usually exponential decays
Morphogens move by passive diffusion throughout the embryo leading to the exponential decay appearance of the gradient
60
How may it be possible to achieve a straight linear morphogen gradient
Active movement of the morphogen may achieve a linear gradient
61
The shape of the morphogen gradient is what encodes the information to direct cell differentiation, T or F
T
62
All molecule involved in patterning of the embryo are morphogens, T or F
F – permissive signals aren’t morphogens
63
Why aren’t permissive signals morphogens
Permissive signals only direct a cell to response to an instructive signal
64
Instructive signals are morphogens, T or F
T
65
What would be seen in a field of cells as a result of ectopic grafting of another source if the secreted factor was a morphogen
A mirror image of cell differentiation would be seen
66
What would be seen in a field of cells as a result of ectopic grafting of another source if the secreted factor was a permissive signal
This would have no effect on cell differentiation, all cells in the field would develop normally as the permissive signal only enables the cells to respond to a morphogen
67
Give an example of an experiment that has proved a gene product is a morphogen by ectopic grafting
Ectopic grafting of an shh soaked bead opposite to its normal site in the chick limb bud leads wing development that defined by a mirror image duplication of the digits etc.
68
What would be the effects of forcing a uniform gradient of a secreted factor across a field of cells if this factor was a permissive signal
There would be no abnormal effects on cell development. The same number of fates will be produced and each cell would adopt the correct fate
69
What would be the effects of forcing a uniform gradient of a secreted factor across a field of cells if this factor was an instructive signal
This would result on the field of cells only adopting one fate as all cells would receive the same ligand concentration
70
Explain what is meant by the bucket brigade signalling mechanism and why this is therefore not used by morphogens
The bucket brigade mechanisms uses sequential signalling of the cells that acts immediately on the adjacent cell. Expression of one signal induces a target cells to produce another different signal. This signal acts on the next cell in the sequence to induce production/secretion of another different factor and so on. As this signalling occurs between adjacent cells and not over distance, this isn’t a type of morphogen signalling
71
How can you experimentally modify a potential morphogenic molecule to make it juxtacrine
Using genetic engineering to add a transmembrane domain region to the protein sequence to make it a membrane bound signal
72
What would be the effects of making a morphogen juxtacrine and how would this differ if the signal was part of a bucket brigade signalling mechanism
If the ligand was a morphogen, making it juxtacrine would lead to only the first cell being induced to adopt its fate as the ligand will be unable to act at a distance and direct differentiation by diffusion. This would be due to it becoming membrane bound and only able to act on adjacent cells. If the factor was part of a bucket brigade mechanism, then the signalling would be unaffected as adjacent cells would still receive their ligand and thus go onto produce the next sequential secreted factor.
73
How would creation of a genetic mosaic lacking a morphogen receptor influence differentiation
The cell lacking the receptor would adopt the same fate as the terminal cell in the field would normally. This would be due to the fact that the terminal cell usually receives the lowest/no morphogen signal. Cells after the one lacking the morphogen receptor would however development normally
74
How would the bucket brigade signalling mechanism be affected by a lacking receptor
Cell fate wouldn’t be effected if the receptor knocked out from a cell was one for a ligand that acts upstream of the cell in the sequential signalling pathway. This is because at this point in the cell field, this ligand is no longer acting and instead a different ligand is acting on the target cell produce by the proximal adjacent cells. However, if the receptor knocked out was the first receptor in the sequential sequence then none of the cells would adopt their fate and would remain undifferentiatiated
75
Shallowing morphogen gradients would encode far less information than steeper ones, T or F
T
76
How can a higher effective concentration of a morphogen be achieved
Preventing the morphogen from diffusing in axes that aren’t desirable
77
How can non-beneficial morphogen diffusion be prevented
Morphogen binding to molecules in the extracellular matrix such as heparan sulphate proteoglycans allows sequestration and facilitation of morphogen diffusion, effectively increasing the size/steepness of the gradient in desirable planes. This acts to increase the amount of information encoded by the morphogen gradient
78
Heparan sulphate proteoglycans are sometimes referred to as co-receptors and bind many different ligands, T or F
T
79
How can rapid degradation of the morphogen at the sink increase information encoding
Increases the steepness of the morphogen gradient
80
Decreasing the range of the morphogen gradient can be achieved by localisation of the morphogen in the desired range, T or F
F – this acts to extend the range
81
How can morphogens move through cells in a tissue and give an example of a molecule for which this is the cane
Planar transcytosis - Repeated cycles of endocytosis and re-secretion. Dpp is transcytosed and antibody staining has revealed its presence in vesicles
82
How is the timing of morphogen gradient establishment critical to cell differentiation
The cells are prevented from responding at an inappropriate time where the morphogen concentration won’t have reached that required by the particular cell to direct its correct fate. This is likely achieved by the cell waiting for a steady state of receptor activation to be achieved but the molecular mechanism by which this occurs isn’t understood
83
Explain how a cell interprets a morphogen gradient before responding and determining its fate
Morphogen concentration is directly correlated to the activation of transcription factors inside the cells. Higher concentration of morphogen often results in a higher concentration of an activated transcription factor. In this model, receptor activation causes transcription factors to enter the nucleus and direct transcription. Levels of activated transcription factor determine the fate of the cell
84
Which morphogen is the only known one which itself is a transcription factor and how does it act
Bicoid is a morphogen and a transcription factor. Bicoid mRNA is localised at the anterior of the egg and is translated into protein during early embryogenesis. Bicoid protein then diffuses through the cytoplasm and accumulates in nuclei of the syncytial blastoderm generating a concentration gradient
85
How do different levels of transcription factors dictate the different fates of cells under the influence of a morphogen
Binding of a transcription factor to the promoter seuequence of genes that confer a particular cell fate is an equilibrium reaction with an on and off rate. The strength of the equilibrium that’s moving gene expression towards its on state will control how strongly the gene is expressed. Increasing transcription concentration factor would increase on state by shifting the equilibrium to the right to counteract this increase in transcription factor levels. The concentration of activated transcription factor determines if it binds to high or low affinity sites. Each of these sites dictate a different cell fate and differential gene expression
86
What happens at low-medium transcription factor levels and how does this confer a particular cell fate
At low-medium concentration of the transcription factor only the high affinity sites will elicit binding which will direct a particular cell fate and specific subset of gene expression
87
What happens at high transcription factor levels and how does this confer a particular cell fate
At high concentrations of the transcription factor the low affinity sites will now allow binding. Binding of the transcription factor to these sites will result in differential gene expression and a different cell fate. More ligand will increase activation of low affinity sites
88
How do the low affinity sites allow differential gene expression if the high affinity transcription factor binding site are also being activated
One of the genes switched on by transcription factor binding to the low affinity sites will turn off by transcriptional repression) the high affinity genes in a process called crosstalk
89
Explain how strict thresholds of cell differentiation are achieved even if the morphogen gradient isn’t steep
Positive feedback helps a cell commit to its specific fate. If one of the genes switched on by high affinity site binding encodes a transcription factor – it can, amongst other things, activate its own expression
90
Drosophila melanogaster is a segmented animal, T or F
T
91
The genome of Drosophila has been sequenced, T or F
T
92
Drosophila can regulate their own body temperature, T or F
F – they cannot
93
What two temperatures are stocks of Drosophila kept
18 and 25°C
94
Which temperature stocks tend to have a lifecycle of around 10 days
25°C stocks
95
Which temperature stocks tend to have a longer life cycle of upwards of 21 days
18°C stocks
96
How much time elapses from laying of a fertilised Drosophila egg to hatching
24 hours
97
Briefly outline Thomas Hunt-Morgan’s contribution to Drosophila research
Hunt-Morgan noticed a white-eyed fly in his lab one day. He bred this fly with wild type flies to investigate the phenotypes produced
98
What was Sturtevants contribution to Drosophila research
Constructed the first genetic map and arranged it in a linear order
99
Bridges showed that chromosomes must contains genes, T or F
T
100
Muller showed that radiowaves caused mutations and chromosomal rearrangements, T or F
F – he showed that X-rays caused mutations
101
Outline Nusslein-Volhard and Wischuas contribution to Drosophila research
Carried out a saturation mutagenesis to identify genes in Drosophila involved in development and patterning. This was an absolutely massive screen and lead to the identification of 139 complementation groups
102
Describe the general arrangement of the Drosophila genome
4 chromosomes consisting of 3 autosomes and 1 sex chromosome. Roughly 17,000-18,000 genes, 14,000 of which code for proteins
103
What was seen as a result of alignment of homologous sequences of genes between Drosophila and other organisms
The protein encoding exons were almost perfectly conserved. The non-coding introns were seen to only be randomly conserved and showed differences between organisms most likely due to not being under any selection pressures. Regulatory sequences in the genes also showed high levels of conservation between species
104
Drosophila can produce 200 progeny in less than 2 weeks, T or F
T
105
Put these Drosophila courtship processes in order, orientation, licking, attempted copulation, tapping, wing vibration
Orientation, tapping, wing vibration, licking, attempted copulation
106
Female Drosophila mate several times in their life cycle while males mate only once, T or F
F – vice versa
107
The strongest Drosophila reproducible behaviour is genetically encoded, T or F
T
108
Outline spermatogenesis in Drosophila
The testes located within the abdomen on the male contain hub cells. These cells secrete factors such as unpaired which maintains stem cell fates in the cells adjacent to the hub. Unpaired is a JAK/STAT ligand and its signalling decreases with distance from the hub. Cells further away from the hub differentiate due to decreased levels of unpaired signalling. These cells divide and give rise to 64 sperm cells
109
What is remarkable about sperm produced by male Drosophila
Spermatids are 25x the length of the adult Drosophila body
110
Explain the interplay between the sexual evolution of male and female Drosophila
The sperm produced by male Drosophila is coated in a sex peptide. This binds to receptors in the brain of female Drosophila and prevents them from being able to respond to subsequent courtship dances by other males. This increases the likelihood of the males passing on their genetic information. However, females evolved the ability to repress the effects of the sex peptide. This caused the males to evolve and produce larger and larger sperm coated in more and more sex peptide to try and increase the females inability to respond to other courtship dances.
111
Outline the process of oogenesis in Drosophila
Stem cells are maintained at one end of the bundled linear structures that make up the ovaries. Cells in the ovaries undergo 4 incomplete cytoblast mitotic divisions. Most cells become nurse cells which undergo endoreduplication making multiple copies of the genome and expressing a huge amount mRNA that supplies the oocyte with the genetic information to synthesise proteins etc. The cells that go on to form oocytes do so by undergoing meiosis to produce cells containing mostly cytoplasm.
112
What is meant by cytoplasmic dumping and how does this occur
The nurse cells that supply the developing oocyte contain massive amounts of genetic information and are transcribing a vast amount of mRNA. This mRNA is then deposited through ring canals into the cytoplasm of the oocyte so that it can synthesise useful products.
113
Explain how polytene chromosomes are produced and what they represent
Polytene chromosomes are chromosomes stained for polymerase activity. Bands represent regions of the genome where there is little activity. Interband regions contain puffs which represent extremely active and transcribed genes.
114
Explain how some polarity is established in the oocyte even before fertilisation
Some of the factors deposited by the nurse cells through cytoplasmic dumping become localised (i.e. through gravity and other mechanisms). This localisation of factors accounts for the inherent polarity of Drosophila oocytes
115
Describe some of the features of the Drosophila egg
Chorion acts as the eggshell surrounding the embryo. The vitelline membrane lies beneath the chorion and provides waterproofing. The micropyle located at one end of the egg is the opening through which sperm enter.
116
Describe what is meant by a syncytial blastoderm and how this is achieved in the Drosophila embryo
Syncytium is a multinucleate cell. This is achieved in Drosophila through nuclei duplication in the centre and then migration to the periphery. After 14 rounds of nuclei duplication and after they have relocated to the edges of the embryo the cell membrane moves in and encapsulated the nuclei creating a cellular blastoderm.
117
What happens to the cells that are retained in the centre of the embryo before cellularisation
These become pole cells and give rise to the gametes and gonads of the adult organisms
118
What are the names given to the two regions of the embryo lower order animals (not humans) that give rise to the three germ layers and extraembryonic structures
Epiblast – gives rise to the germ layers, hypoblast – forms vegetal pole/hemisphere
119
In which regions of the morula would you find the structures that give rise to the embryo and to the extraembryonic structures
Top of the morula gives rise to the epiblast, bottoms gives rise to the hypoblast
120
What changes govern the formation of these two layers in the morula
Governed by morphogens and transcription factors that lead to changes in gene transcription
121
What causes the hollowing of the morula during development alongside squashing
Changes in osmolarity
122
Which region of the morula ultimately ends up undergoing apoptosis
Hypoblast
123
At which stage does the primitive streak in chick embryos form at the posterior of the embryo
Stage 3
124
At stage 4 of chick development, the primitive streak has elongated and Hensen’s node has formed anteriorly, what does this structure then begin to express
Expresses and secretes BMP antagonists such as chordin, noggin and follastatin
125
At what stage in chick development does the notochord begin to form
Stage 5-6
126
What structure then extends forward from the node and is involved in anterior-posterior axis formation
The notochord
127
Why are the events from morulation to gastrulation less studied in mammalian embryos and why is this
Mammalian embryos have been less extensively studied because they are difficult to maintain in culture after the blastula stage due to this being the stage at which they normally implant into the uterus
128
At five days post-fertilisation what structure forms in the mammalian embryo that is similar to the hypoblast
Trophoblast
129
What day corresponds to chick development stage 2 and corresponds to the embryo implantation into the uterus lining
12 days post-fertilisation
130
At what stage in mammalian embryonic development is the embryo referred to as a blastula
12 day stage
131
The processes occurring in chick embryos from stage 2-6 occur in 2-4 week old human embryos, T or F
T
132
At which region of the developing embryo does gastrulation and germ layer formation begin at
Posterior of the embryo
133
What is the first structure to become visible in the initial stages of gastrulation
Formation of the primitive streak due to cells moving into the midline forming a line that elongates anteriorly
134
What forms at the most anterior point of the primitive streak
(Hensen’s – in chicks) node
135
Which regions of the embryo does the primitive streak denote
Posterior/caudal regions
136
The epiblast gives rise to mesoderm, endoderm and ectoderm, T or F
T
137
Explain how signals from the hypoblast layer of the developing embryo accounts for the formation of three germ layers by the overlying epiblast
Signals from the hypoblast (trophoblast) induce some epiblast cells to become mesoderm and definitive endoderm. These cells involute and ingress along the anterior-posterior axis by losing contact with one another and migrating into the embryo. Superficial migrated cells become the mesoderm and the first cells to have ingressed, those now lying deeper within the epiblast will become endoderm.
138
How does the migration of cells into the embryo at the primitive streak account for the formation of both mesoderm and endoderm
Cells that migrated first from the epiblast layer will lie deeper within the embryo and will become the endoderm. Those that migrated later will be lying for superficially and will become mesoderm.
139
Once primitive streak reaches maximum extension and forms Hensen’s node, a similar event takes place whereby cells ingress but begin to move directly anteriorly/rostrally, T or F
T
140
What is different about the gastrulation of Xenopus embryos compared with mammal/human early embryonic development
The Xenopus embryo doesn’t compact to form a blastodisc as mammalian embryos do. It remains morula-like with an animal cap and a vegetal hemisphere
141
What structures in the Xenopus embryo correspond to the epiblast and hypoblast seen in mammalian embryos
Animal cap – epiblast, vegetal hemisphere – hypo/trophoblast
142
Explain how oocytes are already polarised before fertilisation
Particular determinants inside the egg have become localised in the vegetal hemisphere of the cell. This is due to gravity in Xenopus and an interaction with the placenta in mammals
143
What is the result of the inherent polarisation of the egg prior to fertilisation
Different cytoplasmic determinants have sunk to one part of the egg. This leads to the cells that come from this region being different and having specific factors localised/activated
144
In an early cleavage event, the vegetal and animal hemispheres of the now fertilised egg are separated, what is the effect of this on cytoplasmic determinant localisation
Cytoplasmic factors restricted to the vegetal part of the oocyte are now restricted to vegetal hemisphere cells
145
What is the effect of the factors that become localised to the vegetal hemisphere cells of the embryo as a result of early cleavages in the embryo that separate the animal and vegetal cells
These factors localised in the vegetal part of the embryo bind to promoters for particular transcription factors and upregulation. In-turn, these particular transcription factors become solely expressed in vegetal hemisphere cells
146
What is the name of the transcription factor that is specifically expressed and localised to vegetal hemisphere cells
VgT
147
Describe what happens as a result of the localisation of a specific transcription factor in the nuclei of cells in the vegetal hemisphere of the early embryo
Localised VgT in the nuclei of cells in the vegetal hemisphere leads to its binding to the promoter of a gene called Nodal and upregulate its expression. The Nodal morphogen then leaves the cells of the vegetal hemisphere and binds to receptors on cells in the animal hemisphere
148
What is the name of the morphogen expressed in the vegetal hemisphere cells that is upregulated as a result of VgT binding to its promoter
Nodal
149
What is the effect of the morphogen produced by cells of the vegetal hemisphere
Nodal binds to receptor on cells in the animal hemisphere and dictates the mesoderm and endoderm formation via cell proliferation, migration and differentiation.
150
What happens to cells at the top of the animal hemisphere as a result of VgT activity on its promoter
The Nodal morphogen produced as a result of this leaves the cells and binds to receptors in the animal hemisphere. It forms a concentration gradient throughout the animal hemisphere. Cells at the top of the animal hemisphere don’t receive nodal signalling and so remain as ectoderm. This is thought to be due to either not expressing the nodal receptors or by not receiving a threshold concentration of the nodal ligand
151
Nodal is expressed throughout the vegetal hemisphere/hypoblast in the developing embryo. What aspects/axes of early embryonic development does this signalling mechanism not account for
Can't account for anterior-posterior axis formation and the appearance of the primitive streak at the distal region of the embryo
152
Which signalling pathway is therefore responsible for anterior-posterior patterning and formation of the primitive streak
Wnt signalling
153
Wnt signalling mediates a secondary symmetry breaking event but unlike nodal, it is activated on one side of the embryo. Explain how this is achieved and where wnt activity is seen in the early embryo
The Wnt signalling pathway is activated on one side of the embryo, opposite to the point of sperm entry. This region is referred to as the dorsal embryo but also represents the site where gastrulation movements will begin, i.e. the posterior embryo.
154
Nodal is activated throughout the animal cap of the embryo, T or F
F – it is activated throughout the vegetal hemisphere
155
Which gene product accumulates in the nuclei of cells in the posterior/dorsal embryo as a result of increase Wtn signalling and promotes the expression of Wnt target genes
?-catenin
156
What is the Nieuwkoop centre
The region of the embryo where ?-catenin has accumulated in the nuclei and there is activation of Nodal signalling
157
How does ?-catenin and nodal interact
?-catenin and nodal interact directly and result in enhanced activation of Wnt signalling in regions of the embryo where ?-catenin is also expressed. The causes the conversion of what was a uniform gradient of nodal in stage 8 to a clear concentration gradient from anterior to posterior in stage 9 embryos.
158
Where is the highest nodal activity seen as a result of its interaction with ?-catenin
Highest nodal in the dorsal/posterior embryo
159
What is the role of the Nieuwkoop centre
Region of high nodal and ?-catenin signalling that induces the formation of the organiser
160
What happens to regions with low nodal activity
Induction of ventral mesoderm
161
What are the two crucially different types of mesoderm induced in the early embryo
Organiser mesoderm, ventral/lateral mesoderm
162
The mechanism of ?-catenin and nodal interactions is poorly understood, T or F
T
163
What two factors are required for goosecoid expression
Nodal downstream effector known as Smad2/4 binding to its distal region of the promoter and a Wnt/?-catenin downstream effector called Xtwn binding to the proximal element of the promoter.
164
Brachyury is induced in response to high levels of Nodal in the dorsal/posterior embryo, T or F
F – brachyury expression is induced in response to low Nodal signalling in the ventral/anterior embryo
165
What levels of nodal signalling are required for expression of T, another transcription factor involved in anterior-posterior patterning
T is expressed at low Nodal signalling levels
166
Chordin, Xnot and Xlim1 are other genes expressed alongside goosecoid that act as inducers of various mesodermal fates, what is required for their induction
High levels of nodal signalling and wnt signalling activation and subsequent ?-catenin presence in the nucleus
167
Different types of mesoderm have different abilities in terms of proliferation, migration and differentiation etc, T or F
T
168
The combinatorial expression of T, Gsc, Chordin and Tbx6 accounts for the differences seen in the mesodermal tissue, T or F
T
169
What rod-like structure does the organiser/node differentiate into
Axial mesoderm
170
Explain how goosecoid and siamois act in a cell-autonomous way to direct the differentiation of the node/organiser
Siamois and goosecoid expressed in the organiser/node in turn act on the cells that express them in a cell-autonomous, or intrinsic manner to alter their fate. They begin induce the cells of the node/organiser to differentiate into axial mesoderm
171
What is one key property of axial mesoderm and accounts for rolling up of the neural plate into the neural tube during neurulation
Axial mesoderm is able to undergo convergent extension
172
Siamois and goosecoid are expressed at uniform levels but only transiently in different cells. This accounts for the formation of the 3 different types of mesoderm derived from the axial mesoderm, T or F
F – whilst gsc and siamois expression is only transient, they are also expressed at marginally different levels in the cells too
173
What are the three different types of mesoderm derived from the axial mesoderm induced by the transient expression of goosecoid and siamois
Prechordal mesendoderm, prechordal endoderm and notochord
174
Which of the three mesodermal tissues derived from the axial mesoderm are most anterior and most posterior
Prechordal mesendoderm are most anterior and the notochord lies posteriorly
175
Explain the two hypotheses by which Hensen’s node is thought to express chordin and goosecoid
Either cells in Hensen’s node co-express both gsc and chordin or there are individual cells that express each transcription factor
176
Explain how the expression of Gsc and chordin differs between notochord and prechordal mesoderm
Gsc is only expressed in the prechordal mesoderm (and not in the notochord) whereas chordin is only expressed in the more posterior, notochord and not at all in prechordal mesoderm in the anterior embryo
177
Explain how anterior-posterior axis extension is mediated and refined in the posterior embryo
Signals that were initially close together become spatially separated through the anterior-posterior axis. High FGF signalling and the presence of RA in the posterior embryo keep cells in a proliferative state and confers them to a posterior identity. The proliferation of posterior cells causes the addition to the axis as it elongates posteriorly
178
What happens in the anterior embryo
BMP and Wnt antagonises maintain the anterior identities
179
Anterior-posterior identity is through to be realised through the Hox code, T or F
T
180
Neurulation of ectoderm and ventralisation of the mesoderm are both mediated by BMP antagonists, T or F
F – neurulation of ectoderm and dorsalisation are mediated by BMP antagonists
181
BMP antagonists are secreted by which structure
The organiser/node
182
Other than chordin, name four other BMP antagonists
Follistatin, frizbee, cerberus and noggin
183
Where do the BMP antagonists end up and prevent the BMP signal in
Top layer of epiblast
184
How do BMP antagonists act
Block the interaction of BMPs with their receptors
185
What happens to the cells where the BMP signal is inhibited
They will become neural plate
186
Explain how BMP antagonists lead to the refinement of cell fates in the mesoderm
Initially, low levels of Nodal give ventral mesodermal fate. Subsequent inhibition of BMPs in the adjacent mesoderm to the neural tube induces the dorsalisation of what was ventral mesoderm initially. By this process the most proximal mesoderm to the organiser become the somites
187
What feature of the mesodermal tissue leads to the rolling up of the neural plate during neurulation and how does this occur
Convergent extension of the axial mesoderm creates a force in the anterior-posterior direction that is translated to one which drives curling up of the neural plate with somites residing either side.
188
Where does wnt derive its name from
Amalgam of wingless Drosophila gene and Int vertebrate proto-oncogene
189
Int-1 is a proto-oncogene, what causes its activation
Integration of the mouse mammary gland tumour virus
190
Wingless (wg) mutants initially discovered produced wingless but viable flies, T or F
T
191
Explain how wg and hh maintain each other’s expression in an auto-regulatory loop
Wg maintains hh by controlling the expression of engrailed (en), a transcription factor that regulates hh expression. Hh then in-turn maintains and directly upregulates wg
192
What is significant about hh and wg knockout mutants
They exhibit the same phenotype – larvae with a lawn of denticles
193
Unlike similar developmental signalling pathways, wnt expression is highly conserved throughout Kingdom Animalia, T or F
T – even found in sponges
194
Why is it that vertebrates have more wnt genes
Due to genome duplication throughout evolution
195
The wnt protein is produce by a cleavage event that separates its signalling sequence from the initially translated protein, T or F
T
196
What is the role of porcupine in the early modification of wnt
Porcupine is an acyl transferase that adds palmitoleic acid modifications to a serine residue at point 209 in the wnt3a structure
197
What is the hypothesised role of wntless in wnt signalling and what is its basic structure
Wntless is a 7 transmembrane domain protein potentially required for the transport of wnt to the plasma membrane and its subsequent release/presentation to target cells
198
What is the effect of palmitoylation and palmitoleic acid modification of the wnt protein
Addition of these hydrophobic groups makes wnt insoluble in water
199
What components of the extracellular matrix are involved in mediating the diffusion of wnts away from the sending cell
Heparan sulphate proteoglycans (HSPGs)
200
What is significant about the fact that most of the effects of Wnt signalling can be elicited by a membrane bound form of the protein in Drosophila
It suggests that wnts act as juxtacrine signalling molecules or that they don’t diffuse far and act on adjacent cells in Drosophila
201
Recall the two main receptors involved in reception and transmission of wnt signalling in Drosophila
Frizzled and Arrow
202
What is the name of the nuclear factor in Drosophila that is induced as a result of wnt signalling and its corresponding vertebrate homologue
Armadillo (vertebrate homolog – ?-catenin)
203
What are the names of the arrow receptor homologues found in humans
LRP5 and 6
204
Both the frizzled genes and arrow/LRP5&6 act in combination as receptors for wnt signalling molecules, T or F
T
205
Describe the structure of the frizzled receptor and how it interacts with wnts
7 transmembrane domain protein. Wnt binds to the cysteine-rich domain (CRD) in the N-terminus of the Fz protein
206
Describe the structure of the LRP5&6/Arrow receptor for wnt
Single pass transmembrane protein
207
What happens when wnt binds to LRP5/6/Arrow and the Fz receptors
These two receptors come together to form an active wnt signalling complex
208
Which important extracellular wnt inhibitor is overexpressed in order to downregulate wnt signalling in experiments
Dickkopf1 (Dkk)
209
How does Dkk act to downregulate wnt signalling
Dkk is coupled to Kremen. Activation of Dkk by wnt binding promotes the internalisation of the LRP receptors
210
Describe the composition of the degradation complex involved in wnt signalling
Consists of the scaffold protein axin bound to APC, GSK3?, CK1? and slimb
211
Describe what happens in the absence of wnt signalling
Also bound to the degradation complex via an interaction with APC is ?-catenin. In the absence of wnt signalling ?-catenin is phosphorylated by CK1? and then by GSK3?. This poly-phosphorylated ?-catenin is then recognised by the slimb protein which ubiquitinates the ?-catenin marking it for degradation by the proteasome system. With low levels/absence of ?-catenin T cell factor (TCF) transcription factors are bound to the promoter regions of wnt target genes. Also bound to these TCFs is a transcriptional repressor known as groucho. Groucho inhibits the transcription of wnt target genes
212
Describe what happens in the presence of wnt signalling
Wnt binds to its Fz and arrow/LRP 5&6 receptors in the membrane. These receptors come together and form an active complex which recruits the dishevelled protein to the complex. Dishevelled is then phosphorylated and as a result may bind to axin in the intracellular destruction complex. Arrow/LRP is then also phosphorylated this time by GSK3? and the receptor recruits axin also. Binding of the destruction complex to the receptor complex displaces the slimb protein. With slimb lost the destruction complex is inactivated. ?-catenin then accumulates inside the cell due to it not being ubiquitinated and marked for degradation by slimb. It then translocates to the nucleus of the receiving cell and displaces groucho from the TCF DNA binding proteins. In combination with additional downstream transcriptional activators this leads to the transcription and expression of wnt target genes.
213
Explain how ?-catenin degradation is achieved by the destruction complex
?-catenin is phosphorylated by CK1? first, which primes it phosphorylation by GSK3?. Phosphorylation by both kinases is required for ?-catenin recognition by an E3 Ubiquitin ligase complex (which contains b-TrCP/Slimb) and subsequent degradation by the proteasome. The serine/threonine phosphates and surrounding amino acid sequence in ?-catenin as a result of phosphorylation forms an optimal binding site for b-TrCP/Slimb. ?-TrCP/Slimb binds only after GSK3 phosphorylates the 3rd and 4th phosphorylation sites
214
Where does CK1? phosphorylation occur within the ?-catenin/armadillo structure
Sites within the N-terminal tail
215
The S/TXXXS/T(P) is the ideal site for GSK?, what does this mean
A serine or threonine residue followed by 3 residues of any identity and then another serine or threonine that has been phosphorylated by CK1?
216
What is the name of the vertebrate homologue of slimb
?-TrCP
217
Describe the structure of the SCF E3 ubiquitin ligase complex involved in ?-catenin/armadillo degradation
The Skp1-Cullin-F-box E3 ubiquitin ligase complex consists of the ring finger protein Roc1 which binds to an E2 ligase, the scaffold protein cul1 and skp1
218
Explain the role of SCF in ?-catenin/armadillo degradation
The F-box protein interacts with Skp1 via its F-box. The F-box of also interacts with the substrate via the WD40 domain that interacts specifically with phosphorylated targets
219
How does the absence of Wnt signalling lead to no expression of wnt target genes
Without ?-catenin binding to TCF, groucho remains bound. The transcriptional repression by groucho is mediate by its recruitment of histone deacetylases thought to make DNA refractive to transcriptional activation
220
How therefore does wnt signalling lead to expression of downstream target genes
In the nucleus increases levels of ?-catenin displace groucho from the TCF complex. Displacement of groucho leads to the recruitment of histone acetylase CBP/p300 and another transcriptional activator called BRG-1. These lead to transcription of wnt target genes
221
Explain how interactions between TCF/?-catenin and chromatin could also be mediated by legless (Bcl9) and pygopus genes
Mutations in these genes result in wingless-like phenotypes in Drosophila. Both genes also promote wnt signalling in mammalian cell cultures.
222
Wnt signalling components are also involved in directing planar cell polarity and convergent extension, T or F
T
223
Explain the negative feedback mechanism of wnt signalling
Dickkopf1 (Dkk) activation by wnt binding promotes the internalisation of the LRP receptors. This decreases further wnt signalling activation and has important homeostatic roles.
224
Give examples of planar cell polarity events caused by wnt signalling
Wnt signalling aligns all the hairs in the skin in a certain direction
225
Explain how defective wnt signalling can cause cancer, particularly in the gut
Patients who are heterozygotes for APC loss of function mutations suffer from familial adenomatous polyposis. This is where sporadic loss of the other functional wild type APC allele in the gut results in activation of the wnt signalling in such cells. This causes hyperproliferation and culminates in the formation of polyps which may accumulate further mutations and cause colon cancer.
226
The APC gene is a proto-oncogene, T or F
F – it’s a tumour suppressor gene (loss of function results in tumorigenesis)
227
Give an example of another disease phenotype caused by mutation(s) in wnt signalling
Tetra-amelia is a disease where the infant is born without limbs. This is caused by a mutation in wnt3
228
What are the two main problems faced during segmentation
Organism needs to increase from one cells to many cells and makes these cells different from each other
229
Segmentation is highly conserved in all organisms, T or F
T
230
Where is segmentation easily visible in the Drosophila embryo
IN the anterior abdomen
231
When does segmentation occur in Drosophila embryos
Around 24 hours after fertilisation
232
The Drosophila embryo must set up all of the body axes in order to undergo segmentation, T or F
F – the anterior-posterior and dorsoventral axes have already been partially established in the oocyte by the adult fly. The embryo just needs to define and redefine this pattern
233
What was the contribution of Christiane Nüsslein-Volhard and Eric Wischaus to the study of Drosophila
They undertook a saturation genetic screen to identify all the genes involved in development and patterning of the larval cuticle. This lead to the identification of 4332 embryonic lethal mutations and 139 complementation groups involved in patterning
234
Most genes in development require both alleles to be functional to be expressed properly and provide normal function (haploinsufficient), T or F
F – most genes involved in development are haplosufficient
235
Describe how complementation testing can be implemented to determine if mutations lie in the same gene or different ones
If you cross two parent individuals that both have the same mutant phenotype you can determine if these mutations lie in the same gene or different genes. If the progeny produced by breeding these two heterozygotes do not show the mutant phenotype, then the mutations are said to complement each other. If 25% of the progeny do show the mutant phenotype, then the mutations of the parents must lie in the same gene and thus fail to complement each other
236
Recall the hierarchy of genes that dictate patterning in the Drosophila embryo
Maternal genes--> Gap genes--> Paired-rule genes--> Segment polarity genes
237
What type of gene is gooseberry
Segment polarity gene
238
What type of gene is bicoid
Maternal gene
239
What type of gene are knirps, giant, Krupel and tailess
Gap genes
240
Paired and fushi tarazu are hox genes, T or F
F – they are paired-rule genes
241
Where is paired expressed in the Drosophila embryo
In alternating parasegments
242
Gooseberry is expressed in alternating parasegments, T or F
F – it is expressed in all parasegments
243
Genes higher up the patterning hierarchy have a greater influence over segmentation, T or F
T
244
Give an example of another maternal gene, other than bicoid and explain where its expressed and its role in development
Nanos is another maternal gene. It is expressed in posterior embryo and is responsible for patterning the posterior larvae
245
What is seen in bicoid mutants and why is this
Bicoid mutants develop without head structures. This is because bicoid is at its highest concentration at the anterior end and dictates formation of the head structures
246
What is unique about bicoid and its role in development
Bicoid is a morphogen but is also in itself a transcription factor, unlike other maternal and patterning genes that are usually just transcription factors.
247
What is the role of bicoid in establishing the anterior-posterior axis prior to fertilisation
Bicoid mRNA is deposited in the anterior oocyte by the adult female fly in a process called maternal loading. This leads to the localisation of bicoid protein at the anterior region of the embryo
248
Describe the bicoid gradient in the early Drosophila embryo
Bicoid has its highest concentration at the anterior region of the embryo and drops away towards the middle
249
What unique feature of the Drosophila embryo allows the bicoid gradient to be established easily and accounts for the simple early patterning
At this stage in development the Drosophila embryo is a syncytial blastoderm whereby many nuclei are contained in the same cytoplasm. This allows the bicoid protein to diffuse easily through the embryo and establish a gradient easily
250
Explain the transplantation experiments carried out on the Drosophila embryo and what they showed
Transplantation of wild type Drosophila embryo cytoplasm into a bicoid mutant was sufficient to rescue some of the head structures. Transplantation of wild type cytoplasm to an ectopic site in the middle of the embryo lead to a duplication of the embryo with head structures developing in the middle flanked by thoracic segments either side. This shows that bicoid is both necessary and sufficient to dictate head structure formation
251
What happens if both copies of the bicoid gene are mutated, why is this
If both copies of bicoid are mutated then the embryo will try to pattern normally using other maternal genes that are functional such as nanos. However the resultant patterning would still be abnormal
252
What is the effects of forced overexpression of bicoid
Shunting of the segments towards the posterior end
253
With morphogens, the ligand doesn’t act as the signal to dictate differentiation as such but it is in fact the gradient of the morphogen that encodes information on cell fate, T or F
T
254
How can bicoid expression be visualise in situ
Fusing reporter genes for chaperones such as Hsp70 or genes easily stained for such as LacZ to the promoter sequence for bicoid allows easy visualisation. Wherever bicoid is expressed the reporter gene will be to as its under the control of the same promoter sequence. These reporter genes can then be easily visualised
255
What attribute of morphogens accounts for there ability to be used to carry out multiple different functions
The there are different affinity sites for transcription factor morphogens such as bicoid. Some sites in the DNA have low affinity for the transcription factor ligand and so will only elicit binding at really high concentrations. However, other sites will have high affinity and will always elicit binding of the morphogen regardless of concentration
256
What is the role of gap genes
Subdivide the embryo into different parts once the basic pattern is established
257
What is significant about gap gene interactions
The gap genes actually also interact and repress each other as well as their targets
258
Hunchback is a gap gene that regulates points in the embryo where other gap genes are switched on, what is significant about its expression with regards to other maternal gene(s)
Hunchback expression directly mirrors bicoid
259
How are the paired-rule genes expressed in the Drosophila embryo
Expressed in alternating parasegments whereby their expression is controlled stripe by stripe
260
What is paired-rule gene expression dependant on
Interactions of positively and negatively acting transcriptional regulators, many of which are gap genes
261
Even-skipped is a paired-rule gene only expressed in parasegments 3. What conditions are required for even-skipped expression
Very low concentrations of giant and kruppel, high concentrations of hunchback and a little bit of bicoid
262
Describe the expression pattern of segment polarity genes
Segment polarity genes are expressed in all 14 parasegments
263
What has happened in the Drosophila embryo by the time that the segment polarity genes are expressed, and what are the implications of this
Cellularisation has occurred meaning that signalling pathways dictating patterning and development now need to get more complicated. Factors used for signalling now need to be secreted
264
Engrailed is a segment polarity gene that is always expressed in the very anterior part of the segment, T or F
F – this was initially thought to be the case but in fact, engrailed is aways expressed most posteriorly in the segment
265
What common phenotype is seen in wingless and hedgehog mutants in Drosophila and why is this
Both mutants are said to have a lawn of denticles in the larvae. This is because both genes maintain each other, hh maintains wg which supresses denticle development
266
What is the function of wingless and hedgehog together
They work together to inhibit the formation of the denticles
267
The segment polarity gene engrailed is switched on by wingless, what is the role of engrailed
Engrailed then switches on hedgehog
268
Explain how wingless and hedgehog interact
Hedgehog signals in a paracrine manner to the adjacent cell to switch on wingless expression. Wingless then acts on its own receptors in an autocrine manner to upregulate is expression but also acts via engrailed to upregulate hedgehog too
269
Hh and wg feedback onto each other to maintain their expression and refine segment borders, T or F
T
270
The hedgehog-wingless signalling loop is present in in the posterior parts of the segments, T or F
T
271
Wg is expressed uniformly throughout the Drosophila embryo, T or F
F – its expressed as a gradient
272
What is the role of the Hox genes (homeobox domain containing genes)
Hox genes provide identity once the segments have already formed
273
Hox genes are transcription factors, T or F
T
274
From which genes do the hox genes receive input from
Pair rule and gap genes
275
What is unique about the expression of the hox genes
They are expressed along the anterior-posterior axis in the same order in which they lie in the genome
276
What happens due to a of loss of function in a hox gene
The segment will adopt a different identity, this is known as a homeotic transformation
277
What is meant when Drosophila are referred to as long germ band organisms
All 14 segments are defined at once. All tissues are present they just need defining during development. This process is relatively quick (24hours in Drosophila)
278
How do short germ band organisms develop differently to long germ bands
Similar process to short band organisms in the anterior head and thoracic segments. However, the abdominal segments are added sequentially by a region known as the posterior proliferative disc budding off segments as it gets smaller
279
Short germ band segmentation is more complex and slower than that of long band organisms, T or F
T
280
What signalling pathway mediates the segmentation of short band organisms
Delta-Notch
281
Explain how the segmentation clock of delta, notch and Hes1 accounts for segmentation in short germ band organisms
Notch and delta signal to each other in a juxtacrine manner. Activation of the Notch receptor leads to the expression of Her/Hes1 which inhibits delta expression in the nucleus of that cell. At high concentrations Her also inhibits itself. This mechanisms acts as a molecular oscillator or clock whereby notch activity rises and then triggers the expression of Her. Her then switches off delta and then itself. This causes notch to rise again and the process repeats. This pattern the segments at exactly the right time as the organism grows based on the time taken to produce Her mRNA and then translate it and switch delta off and then itself
282
How is the segmentation clock relevant to vertebrates
Humans are short band organisms. The primitive streak in vertebrates also use the notch pathway and its oscillations to produce and pattern body segments
283
Which axis is the first to be laid down during embryonic development
Anterior-posterior axis
284
Which germ layer forms at the primitive streak during gastrulation
Mesoderm
285
What experimental method lead to the identification of the origin of mesodermal tissue development
Lineage tracing
286
What determines the adoption of different mesodermal fates in the developing embryo
Where the cells ingress along the anterior-posterior axis into the primitive streak
287
Put these different mesodermal tissue types in order from most anterior to most posterior. Intermediate mesoderm, axial mesoderm, lateral mesoderm, paraxial mesoderm
Axial mesoderm, paraxial mesoderm, intermediate mesoderm, lateral mesoderm
288
The axial mesoderm is one of the mesodermal tissues formed by the ingression of cell into the primitive streak, what two subtypes of mesoderm does it give rise to and where
Prechordal mesoderm (anteriorly) and the notochord (posteriorly)
289
Which mesodermal tissue gives rise to the somites
Paraxial mesoderm
290
Cells that ingress posteriorly to the paraxial mesoderm give rise to which tissue
Intermediate mesoderm
291
The more posterior a cell ingresses into the primitive streak, the more posterior the mesoderm it will give rise to will be, T or F
F – more posterior ingress, the more lateral the mesoderm
292
Alike the axial mesoderm, the posterior paraxial mesoderm is also subdivided, what are these subdivisions
Unsegmented posterior paraxial mesoderm and the segmented posterior paraxial mesoderm that give rise to somites
293
What structures do the intermediate mesoderm give rise to
Kidneys and gonads
294
How many components is the lateral mesoderm divided into and what do these divisions give rise to
The lateral mesoderm divides into 3 components, two of which give rise to the circulatory system and the other which contributes to extraembryonic structures and limb bones
295
List the structures that the paraxial mesoderm give rise to
Axial skeleton, heart, somites, cartilage and tendons
296
The formation of somites occurs in a sequential manner on both sides with the size of somites being preserved throughout, T or F
T
297
The pre-somitic mesoderm is unsegmented, T or F
T
298
Describe the rough structure of the somites viewed as a horizontal cross section
The somites show a clear metameric structure with a clear repeating pattern and defined anterior and posterior boundaries
299
Somites are the earliest evidence of segmentation in vertebrates, T or F
T
300
How do the number of somites relate to segmentation in vertebrates such as humans
Somite number dictates the number of vertebrae
301
How many somites are present in humans and how does this relate to their segmentation
The human embryo has between 38 and 44 somites, this correlates to the 33 vertebrae which we are born with
302
The number of somites is fixed for any given species and the timing of somite formation remains constant, T or F
F – whilst the number of somites does differ between species, so too does the timing of somite generation
303
What five factors must cells in the paraxial mesoderm be able to respond to
Positional information, mechanisms that coordinate left and right, anterior and posterior boundary formation and the formation of the cleft
304
What structure separates the left and right somites
Spinal cord
305
What model describes the periodicity of somite formation and how is this achieved
Clock and wavefront model. The clock explains the temporal component whilst the wavefront provides spatial information to drive somite formation. Where cells hit the travelling wavefront an abrupt change of property leads to the decision to form somites
306
Explain how a molecular oscillator drives mesodermal segmentation in chick embryos
In the embryo levels of the helix-loop-helix transcription factor C-hairy was found to fluctuate at different embryonic stages. Later genes were discovered that regulate the timing of this clock oscillation and are members of the notch, wnt or FGF signalling pathways
307
What is meant by the determination front
Wavefront that travels from the anterior part of the presomitic mesoderm towards the posterior embryo
308
How does the determination front dictate somitigenesis
When the cells of the paraxial mesoderm encounter oscillations from the molecular clock an abrupt change determines their formation of the next somite pair
309
What aspect of the paraxial mesoderm determines somite boundary formation
Position of the somite minus II (S-II)
310
Explain the results of ectopic grafting of boundary cells
Somite boundary cells isolated from one embryo transplanted into another embryo is sufficient to induce the formation of a new boundary. Where you’d expect to see one somite you would now get two. This shows that boundary cells instruct cells that are anterior to it to form a boundary
311
What family of genes were discovered to be expressed at the somite boundaries
Notch family genes. They are selectively expressed in the anterior or posterior part of the somite
312
Explain the results of forced lunatic fringe expression in the paraxial mesoderm and the significance of this
Lunatic fringe is a gene that blocks notch activity and thus forced expression results in an inhibition of notch signalling. This results in the formation of a new boundary and hence an additional somite
313
Give an example of a human disease that proves a role of notch signalling in mesodermal segmentation
Jarcho Lewin syndrome causes spondylocostal dysplasia due to problems with the segmentation of the axial skeleton. This occurs due to a mutation in the delta 3 ligand that alters notch signalling and problems with somite segmentation
314
Explain how the determination front is positioned
The determination front is determined at the interface of two opposing gradients. Retinoid acid which is high anteriorly and fibroblast growth factor 8 which is high posteriorly. When these gradients are equal the determination front forms.
315
Where is retinoic acid produced to dictate determination front positioning
Somites
316
RA and FGF8 agonise eachother, T or F
F – they antagonise each other
317
Explain how RA and FGF8 interact to dictate determination front positioning
High levels of FGF8 result in high levels of Cyp26 which inhibits RA synthesis. High levels of FGF8 also inhibit the production of the Rhald2 enzyme that is normally required for RA synthesis
318
What transcription factor do RA and FGF8 regulate the expression of
Mesp2
319
How do RA and FGF8 interact to regulate expression of transcription factor(s) involved in somite boundary formation
RA activates Mesp2 expression whilst FGF8 inhibits it. Mesp2 expression in turn blocks local Notch signalling. Lower Notch signalling on one side of the border results in high notch signalling in adjacent cells on the other side of the boundary.
320
What are the downstream effects of opposing notch signalling in cells either side of the boundary
High notch acitivity in one side and low activity in the cells opposite leads to formation of the somite boundary. The boundary itself forms from physical formation of cleft within the mesenchymal tissue. Downstream extensive changes in cell morphology and adhesion leads to creation of this cleft and is mediated by ephs and ephrins
321
Whose saturation mutagenesis experiments lead to the discovery of the hedgehog signalling pathway
Nusslein-Volhard and Wieschaus
322
What was seen in hh mutant Drosophila embryos
Defects in segmentation. The segments contained a lawn of denticles and no naked cuticles
323
What type of gene is hedgehog
Segment polarity gene
324
Explain how hh and wg interact
Hh directly upregulates wg. Wg then controls the expression of the transcription factor, engrailed (en) which in turn then regulates hh expression
325
Hh and wg are said to be dependent on each other, what does this mean
These genes require each other to be expressed. Loss of either gene will lead to the loss of the others expression. Thus mutations in either gene will give rise to similar phenotypes
326
Discuss the genetic conservation of the hh signalling pathway
Hh signalling is relatively conserved amongst metazoans and kingdom Animalia however not to the extent that wnt signalling is. For example C.elegans lacks hh signalling mechanisms
327
Recall some of the vertebrate homologues of hedgehog
Sonic hedgehog, Indian hedgehog and desert hedgehog
328
What accounts for the increase in hedgehog genes seen in vertebrates compared to invertebrates
Vertebrates contain more homologues of the hedgehog family genes due to genome duplication throughout evolution to account for the importance of the pathway in patterning
329
What is the significance of the N-terminal signal sequence of the hedgehog ligand when first synthesised
It targets the protein to the secretory pathway
330
What happens to the hedgehog ligand once it reaches the membrane
The N-terminal signal sequence is cleaved off by an autoproteolytic cleavage catalysed by the C-terminus of the protein
331
What enzyme carries out the autoproteolytic cleavage of the N-terminus of the hedgehog protein
Hedgehog acetyltransferase in vertebrates and skinny hedgehog in Drosophila
332
Cleavage of the N-terminal part of the hedgehog protein is coupled to a cholesterol molecule addition. This occurs as well as palmitoylation. What are the combined effects of these modifications and the effects on the signalling pathway
Addition of these groups makes the hedgehog protein very hydrophobic. This renders the molecule insoluble in water and acts to target its localisation to the membrane. In addition, it makes hedgehog unable to leave the membrane and hence restricts it to signalling only to neighbouring cells
333
What proteins are required for hedgehog release from the signalling cell and its long-range diffusion
Dispatched, Scube and other HSPGs
334
Recall some of the hedgehog receptors in vertebrates
Patched 1 and 2, Hedgehog interacting protein, Smoothened, CDO, Brother of CDO (BOC)
335
How many transmembrane domains does patched have
12
336
What is the normal action of ptc in the absence of signalling
Patched constitutively inhibits smoothened in the absence of the hedgehog ligand
337
What happens when hedgehog binds to its ptc receptor
Leads to an inhibition of the inhibitory action of the ptc receptor
338
A single ptc receptor inhibits a single smo receptor, T or F
F – a single patched molecule can inhibit the activity of several smo receptors
339
Explain the effects of ptc on smo both when the hedgehog ligand is absent and present
Patched regulates the subcellular localisation and stability of smoothened. In the absence of HH, Ptc keeps Smo from getting to the cell surface by causing its trafficking to a compartment where its degraded. When Ptc binds to HH, they both get internalized and degraded. This allows and Smo trafficking to the cell surface
340
Explain how cilia are involved in mammalian hedgehog signalling
In the absence of the hedgehog ligand, ptc1 is localised to the primary cilium of the cell and smo is excluded from this region. Hedgehog binding to ptc causes its removal from the cilium which allows smo to accumulate there and initiate signalling
341
How have mutations in cilia identified their role in hedgehog signalling
Mutations that disrupt cilia formation were found to impair hedgehog signalling too
342
By explaining its homology to other similar proteins, describe to evidence for ptc action as a pump
Ptc has homology to RND permeases which pump out toxins and are involved in multi-drug resistance. Ptc also has homology with NPC1 which can transport some molecules across membranes as well as move cholesterol containing vesicles.
343
What is Niemann-Pick Disease and how is this related to ptc
NP disease is caused by a mutation in the niemann pick protein C1 and is a lipid storage disorder that leads to the accumulation of harmful quantities of lipids in the visceral organs and brain. Ptc contains regions homologous to the structure of NPC indicating its potentially similar roles
344
Explain the hypothesised action of Ptc on small molecules in order to exerts its inhibitory effect on Smo
Ptc either pumps a small inhibitory molecule into cells or a small excitatory molecule out. Or Ptc may pump a small molecule into or out of the cells that effects Smo trafficking or localisation in the membrane
345
Explain the hedgehog signal transduction pathway in the absence of the ligand
In the absence of the ligand the activator Ci transcription factor is kept out of the nucleus by the combined action of two complexes. The first complex contains costal2 (Cos2) which is a kinesin like molecule that acts as a scaffold protein, and fused, a serine-threonine kinase. The other complex contains the Ci and the suppressor of fused (SuFu) gene. The Cos2-fused complex binds to the smo receptor and causes three other genes to act on Ci, casein kinase I, protein kinase A and glycogen synthase kinase 3?. This kinase complex processes the Ci transcription factor by phosphorylation and creates binding sites for the slimb protease machinery. Slimb protease binds and partially degrades the Ci transcription factor from its full-length activator form, CiA to its repressor form, CiR. CiR then translocates to the nucleus and represses hedgehog target gene expression
346
What happens in the hedgehog signalling transduction pathway at low concentrations of the ligand
At low concentrations of hedgehog, the PKA/GSK3?/CKI complex dissociates from the complex containing Ci, fused and cos2. As a result, active repression by CiR is lost
347
Explain the signal transduction mechanism at high levels of hedgehog ligand
Hedgehog acts through both the CKI/GSK3?/PKA and Cos2/Ci/Fused/SuFu complex by leading to phosphorylation of full-length CiA and/or SuFu. This prevents partial degradation of CiA and allows its translocation to the nucleus in its activator form. Thus, resulting in the promotion of hedgehog target gene transcription.
348
Explain the mechanism by which full-length Ci (CiA) is partially degraded by an intracellular protease in the absence of hedgehog signalling
PKA initially phosphorylates CiA which primes it for subsequent phosphorylation by GSK3? and CKI. These multiple phosphorylations create a binding site for Slimb protease binding. Slimb binds and partially degrades CiA by removing the activator domain.
349
What is the result of mutations in sites within the Ci transcription factor that are usually phosphorylated by the CKI/PKA/GSK3?
Reduction in phosphorylation of Ci and processing. This reduces Slimb binding and may lead to increase hedgehog signalling
350
What type of protein is slimb referred to as
F box domain containing protein
351
What is the name of the complex to which slimb belongs
SCF ubiquitin E3 ligase complex
352
Which family are the vertebrate homologues of the Ci transcription factor
Gli family proteins
353
What is significant about the three different vertebrate homologues of Ci
Gli1 cannot be cleave by slimb due to missing one phosphorylation site. This means that it isn’t recognised by the degradation machinery and hence is always a transcriptional activator. Gli2 and 3 however can be transcriptional activators or repressors
354
Explain the hedgehog signalling negative feedback loop mediated by ptc/ptc1
One of the downstream targets of hedgehog signalling is the ptc protein. This acts to inhibit subsequent hedgehog intracellular signalling and limits the level of activation of the pathway by reducing the range of movement of the hedgehog signal
355
What are the two effects of ptc1 induction by hedgehog
Suppression of the intracellular signalling within the cell and restricting further diffusion of the hedgehog ligand by receptor binding. The latter acts to steepen the gradient of hedgehog expression
356
Explain the negative feedback mechanism mediated by hedgehog interacting protein
Hedgehog binding signalling leads to hedgehog interacting protein induction. This acts to limit diffusion of the ligand and the level of subsequent signalling by the downregulation of hedgehog signalling stimulators CDO, BOC and GAS1
357
Give an example of a positive feedback mechanism involved in hedgehog signalling
Gli1 is a downstream target gene of hedgehog signalling. Gli1 was is a constitutive activator of hedgehog gene expression leading to a feedforward response and subsequent increase in hedgehog signalling
358
Explain the role of non-canonical hedgehog signalling in adipocyte and myocyte cellular metabolism
Activation of smo by decrease inhibition by ptc leads to the stimulation of aerobic glycolysis mechanisms in the target cells. This results in a partial metabolism of glucose and leads to the production of lactate which leaves the cells and acidifies the extracellular environment
359
What is the significance of the non-canonical hedgehog signalling in adipocytes/myocytes in tumorigenesis
Aerobic glycolysis is a feature of many tumour cells and is known as the Warburg effect
360
What is unusual about antagonists of the canonical hedgehog signalling pathway
They were found to be agonists of the non-canonical hedgehog signalling pathway
361
Explain the role of hedgehog signalling in wing patterning
Hedgehog signalling is responsible for anterior-posterior patterning in the wing imaginal disc. Hedgehog is secreted from cells in the posterior wing imaginal disc and diffuses anteriorly to induce the expression of decapentaplegic. At the boundary between the presence and absence of hedgehog, a signalling centre forms which patterns the wing.
362
Explain the role of hedgehog signalling in neural development
Sonic hedgehog is secreted from the ventral floor plate and notochord and is responsible for patterning of the neural tube. Once released sonic hedgehog diffuses into the neural tube and patterns cells within it. Depending on the concentration and duration of hedgehog exposure by neural progenitors determines their subsequent fate, this is mostly related to their position
363
Describe the role of sonic hedgehog signalling in patterning of the limb
Sonic hedgehog is expressed in the zone of polarising activity in the posterior limb bud. It is responsible for patterning posterior regions of the limb
364
What was the result of ectopic sonic hedgehog signalling in the anterior limb bud, either by ZPA cell grafting or implantation of sonic hedgehog soaked beads
Mirror image duplication of limb structures
365
Describe a non-tumour related disease associated with abhorrent hedgehog signalling and what causes it
Cyclopia and holoprosencephaly are caused by a lack of or inhibition of hedgehog signalling in the brain. Sonic hedgehog is required to pattern the ventral midline of the embryo. This diseased is characterised by a failure of the forebrain to separate into two hemispheres and results in fused features, such as eyes etc.
366
Gain of function mutations in hedgehog cause cancers, T or F
T
367
What three cancers are directly caused by ectopic hedgehog signalling
Basal cell carcinoma, medullablastoma and rhabdomyosarcoma
368
Inactivation of ptc1 or SuFu can cause cancers, what type of genes are these
Tumour suppressor genes
369
Excess activation of smoothened caused by an insensitivity to ptc1 inhibition can cause cancer, thus what type of gene is smo
Proto-oncogene
370
Gorlin syndrome or Nevoid Basal Cell Carcinoma is a disease state caused by problems with hedgehog signalling that leads to massive numbers of BCCs. Explain how this disease is caused and leads to systemic tumour formation
Patients with Gorlin syndrome are heterozygous for a loss of function mutation in ptc1. Hence sporadic inactivation of the other, functional, copy of the patched1 gene leads to tumorigenesis
371
Ptc is involved in triggering stem cell differentiation, T or F
F – it maintains stem cells in the skin via proliferation
372
Explain how smo inhibitors have been looked at as potential cancer treatments
GDC-0449 is an antagonist of the hedgehog signalling pathway that inhibits hedgehog signalling and continuous abhorrent proliferation in tumour cells
373
Currently smo inhibitors fail after long-term use, why is this
The tumours pick up additional mutations that render the smo inhibitors ineffective due to tumour resistance
374
Both wnt and hedgehog signalling molecules are modified by addition of hydrophobic moieties, T or F
T
375
What similarities are seen in the frizzled and smoothened receptors involved in wnt and hedgehog signalling
They are both 7 transmembrane domain proteins where the heteromeric G-proteins don’t play a central role in signalling
376
Both wnt and hh signalling use scaffold proteins, recall the proteins involved in each signalling pathway
Wnt signalling – uses axin. Hedgehog signalling – uses costal2
377
Phosphatases are involved in both hedgehog and wnt signalling pathways, T or F
F – kinases are involved in both
378
What is the main different in transcription factor degradation reactions that occur in wnt and hh signalling
Ci is only partially degraded, whereas ?-catenin is fully degraded
379
Which degradation machinery protein(s) are involved in both wnt and hedgehog signalling
Slimb/TCRP-?
380
In absence of the ligands in both wnt and hedgehog signalling there is transcriptional repression. Which transcription factors mediate this in each pathway
Transcriptional repression is mediate by groucho/TCF in wnt signalling whereas in hedgehog signalling this is carried out by Ci-75/CiR
381
What are the main roles of skeletal muscle
Control of coordinated movement and posture, communication through speech expression and writing, maintenance of temperature by heat release during contraction, and respiration
382
Recall the pathway of muscle development
Stem cells --> myoblasts --> myotubes --> myofibres
383
Describe the mature muscle cell
The mature muscle cell or myofibre is a multinucleate contractile cell
384
Muscle cells aren’t differentiated until they become myofibres, T or F
F – the differentiated muscle cell is called a myotube, the myofibre is a mature muscle cell
385
Myotubes are mononucleate, T or F
F – they are multinucleate
386
How do myotube form
Fusion of mononucleate myoblasts
387
At what stage in muscle development do the cells become committed to the muscle cell lineage
When they become myoblasts
388
What are C3H10T1/2 cells
These are a population of fibroblast cells that are already capable of giving rise to myocytes under certain conditions
389
What is the result of exposure of certain types of fibroblasts to 5aza and how does this occur
Exposure of the C3H10T1/2 cells to 5aza causes the fibroblasts to commit to a muscle cell fate. This is because 5Aza is a demethylating agent that demethylates histones and CpG nucleotides which results in a change in conformation of the chromatin. This provides a favourable environment for transcription and gene expression
390
What does MyoD stand for
Myogenic determinant factor
391
Explain the experimentation that lead to the isolation of MyoD
The transcriptomes of fibroblasts cells that have been exposed to 5Aza were compared to a population of the same cells that hadn’t been exposed to it to identify the different expression profiles of the cells that conferred their commitment to the muscle cell lineage. These mRNA transcripts were used to create cDNA which allowed for the isolation of MyoD
392
What is meant by MyoD being a master regulatory gene and how was this discovered
MyoD can induce a transition of a fully differentiated cell into a fully differentiated cell. This was carried out by placing the MyoD gene under a constitutively active promoter and introducing the construct into terminally differentiated cells. This results in the cells switching to a fully differentiated muscle cell fate
393
What type of protein is MyoD and how does it interact with DNA
MyoD is a basic helix-loop-helix protein that acts as a transcription factor. Its basic amino acid domain is responsible for binding to the DNA whilst the helix is involved in dimerization with E12 and E47 proteins. MyoD binds to E-box regions contained in the promoter and enhancer regions upstream of muscle related genes
394
Other than MyoD what 3 other genes in the same family act to confer muscle cell fate
Myf5, myogenin, MRF4
395
What is meant by the term myotome
The myotome is the dorsal region of the somite that will give rise to the skeletal muscles of the ventral trunk
396
Initially the somitic mesoderm is pluripotent, T or F
F – it is multipotent and can give rise to cartilage, bone and skeletal muscle
397
What is meant by the sclerotome and how is this region formed in the somites
The sclerotome is the region in the ventral somite that will give rise to the axial skeleton. It is formed when cells in the ventral somite undergo an EMT and delaminate
398
How is the myotome formed
The myotome forms when dorsal epithelial cells in the somites undergo an EMT and drop beneath the somite
399
What structures do the myotome give rise to
Skeletal muscle of the trunk (and limbs)
400
Myotome cells become sandwiched between cells that remain epithelial in the dorsal somite called the dermomyotome and the cells of the ventral sclerotome, T or F
T
401
What are the two definitive regions of the myotome and which specific structures do these give rise to
Cells in the medial myotome give rise to the epaxial muscles of the back. Cells in the lateral myotome will give rise to the hypaxial muscles of the abdomen and limbs
402
MRFs are factors expressed in the myoblasts during embryogenesis with important functions in the formation of skeletal muscle, what region is their expression restricted to
The myotome
403
What happens when you knockout Myf5 in mice
Mice produced are still viable but do show delayed myotome formation until MyoD is expressed
404
What is seen in MyoD knockouts
Mice are born viable with no obvious muscle defects at birth. There is a slight delay in limb muscle development and a deficit in muscle regeneration in adults
405
What is meant by functional redundancy
One gene can restore the function of another gene if that gene is knocked out
406
What can be done to determine if Myf5 and MyoD act in functional redundancy
Double knockout mice
407
What is seen in the MyoD and Myf5 -/- mice
Complete absence of skeletal muscle and no presence of myoblast cells
408
What can be determined about how Myf5 and MyoD interact and dictate muscle generation
Myf5 and MyoD act in functional redundancy and are required to generate myoblast cells
409
What is seen in myogenin knockout mice
The offspring die shortly after birth from a diaphragm defect. They also have reduced density of myofibres. The mice are capable of producing myoblasts but not myotubes
410
What can be inferred about myogenin from its knockout in mice
Myogenin is required downstream of MyoD and Myf5 to confer muscle differentiation from myoblasts to myotubes
411
Which genes are expressed during each stage of muscle cell development
MyoD, Myf5 and MRF4 expressed during specification of stem cells to a muscle lineage. Myogenin expressed during differentation. MRF4 expressed again during maturation
412
Which region of the myotome gives rise to the muscles of the back
Epaxial myotome
413
Which region of the myotome gives rise the muscles of the abdominal wall
Hypaxial myotome
414
Which cell signalling events are required for formation of the epaxial myotome
Sonic hedgehog signalling from the notochord and floor plate and wnt signalling from the dorsal neural tube
415
Which cell signalling events are required for formation of the hypaxial myotome
Wnt signalling from the ectoderm and BMP4 from the lateral mesoderm
416
Which region contains cells that will give rise to the muscles of the limbs
Lateral myotome
417
MyoD and Myf5 expression if delayed in the cells that become the muscles of the limb, explain the events that lead up to this delay
Cells in the lateral myotome undergo and EMT to initiate their migration. It is only once they have reached the dorsal and ventral positions in the limb bud where they begin to differentiate
418
Describe the splotch mouse phenotype
Splotch mice have a loss of function of the Pax3 transcription factor and have neural tube and brain defects. Importantly for muscle development however, they have muscles of the trunk that develop normally but have a complete lack of skeletal muscles in the limbs
419
What can be inferred about the action of Pax3 from splotch mice data
Pax3 acts upstream of MyoD in the cells of the somite that are to migrate and give rise to muscles of the limb
420
How does Pax3 act
Pax3 is a transcription factor that is expressing in somitic cells and drives the expression of the c-Met receptor. c-Met binds to its ligand hepatocyte growth factor (HGF) which is synthesised by the mesenchymal cells of the limb. This acts as a chemoattractive signal to direct these cells to migrate to the limb bud
421
Depletion of Pax3 results in a failure of limb skeletal muscle cell progenitors to migrate to the limb bud, T or F
T
422
What is the name given to muscle stem cells that are retained in adult muscles and dictate muscle regeneration
Satellite cells
423
What is significant about the number of muscle stem cells during development and what role does this indicate
Satellite cells make up 32% of muscle nuclei at birth but this decreases to only 5% by adulthood. This indicates the role of these cells in directing postnatal muscle growth
424
What stimuli can trigger the induction of muscle stem cells in adults
Injury, exercise, denervation and stretching. They can also become more active in muscle wasting disease such as Duchene muscular dystrophy
425
What is significant about the gene expression by muscle specific stem cells in muscle regeneration
They express the same sequence of genes as is seen in development and embryogenesis firstly expressing Pax7(duplicate of Pax3) then Myf5, MyoD and Myogenin
426
What signalling pathway is responsible for induces MyoD expression and causing muscle differentiation
Sonic hedgehog
427
What does TGF-? stand for
Transforming growth factor-?
428
What are the four main branches of TGF-? signalling
BMPs/GDFs, TGF-?, Activin and Nodal signalling
429
Give an overview of the TGF-? signalling pathway
The TGF-? ligand binds to the extracellular domain of the Type II TGF-? receptor and promotes its further binding to the Type I TGF-? receptor via the ligand also. The activated TGF-? receptor dimer undergoes transphosphorylation of the Type I receptor by the action of the intracellular serine-threonine kinase domain of the Type II receptor. This transphosphorylation causes the binding of a cytosolic Smad proteins which then get phosphorylated, dimerise with other Smads and translocate to the nucleus where they regulate gene transcription.
430
What are the four ligand subfamilies of TGF-? signalling
Activin, Nodal, BMPs/GDFs and TGF-?
431
Which TGF-? ligand family have an important role in left/right patterning and splitting of bilateral symmetry
Nodal
432
Which TGF-? ligand family have an important role in proliferation and differentiation of cells
TGF-?
433
Which TGF-? ligand family have an important role in hormonal regulation
Activins
434
What is meant by the ligand and receptors involved in TGF-? signalling being referred to as promiscuous
There are multiple ligands and receptors that can interact in different combinations
435
The TGF-? precursor is cleaved into two parts by the action of a protease, what are these two regions and what is their role
The C-terminal region is responsible for the formation of the active ligand by dimerisation with other TGF-?s. The N-terminal part binds to TGF-? and is called Latency Associated Protein (LAP)
436
What kind of bonding hols the LAP and TGF-? dimers together in a complex
Disulphide bridges between adjacent cysteine residues
437
What is the role of Latent TGF-? binding protein (LTBP)
LTBP tethers the LAP part of the complex to components of the extracellular matrix to increase the effective concentration of the ligand by restricting its diffusion
438
Explain the processes that lead to the release of the active ligand from the complex
Release of the TGF-?/LAP/LTBP complex from the extracellular matrix involves cleaves by the proteases plasmin and calpain that releases the complex from the extracellular matrix. Subsequent binding of thrombospondin to LAP releases the active TGF-? ligand
439
An in situ hybridisation experiment revealed the expression of TGF-? in a developing chick embryo, it was later determined however that these cells weren’t signalling. Explain how this could be
Expression of a ligand by a cell doesn’t necessarily mean that the cell can signal. If a cell fails to make the proteases for example, then it will fail to release the active ligand
440
The BMP family of TGF-? ligands are negatively inhibited by several different proteins that act as extracellular antagonists. List 5 of these such BMP antagonists
Noggin, Chordin, Follistatin, Cerberus, Gremlin
441
How many classes are there of general TGF-? antagonists and what is the purpose of these different families
There are five different families of TGF-? negative regulators these allow for distinct expression profiles allowing for greater refinement of the signal by binding of different subsets with different affinities.
442
What are the two ways in which TGF-? signalling antagonists can act
They can either compete for binding at one site on the ligand (I.e. gremlin, noggin, cerberus and DAN on BMP2) or, antagonists can bind to independent sites allowing for multiple inhibitor binding
443
The extracellular TGF-? antagonists all have the same rate of diffusion, T or F
F – they all have different diffusion rates, for example chordin is the slowest as it’s the largest protein (120kDa)
444
Some extracellular TGF-? signalling antagonists can bind to multiple ligands whilst others can only bind to one, T or F
T
445
Describe the activation of the TGF-? receptor following ligand binding
Activation of the receptors comes from the formation of a dimer receptor complex. The Type II TGF-? receptor binds to the TGF-? ligand homodimer and then recruits and phosphorylates the Type I receptor. This leads to the phosphorylation of Smads and further downstream signal transduction
446
Structurally, Type I and Type II TGF-? receptors are very similar. What action do they both have
They are both serine-threonine kinases but have different functions in the pathway
447
Lefty and antivin are TGF-? antagonists that inhibit Nodal signalling, explain how these pseudo-ligands act
These antagonists bind to the Type II receptors but lack an ?-helix loop regions that allows for dimerisation and the cysteine resides responsible for disulphide bridge formation. This ultimately results in the inability of the Type II receptor to bind to the Type I receptor
448
Decoy receptors can also inhibit TGF-? signalling, give an example of this
The BAMBI pseudo-receptor resembles the Type I receptor but lacks the intracellular kinase domain. It forms an inactive complex with the ligand and the Type II receptor and inactivates BMP, activins and TGF-? signalling
449
Explain the TGF-? signal transduction pathway following ligand binding
The activated Type I receptor phosphorylates Smad proteins. The type of TGF-? ligand that binds to the receptor determines the specific smads phosphorylated by the activated receptor. BMP ligand binding leads to phosphorylation of Smad1,5 and 8 whilst TGF-? binding results in Smad2 and 3 phosphorylation. Smads1,2,3,5 and 8 are collectively known as receptor-regulated or R-Smads. R-Smads are maintained near the plasma membrane close the kinase domains of the receptors. When the pathway is inactive the R-Smads are anchored at the cells membrane by Smad anchor for receptor activation proteins (SARA). Once R-Smads have been phosphorylated by the activated Type I receptor they oligomerise with Smad4 to form heterodimers. This Smad complex then migrates to the nucleus where its binds to the DNA to regulate gene expression
450
What is significant about Smad4
Smad4 is constitituvely expressed and unlike the other Smads, it is common to all TGF-? signalling pathways. It is therefore referred to as the common-mediator or Co-Smad
451
What are the results of mutations in Smad4
Disruption of all TGF-? signalling pathways
452
Smads are transcription factors, T or F
T – they can be transcriptional activators or transcriptional repressors
453
How do Smads lead to an increase in gene transcription
Smads can recruit and bind to histone acetylases one the DNA. These histone acetylases add acetyl groups to the histones which loosens the structure of the chromatin and increasing gene transcription
454
How can Smads lead to a decrease in gene transcription
Smads can also bind to co-repressors and recruit histone deacetylases (HDACs) which remove acetyl groups from the histones and decrease gene transcription by tightening the chromatin structure.
455
Explain the role of Smad6 and Smad7 in the Smad pathway
Whilst smad2 and smad3 are positive regulators of the TGF-? signalling pathway, Smad6 and 7 are negative regulators. These are also known as inhibitory I-Smads and antagonise TGF-? signalling by binding to the activated Type I receptor and activated R-Smads, blocking their activity.
456
Explain the two attributes of I-Smads and how this influences TGF-? signalling
I-Smads still bind to type I receptors but are not substrates so aren’t release. They also still bind to R-Smads but do not work as transcription factors
457
What are the three key domains of an R-Smad and what is their function
The MH1 domain in the amino terminus is responsible for binding to the DNA. There is an MH2 interaction domain that controls protein-protein interactions and regulates the homo/heterodimerisation of Smads. Finally, R-Smads also contain a domain in the carboxyl terminus that acts as a recognition site for phosphorylation by the activated Type I receptor
458
How does the structure of an I-Smad differ from that of an R-Smad
Whilst I-Smads have retained the MH2 interaction domain they lack the functional MH1 DNA binding domain as well as the phosphorylation domain
459
TGF-? is expressed prior to apoptosis and acts as a pre-requisite to indicate the programmed cell death. Give an example of a role of TGF-? signalling in apoptosis
Mouse mammary glands upregulation TGF-?3 within 9 hours of inactivity after weening. This results in death of the milk-secreting cells. After 3 days these cells will begin to express genes characteristic of apoptotic cells
460
What type of mutations related to TGF-? signalling are often found in patients with certain cancers
Hypomorphic (partial loss of function) mutations
461
If mutations in TGF-? signalling lead to some cancers, what can we infer about the role of this pathway in tumour formation
It is likely that TGF-? signalling supresses tumour formation
462
What is often seen in the cancer causing TGF-? signalling mutations
Often involve microsatellite instabilities characterised by increases or decreases in the length of microsatellite repeat sequences in the DNA. Unstable microsatellites near to or in genes is thought to affect their transcription and may cause problems during DNA replication. This may indicate that the DNA repair machinery is malfunctioning
463
It was later found that mutations that abolish TGF-? signalling (amorphic mutations) are less dangerous than hypomorphic mutations, why is this
Later in cancer progression TGF-? signalling promotes tumorigenesis
464
Explain the dual role of TGF-? signalling in cancer progression
TGF-? signalling inhibits the over proliferation of mutated cells to form benign legions in early stages of cancer. However, later in the progression of the cancer TGF-? signalling promotes the EMT and angiogenesis ultimately resulting in metastasis
465
Explain how both hypomorphic and amorphic TGF-? signalling mutations impact cancer and tumorigenesis
Null/apomorphic mutations cause benign lesions which cannot go through EMT. Hypomorphic mutations increases rate of benign lesions, by decreasing inhibition of clonal expansion, with enough signalling still conserved to trigger later stage EMT and metastasis.
466
The neural plate cells are a neuronal population of cells, T or F
F – they are a glial population
467
Which structure begin to express sonic hedgehog in the axial mesoderm as is extends forwards
Prechordal mesoderm, notochord
468
At what chick embryonic stage is sonic hedgehog first expressed at and what does the correspond to
Sonic hedgehog is first expressed in the axial mesoderm at stage 4 where the primitive streak has reached its maximum extension
469
In vertebrates sonic hedgehog is expressed in the axial mesoderm and the floor plate simultaneously, T or F
F – sonic hedgehog is first expressed in the notochord and prechordal mesoderm and then induces its own expression in the floor plate of the ventral neural tube
470
Cells of the axial mesoderm and floor plate are responsible for ventralising the neural tube, T or F
T
471
Explain the differences between experiments visualising sonic hedgehog mRNA
In situ hybridisation experiments aimed to visualise where sonic hedgehog mRNA is transcribed shows a clear define pattern. Initially this shows a clearly defined, round expression pattern indicating its expression in the notochord/prechordal mesoderm. Later experiments would indicate another clearly defined triangular region of expression lying dorsally to the notochord and in the ventral neural tube. This identifies the newly induced sonic hedgehog expression by the floor plate.
472
How do experiments looking at the expression of sonic hedgehog protein differ from those visualising mRNA
Immunohistochemistry experiments using antibodies to identify where sonic hedgehog is localised does show these round and triangular regions representing the axial mesoderm and floor plate. However, the expression is punctuate and more diffuse indicating a morphogen gradient. Sonic hedgehog is highest at the axial mesoderm and floor plate and decreases dorsally throughout the neural tube
473
How does experimental evidence indicate sonic hedgehog is a morphogen
Immunohistochemistry to visualise where sonic hedgehog expressed reveals an expression pattern that indicates a morphogen gradient
474
What molecules are attached to antibodies to enable you to visualise proteins in a cell/tissue
Fluorochromes
475
Genes encoding different transcription factors begin to be transcribed and translated in different cells along the D-V axis in a precise spatial manner, T or F
T
476
Which gene is expressed most dorsally in the neural tube having initially been expressed throughout the axis prior to sonic hedgehog expression
Pax6
477
What happens later in development as cells of the neural tube begin to differentiate
As progenitor cells differentiate into neurons that differentiating progeny move laterally so that mature neurons are present in the mantle zone. The other daughters remain as progenitors near the lumen of the neural tube
478
Sonic hedgehog patterns the ventral portion of the developing neural tube, T or F
T
479
Below are a list of regions contained in the ventral neural tube that are formed as a result of sonic hedgehog signalling, put them in order of dorsal to ventral. A - floor plate, B - Motor neuron progenitor, C - ventral progenitor 1, D - ventral progenitor 0, E - ventral progenitor 3, F - ventral progenitor 2
d, c, f, b, e, a
480
What transcription factors are characteristic of motor neuron fates
Olig2 and Nkx6.1
481
FoxA2 is a transcription factor characteristic of the floor plate, T or F
T
482
Dbx 1 and dbx2 are expressed in which regions of the ventral neural tube
Ventral progenitors 0, 1 and 2
483
Sonic hedgehog acts as a morphogen to regulate transcription factor expression by directly inducing their expression, T or F
F – sonic hedgehog activates transcription factor expression through de-repression
484
Explain how sonic hedgehog signalling leads to transcription factor expression in the ventral neural tube
Genes in the neural tube are initially expressed throughout the structure due to Wnt and BMP signalling. Shh then suppresses their expression and confines them to the dorsal part of the spinal cord. These genes are differentially sensitive to Shh (Pax7, Dbx1, Dbx2, Irx3) and are referred to as class 1 genes, transcription factors which act to repress other genes. As class 1 genes are transcriptionally repressed by Shh class 2 genes are turned on because they are de-repressed (Nkx6.1, Nkx2.2, Olig2)
485
Give examples of class 1 genes transcriptionally repressed by sonic hedgehog
Pax7, Dbx1, Dbx2, Irx3
486
Give examples of class 2 genes switched on by sonic hedgehog signalling
Nkx6.1, Nkx2.2, Olig2
487
GliA is an example of a gene that is switched on by sonic hedgehog. What is the role of GliA in patterning the neural tube and how is the mechanism of its activation different to most other genes switched on by sonic hedgehog
GliA is a transcription factor that defines the floor plate and turns on the expression of other characteristic floor plate genes. It, along with a few other genes, is directly activated by very high concentrations of sonic hedgehog
488
BMP signalling is responsible for patterning the dorsal neural tube. How is a BMP gradient established dorsally in the neural tube
BMPs from the surface ectoderm induce their own expression in a dorsal group of cells called the roof place cells. BMPs then establish a dorsal morphogen gradient
489
What other key signalling pathway is active in the dorsal neural tube and is thought to maintain proliferation
Wnt signalling
490
What is the name of the border that forms at the neural-ectoderm boundary
Neural plate border
491
What causes the formation of this border at the neural-ectoderm boundary
Cells have received an intermediate level of BMP and have begun to go down but not fully towards a neural fate
492
What structures does the neural plate border give rise to
Peripheral nervous system, roof plate cells
493
The early border begins to express msx which acts with Wnt and FGF to turn on transcription factors Pax3, Zic1 and Pax7, T or F
T
494
What transcription factors upregulated by NBP and Wnt cause proliferation and multipotency and characterise neural crest cells
C-Myc, Id and Snail
495
What cell types do the neural plate border cells form
Neural crest cells
496
All of the neural plate border cells form neural crest cells, T or F
F – some neural plate border cells are retained and form roof plate cells
497
What is the roles of roof plate cells
Important in the final step of neurulation and dorsal neural tube patterning
498
BMPs and Wnts released by the roof plate cells diffuse into dorsal neural tube and induce expression of which set of transcription factors
Pax6, 7, 3 and Lim1
499
What is the role of Pax3, 6, 7 and Lim1
Cause neural tube progenitors to acquire dorsal identities
500
What factors help determine which cell types neural progenitors differentiate into
Position or origin of neural crest cells, time of generation and migratory pathway
501
How many different progenitor domains are established along the DV axis that give rise to different neuronal populations
10-12
502
What neuronal fates with the most ventral cells in the neural tube adopt and which transcription factors are involved in this
The most ventral neurons will become motor neurons of the spinal cord reflex arcs and will begin to express Olig2 and Nkx6.1
503
What will the most dorsal neurons in the neural tube give rise to
These neurons will become the commissural sensory relay neurons that relay information from the spinal cord to the brain
504
What do the middle neurons between the most dorsal and ventral progenitors become
These will become interneurons that mediate interactions between the other neurons
505
Below is a list of the embryonic origins and aspects of the skeleton that they give rise to. Match up the embryonic origin with the bone structure. A - cranial neural crest cells, B - lateral mesoderm, C - somites. i - limb skeleton, ii - axial skeleton, iii - craniofacial skeleton
a – iii, b – ii, c – I
506
Each different embryonic precursor gives rise to a different bone structure using completely different mechanisms, T or F
F – the process of forming the bones is very similar in all regions, with the extracellular matrix playing a big role
507
What structure gives rise to the somites that go on to give rise to the vertebral column and ribs
Paraxial mesoderm
508
What embryonic precursor structure gives rise to the appendicular skeleton
Lateral mesoderm
509
Hox gene expression provides the positional information responsible for the adoption of different cell fates along the anterior-posterior axis. What is the effect of this on formation of bones
The morphological structure of the axial skeleton throughout the anterior-posterior axis (i.e. ribs and vertebrae)
510
What is meant by the collinearity of hox gene expression
Hox genes are expressed along the anterior-posterior axis in the same sequence that they occur in the genome. 5’ genes are expressed most posteriorly and 3’ are expressed anteriorly
511
What do the boundaries between the expression of different hox genes/hox profiles indicate in the axial skeleton
Boundaries between different hox expression correlates with a transition in the type of vertebrae
512
What are the three stages of axial skeleton formation
Sclerotome induction, cartilage formation and ossification
513
What is meant by chondrogenesis and osteogenesis
Chondrogenesis is the generation of cartilage and osteogenesis is the formation of bone
514
Below is a list of cells in the chondrogenesis pathway and the corresponding processes they undergo. Put the cells in chronological order in which they appear and determine which process each undergoes to produce the next cell. A - Chondroblasts, B - Stem cells, C - Specification, D - Hypertrophic chondrocytes, E - Differentiation, F - Sclerotomal cells, H - Maturation, I - Chondrocytes, J - Determination
b(c), f(i), a(e), h(g), d
515
Which two paired-box genes with homology to the Pax3/7 genes involved in myogenesis play a key role in chondrogenesis
Pax1/9
516
Which regions of the somite are the specific pax genes involved in axial skeleton development localised in
Ventral somite – Pax1 tends to be medial whereas Pax9 is lateral
517
What is seen in mice knockouts for the Pax genes involved in chondrogenesis
Pax1 knockout produces viable mice that do show abnormalilites in vertebral column, sternum and scapula. Pax9 knockout produces mice that die shortly after birth due to abnormal craniofacial, visceral and limb skeletogenesis
518
What is the result of knocking out both pax genes involved in skeletogenesis in mice and what does this show
The mice completely lack derivatives of the medial sclerotome including the vertebral bodies, interverbal discs and proximal rib regions. The distal and sternal rib portions are maintained however. This shows that Pax1 or Pax9 are required for medial sclerotome development
519
Explain the role of sonic hedgehog signalling in the control of sclerotome formation
Sonic hedgehog secreted from the ventral floor plate and notochord activates Pax1/9. Shh (-/-) fail to activate Pax1/9 and don’t development axial skeletons
520
Explain how BMP4 signalling acts to control Pax gene expression in the ventral somite
BMP4 from the lateral mesoderm prevents Pax1 expression from expanding into the Pax9 expression domain.
521
What four processes to sclerotomal cells undergo as they give rise to chondroblasts
Migration of cells around the notochord, downregulation of Pax1 and Pax9, the condensation of the cells and expression of extracellular matrix proteins
522
As chondroblasts differentiate into chondrocytes, proliferation is induced by expression of BMP2, BMP4 and BMP5 and a cartilaginous matrix is produced. What gene expression is required for this process
SOX9 HMG-box transcription factor
523
What method of ossification is seen in the bones of the skull
Intramembranous ossification – this doesn’t involve chondrocytes or chondroblasts and instead includes nodules (mesenchymal cells --> nodules --> bone)
524
Endochondral ossification is the other main type of ossification, when is this kind used and how does it work
Used for ossification of most bones other than those of the skull. It involves the development of bones by the replacement of a cartilage model
525
What is the first stage of endochondral ossification
Chondrogenesis the formation of a cartilaginous model of the bone
526
What happens when chondrocytes mature and stop dividing
They become hypertrophic and increase their cell volume
527
As a result of a change in area at the periphery of the future bone, the region becomes known as the perichondrium. What is the significance of this region
The perichondrium is the precursor of osteoblasts
528
Following the maturation of chondrocytes, they then undergo apoptosis. What fills the space left behind by this cell death
Blood vessels and osteoblasts from the perichondrium enter the space left by chondrocyte death and begin to secrete the bone matrix. This region will become the bone marrow.
529
What is meant by the term epiphyses
The ends of the bones
530
Explain how primary and secondary ossification centres form
Osteoblasts begin to replace the disappearing cartilage and form a primary ossification centre where the centre of the bone is devoid of hypertrophic chondrocytes and full of a bone matrix. Blood vessels enter at the epiphyses which have remained cartilaginous during bone development. These epiphyses form the secondary ossification centres and leaves a cartilage growth plate between the epiphysis and diaphysis
531
What is significant about the need to ensure that chondrocytes of the secondary ossification centre don’t differentiate into osteoblasts too early
This is vital to ensure normal growth of the bone which would be prevented if the chondrocytes differentiated prematurely
532
A balance between which two genes allows the bones of the axial skeleton to grow but not maturate and differentiation too early
SOX9 and Runx2
533
How has the study of the disease campomelic dysplasia implicated the role of a gene in regulation cartilage and bone production
Campomelic dysplasia (CD) is a disease that causes deformations of the long bones and axial skeleton. It is a dominant disease also characterised by defective airway cartilage and a smaller rib cage with fewer ribs. These defects are like those caused by mutations in the cartilage collagens II and XI. It was identified that CD is causes by a loss of function mutation in the SOX9 gene which is important for the formation of cartilage.
534
What can be seen in conditional SOX9 mouse knockouts
Early inactivation of SOX9 lead to a failure of the embryo to form chondrocytes. Later SOX9 inactivation lead to a defected ability of the already formed chondrocytes to go hypertrophic
535
What are the role of SOX9 in skeletal development implicated by conditional mice knockouts
Early inactivation of SOX9 reveals its role as a transcription factor that controls the expression of cartilage extracellular matrix proteins such as collagen II and XI. Later SOX9 knockouts indicated a second role for SOX9 in balancing the proliferative and hypertrophic chondrocyte levels.
536
Explain how studying cleidocranial dysplasia revealed another key gene involved in skeletal development
Cleidocranial dysplasia is a dominant disease caused by a mutation in Runx2. More than one mutation in the Runx2 gene is embryo lethal however patients with one mutation will show defective bone formation.
537
What is seen in mouse models of Cleidocranial dysplasia
Runx2 knockout mice show cartilage formation but this cartilage fails to form bone and never ossifies
538
What is the role of Runx2 in skeletal development
Runx2 is a transcription factor that is required to drive expression of the osterix gene that is required for bone development and ossification.
539
What is the growth plate of long bones
The growth plate is a region of long bones that never ossifies and is located between the primary and secondary ossification centres. It is a stratified structure containing cells in all stages of bone development; pluripotent cells, chondroblasts, chondrocytes, pre-hypertrophic chondrocytes and hypertrophic chondrocytes
540
What is the significance of the growth plate
Disruption of this area leads to dwarfism
541
Explain the negative feedback loop between Indian hedgehog and parathyroid-related hormone that controls chondrocyte differentiation and maturation
As bone grows the chondrocytes become hypertrophic and release Ihh which signals in the same way as Shh. Cells in the perichondrium will respond to Ihh binding to their receptors and release PTHrP. The PTHrP receptor is only expressed in the chondroblasts and the pre-hypertrophic chondrocytes and hence these cells are the only ones that can respond to PTHrP. PTHrP signalling then results in the promotion of proliferation and inhibition of progression of the cells towards differentiation. As the cells differentiate they secrete a signal that will promote the proliferation of progenitors. This negative feedback loop ensures that as differentiation continues a pool of proliferative progenitors are maintained to cater for future growth
542
Recall the signalling pathway that regulates bone formation
FGFR3 --| Ihh --> PTHrP
543
Explain how Ihh or PTHrP knockout results in dwarfism
This leads to a loss of promotion of proliferation chondroblasts and pre-hypertrophic chondrocytes and the progenitor cells are exhausted quickly hence resulting in smaller bone growth
544
What class of receptors are the receptor tyrosine kinases
Enzyme-linked receptors
545
What sorts of cell behaviours are RTKs involved in regulating
Proliferation, differentiation and migration
546
There are 20 different families of RTKs, how many human RTK genes have been identified
58
547
Each ligand receptor pair involves one specific ligand and one unique receptor, T or F
F – whilst some ligands are specific for one receptor and vice-versa, some ligands and receptors can be promiscuous and bind to various other components
548
What is the overall function of RTKs
Receptor tyrosine kinases phosphorylate tyrosine residues in target proteins
549
Give some examples of RTK ligands
Ephrins, Nerve Growth Factor, Fibroblast Growth Factor, Epidermal Growth Factor
550
The ligands for RTKs all tend to be extracellular, T or F
T
551
What sort of concentration levels do RTK ligands act at
Very low concentrations in the nM or pM range
552
Most RTKs are monomers with one major exception, which receptor is this
The insulin receptor is an RTK which is present as a dimer
553
What can be said about the extracellular domains of RTKs throughout the family
The EEC domains vary greatly along with the ligands. They do however share features such as Ig-like and fibronectin-like domains
554
Describe the structure of the intracellular domain of RTKs
The intracellular domains possess the kinase activity. These are present as a single domain or split into two
555
What can be said about the transmembrane domain in RTKs
The transmembrane domain is short and string like, consisting of between 25 and 38 amino acid residues
556
Outline canonical RTK activation
Following ligand binding, either as a dimer or monomer, the monomeric RTK receptor will dimerise by recruitment of the other receptor monomer. Activation of the RTK causes a change in conformation of the receptor dimer. This starts with the extracellular and transmembrane domains and is then translated to the intracellular kinase domain. This change in conformation of the intracellular domain unmasks the tyrosine kinase domain and exposes important residues for this process. The activated receptor then undergoes auto and crossphosphorylation. This increases the activity of the kinase domains, stabilises the active state of the receptor and causes the kinase domain to phosphorylate other tyrosines in the receptor to create docking sites. These kinase domains are now able to phosphorylate target proteins that bind to the docking site to transduce the signal.
557
What are the effects of auto and cross-phosphorylation of the active RTK
Increased kinase domain activity, stabilisation of the receptor active state (ligand independent) and the creation of docking sites for target proteins
558
Explain the role of dimerisation in RTK activation
The RTK ligand can bind as a ligand leading to recruitment of the other receptor monomer. However, this dimerisation of the receptor monomers is the essential stage in RTK activation
559
Explain the gain of function approach that can be used to investigate RTK signalling
Genetically engineer DNA to generate a gene encoding an RTK whose extracellular ligand binding domain has been replaced with a homodimerization domain. Expression of this gene in an organism by incorporation of a transgene will results in the production of an RTK capable of dimerising in the absence of ligand binding. This receptor tyrosine kinase will be activated independently of the ligand and known as constitutively active
560
Explain the loss of function approach that can be used to investigate RTK signalling
Genetically engineer DNA to generate a gene encoding an RTK whose intracellular kinase domain is mutated. This will lead to a loss of kinase activity and thus no auto and crossphosphorylation. Hence the RTK will be unable to activate in response to ligand binding. This DNA can then be expressed at high levels to result in a dominant negative or antimorphic mutation whereby the mutant RTK will poison the endogenous receptor
561
Proteins containing what type of domain can bind to the phosphorylated tyrosine residues in activated RTKs
SH2 domain containing proteins
562
Proteins that bind to phosphotyrosines in RTKs by particular domains also recognise adjacent residues. What is the recognition sequence which they recognise
Phosphotyrosine-Glutamate-Glutamate-Isoleucine
563
Give some examples or proteins that bind to RTKs
GTPase-activating proteins (GAPs), phospholipase C-? and PI-3 kinases
564
What change in the intracellular environment is a common theme in signal transduction
Changes in the subcellular localisation of components leads to activation of the pathway
565
Below is a list of several proteins involved in signal transduction and RTK activation, determine if they are a GTPases, GEFs, Kinases, GAPs or adapter proteins. Sos, Gbr2, Ras, MAP-KKK, PI-3, Erk, Mek and Raf
Sos - GEF, Gbr2 - Adapter Protein, Ras - GTPase and MAP-KKK, PI-3, Erk, Mek and Raf - kinases
566
Explain how activation of RTKs leads to signal transduction by the Ras pathway
Ras is a smallGTPase that is present in the membrane of the cell. The activated RTK contains phosphorylated tyrosine residues in its intracellular kinase domain that have occurred because of autophosphorylation. These phosphotyrosines are recognised by proteins that contain an SH2 domain. In the Ras pathway, this protein is Gbr2 which binds to the activated receptor by its SH2 domain. Gbr2 then recruits another protein to the complex called sos by its SH3 protein-protein interaction domain. Sos is a guanine nucleotide exchange factor (GEF) that is bound to the Ras GTPase. Binding of Gbr2 to sos couples the activated RTK to the inactive Ras. Sos also promotes dissociation of GDP from Ras which is displaced by GTP. Now that it’s bound to GTP Ras dissociates from sos and phosphorylates MAP-KKK to transduce the signal further.
567
How does the MAP kinase pathway rely the signal transduction further from activation of the Ras GTPase
Activated Ras phosphorylates MAP-KKK which then binds to and activates MAP-KK by phosphorylation. Activated MAP-KK then goes onto phosphorylate and activate MAP-K. Activated MAP-Kinase can then phosphorylate transcription factors and other proteins leading to the regulation of gene transcription
568
What are the mammalian homologues of MAP-KKK, MAK-KK and MAP-K
MAP-KKK --> Raf, MAP-KK --> Mek and MAP-K --> Erk
569
What is the effect of having multiple stages in the MAP Kinase pathway
One activated MAP-KKK can phosphorylate and activate several MAP-KK proteins which in turn can phosphorylate and activated multiple MAP-K proteins. This acts as an amplification step in signal transduction
570
Cyclins are an example of downstream targets of MAP-Kinases, T or F
T
571
MAP-K is regulated by its phosphorylation by MAP-KK, describe how MAP-K is activated
In order to be activated MAP-K must have both of its phosphorylation sites on threonine and tyrosine residues phosphorylated by MAP-KK. These amino acids are interspaced by only one residue and lie in close proximity.
572
How long after RTK activation is gene transcription influence
Within minutes
573
One member of the superfamily of RTKs is the FGF receptor family. What are the three different subgroups of FGF ligands
Paracrine, Endocrine and Intracrine
574
Which subgroup of FGF receptor ligands are the largest
Paracrine FGFs
575
Give example of roles that FGF signalling plays in development
FGF8 is involved in formation of the limbs. It is also expressed in the somites particularly, in the myotome. FGF8 also plays a part in midbrain-hindbrain patterning
576
There are only 4 different genes for all of the FGF receptors. How is it then that these genes can account for 48 different receptors
The four FGF receptor genes have different splice variants that creates the 48 different isoforms of FGF receptors.
577
Which domains are the ligand binding domains of the FGF receptor
D2 and D3 domains
578
What is the role of the acid box domain of the FGF receptor
The acid box domain is involved in negative regulation of the receptor by preventing its activation in the absence of ligand binding
579
What component of the extracellular matrix does the FGF receptor-ligand often form complexes with
Heparan sulphate proteoglycans (HSPGs)
580
The FGF receptor-ligand complex can become activated in the absence of binding to components of the extracellular matrix, T or F
F – the receptor can only become activated when in a complex with HSPGs
581
What species attached to the proteoglycan backbones can be sulphated to trigger ligand binding to the FGF receptor
Glycosaminoglycans
582
HSPGs are important extracellular modifiers of cell-cell signalling, what is their role in the extracellular environment
They are important in organising the extracellular matrix into basal lamina
583
Give some examples of HSPGs
Glypican, Syndecan and Perlecan
584
Describe the structure of HSPGs
Consist of a proteoglycan core with glycosaminoglycan side chains
585
The protein core of HSPGs tethers them to the membrane, T or F
F – whilst the protein core is the unit responsible for membrane tethering, this doesn’t occur always. Some protein cores are transmembrane domains or can direct the HSPG for secretion
586
Other than Heparan Sulphate, give some examples of sugar side chains present on proteoglycans
Aggrecan, Betaglycan, Decorin and Perlecan
587
Modification by sulphation of GAGs can provide a code which creates binding sites for specific proteins and sequences that carry information, T or F
T
588
Describe the effects of HSPGs on the gradients of secreted molecules
HSPGs control the steepness of a secreted molecule gradient and how far a growth factor can diffuse through the extracellular space
589
Give an example of a disease caused by mutations in FGF signalling and describe how this accounts for the symptoms
Achondroplasia is a dwarfism disease caused by an activation mutation in the transmembrane domain of the FGF receptor. This means that the receptor is constitutively active independent of ligand binding. FGF signalling then leads to a repression of Ihh which in turn leads to a loss of expression of PTHrP. PTHrP is required to stimulate proliferation and chondrogenesis and hence increased FGF signalling leads to the decreased bone growth characteristic of dwarfism.
590
What is the results of ectopic hedgehog signalling in the Drosophila appendage structures
Ectopic hedgehog signalling creates a mirror image duplication of limb structures
591
What are the Drosophila imaginal discs
Columnar epithelial cells of the ectoderm that give rise to adult structures
592
Other than limbs/appendages, what others adult structures are the imaginal discs for in the Drosophila embryo
Mouthparts, eyes/antennae, genitals
593
Imaginal discs are set up early in embryogenesis, T or F
T
594
Which segment of the Drosophila embryo contains the haltere imaginal disc
(Thoracic) T3
595
Which segment of the Drosophila embryo contains the leg imaginal disc
(Thoracic) T3
596
Which segment of the Drosophila embryo contains the wing imaginal disc
(Thoracic) T2
597
What is meant by a haltere
The haltere is a paired structure of the adult fly that is essentially its second pair of wings. However, over time, this structure has evolved a new role as a balance organ that counteracts the movement of the wings
598
The wing imaginal disc grows massively throughout development, increasing from 20/30 cells to 50,000+ cells. What is surprising about the structure during this period
The structure remains surprisingly similar throughout
599
There are three regions of the wing imaginal disc, what three adult structures do these regions give rise to
Notum, proximal wing and hinge, and the wing blade
600
What is mean by the wing imaginal disc developing akin to how an extendable aerial extends
The three concentric regions of the wing imaginal disc extend outwards forming the proximal-distal axis. The dorso-ventral axis is defined by a line the passes horizontally through the disc
601
The muscles that move the adult fly wing are found in the hinge, T or F
F – the wings themselves are stiff with all movements controlled by the muscles of the notum. The twisting motion of these muscles is translated by the hinge into the up and down action of the wing blade
602
How many cells thick is the adult fruit fly wing
2 cells thick
603
Bands of expression of which genes dictate the anterior-posterior and dorso-ventral axes
Dorso-ventral is defined by a band of wingless expression whilst anterior-posterior boundary is indicated by a band of ptc expression
604
Which axis forms the edge of the wing
Dorso-ventral
605
What three key genes are involved in dividing the anterior-posterior wing imaginal disc
Engrailed, hedgehog and decapentaplegic
606
What type of gene is engrailed
Engrailed is a segment polarity gene encoding a transcription factor
607
What is the effect on gene expression of engrailed
Engrailed switches on hedgehog which in turn induces wingless expression
608
Wingless and hedgehog work together to inhibit the formation of denticles, T or F
T
609
In early embryogenesis engrailed in involved in polarising segments, which region of the segment is indicated by engrailed expression
Engrailed defines the posterior compartment of each segment
610
What is similar about engrailed expression in the body segments and wing imaginal discs
Like the body segments, the wing imaginal discs also express engrailed only in the posterior. The presumptive imaginal discs inherit their anterior-posterior identity from the segmentation machinery.
611
Engrailed expression in the posterior segments pre-dates the regions where the imaginal discs form, T or F
T
612
Engrailed expression in the posterior wing, thoracic and abdominal segments is switch off in the adult fly, T or F
F - Engrailed expression remains on in the adult fly and it remains expressed in the posterior wing, thoracic and abdominal segments
613
What experimental evidence is there that engrailed is expressed in the posterior thoracic and abdominal segments as well as posterior wing
Creating a reporter construct encoding a engrailed-LacZ fusion protein drives expression of ?-galactosidase under the control of the engrailed promoter. Introducing the LacZ substrate X-galactose results in the production of a blue precipitate in the posterior segments etc.
614
What phenomena accounts for the distinct boundaries between engrailed expressing cells and those not expressing it
Cells expressing engrailed only having affinity for other cells expressing engrailed and vice versa. This is due to differences in intercellular adhesion
615
What is the result in hedgehog signalling in mutants with loss-of-function mutation in smo
Smo mutants lack hedgehog target gene repression and thus increased expression of target genes even in the absence of a hedgehog ligand
616
What is the effect of engrailed on Ci
Engrailed suppression Ci expression
617
What is the effect of engrailed on hedgehog expression
Engrailed induces hedgehog expression
618
What is the effect of engrailed on wingless
Engrailed upregulates expression of wingless. However, this isn’t direct and in fact, acts through hedgehog which upregulates wingless
619
Is Ci is only expressed in the absence on engrailed, where in the wing imaginal disc is it expressed
In the anterior compartment
620
As well as being the hedgehog receptor, Ptc is also a hedgehog signalling pathway gene upregulated by ligand binding, T or F
T
621
What can be said about the ptc expression pattern in the wing imaginal disc
Ptc is expressed in a band along the dorso-ventral axis
622
If ptc is a target gene of the hedgehog signalling pathway, then which cells is its expression confined to
Ptc can only be expressed in cells where hedgehog is present and Ci is not being repressed by engrailed
623
As hedgehog is only expressed in response to engrailed, its transcription is confined to the posterior wing imaginal disc, T or F
T
624
Explain using what you know about hedgehog why an expression band of ptc only occurs in the midline and initial anterior cells of the wing imaginal disc
As hedgehog is a morphogen it is made in the cells of the posterior wing imaginal disc and then is secreted. Hedgehog ligands then diffuse anteriorly to regions of cells that don’t express engrailed and thus haven’t had their Ci expression switched off. This means that the only cells capable of responding to the hedgehog induced in the posterior wing disc are those lying anterior of the midline where Ci is still being expressed. These cells will thus express ptc as a result of hedgehog target gene expression.
625
Decapentaplegic is another gene product involved in patterning the Drosophila wing. What causes dpp expression
Dpp is a hedgehog pathway target gene expressed as a result of hedgehog ligand binding
626
What type of patterning molecule is dpp
Dpp is a morphogen
627
Dpp is an invertebrate gene, what is its vertebrate homologue
TGF-?
628
Dpp is expressed exclusively in the midline of the wing imaginal disc, explain how this occurs
As dpp is a transcriptional target of hedgehog expression it can only be expressed in cells receiving hedgehog from the posterior wing disc cells where engrailed is being expressed. However these cells themselves need to not be expressing engrailed so that Ci expression isn’t inhibited. These cells occurs at the midline and slightly anteriorly and begin to express dpp. However, as dpp is a morphogen it diffuses both anteriorly and posteriorly creating a uniform decreasing gradient either side of the midline
629
Remembering that dpp is a morphogen and a homologue of the vertebrate TGF-?, what are the specific Type I and Type II receptors which it binds to
Tkv and Pnt
630
What invertebrate homologues of Smad proteins are activated as a results of dpp binding to type I and II receptors
Mad proteins
631
Brinker, omb and sal are target genes of dpp signalling, what are the specific effect of decapentaplegic on these genes
Dpp inhibits brinker expression and upregulates omb and sal genes
632
How can dpp’s action as a morphogen be visualised experimentally
Raising an antibody specific for phosphorylated mad proteins
633
Why can dpp protein be detected at a distance, using immunohistochemistry, in cells where in situ hybridisation experiments revealed no mRNA for that gene
Dpp is a morphogen
634
Experimental evidence for dpp’s effects on sal and omb expression revealed a much wider band of omb throughout the imaginal disc whereas sal expression was refined to a thinner band closer to the midline. Explain these experimental results
Sal requires a much higher concentration of dpp to be expressed by the cells. Hence sal isn’t expressed more distant from the midline because the concentration of dpp isn’t sufficient. In contrast there is sufficient dpp to induce omb expression throughout
635
What is meant by a morphotrap
A morphotrap is a membrane-tethered anti-GFP antibody that is a single chain antibody made by cammelids
636
In wild type embryo wing imaginal discs, where are the highest levels of brinker (brk) expression seen and why
The highest brk expression is seen at the periphery where ddp expression is at its lowest levels – dpp negatively regulates brk
637
What would be the effect of creating a morphotrap for GFP if you expressed it in cells under the control of the dpp promoter that also have a GFP motif knocked into the GFP gene
As morphotraps are membrane-tethered they will restrict the expressed dpp-GFP fusion protein to the cell surface effectively making it paracrine.
638
What would be seen experimentally if you restricted the diffusion of dpp away from the midline
A more refined band on dpp expression in the midline and brk expression projecting much more medially and forming a clear boundary rather than a gradient
639
Explain what would happen as a result of cloning cells in the posterior wing imaginal disc that have engrailed knocked out
These cells will express ptc due to a loss of repression of hedgehog target genes. The cells will be produce Ci because it isn’t being repressed by engrailed which has been knocked out. Hedgehog produced by neighbouring cells will bind to ptc receptors leading to expression of hedgehog target genes. These cloned cells will therefore express dpp because hedgehog is present and Ci is expressed
640
Explain what would happen as a result of cloning cells in the anterior wing imaginal disc that have forced engrailed expression
This will result in a border of dpp expression around the cells with forced engrailed expression. This is because Ci expression in those cells will be repressed by engrailed. Also engrailed expression causes hedgehog expression in those cells. This hedgehog will diffuse out of the cells where engrailed is expressed to the surrounding cells of the anterior wing imaginal disc. These anterior cells are wild type and do not express engrailed and thus are expressing Ci and can respond to hedgehog signalling. This will result in the activation of hedgehog signalling and expression of dpp
641
Explain what would happen as a result of cloning cells in the anterior wing imaginal disc that have patched knocked out
This will have the same effect as ectopic hedgehog signalling and will lead to dpp expression in those cells with ptc -/-. This is because ptc -/- will have decrease inhibition of smo. This means smo will be constitutively active even with the ligand absent. As Ci is also present because there is no engrailed anteriorly, dpp is expressed as well as other hedgehog target genes
642
What would be the result of a Ptc and Ci double knockout experiment in a region of cells in the midline, where dpp is normally expressed
Ptc and Ci double knockout in the midline will result in a further hedgehog diffusion anteriorly from those cells in the posterior where it is expressed. This is because hedgehog is not binding to any ptc receptors in the midline and so more is available to diffuse further.
643
What type of signalling protein is wingless
A morphogen
644
Wingless expression drives expression of pathway target genes in a concentration dependent manner and defines the dorsal ventral boundary in the same way that engrailed does the anterior-posterior axis, T or F
T
645
Below is a list of limb bone structures and their embryonic precursors regions. Match up the corresponding bones and embryonic structures. A - humerus, B - Metacarpals, C - Radius, D - Carpals, E - Phalanges, F - Ulna. i - Stylopod, ii - Autopod, iii - Zuegopod
A - i, B - ii, C - iii, D - ii, E - ii, F - iii
646
What are the names given to the structures that initially form and demarcate where the limb buds will form
Limb fields
647
What set of genes dictate where the early limb regions are positioned
Hox genes
648
The anterior border of Hox6 expression was found to correlate with limb bud formation. As Hox 6 is a transcription factor, what type of experiment would be used to prove that at the anterior border of its expression, the limb fields would form
In situ hybridisation – identify the cells where the Hox6 mRNA transcript is produced
649
Tbx4 and tbx5 were wound to be the downstream genes activated that specify the limb fields. What type of genes are tbx genes
Tbx genes are T-box containing transcription factors
650
Which tbx gene is responsible for specifying the anterior or forelimb bud
Tbx5
651
Which tbx gene is responsible for specifying the posterior or hindlimb bud
Tbx4
652
What is the result of forced tbx4 expression in the forelimb field at the anterior limb bud in chicks
This will lead to the development of a leg (or hindlimb) instead of a wing (or forelimb)
653
Explain the interaction between tbx4 and 5 that act to restrict the limb fields
There is a negative regulation mechanism between the tbx 4 and 5 genes. Tbx5 can block the expansion of tbx4 expression which prevents the hindlimb field from progression anteriorly into the forelimb field
654
Hox genes directly induce the expression of tbx transcription factors in the process of determining the limb field regions, T or F
F – Hox proteins lead to the expression of retinoic acid which in turn upregulates tbx transcription factors
655
In vertebrate limb development, hox genes are expressed in the lateral and paraxial mesoderm, which ultimately leads to the expression of tbx4 and 5, T or F
T
656
FGF was considered to have a role in formation of the limb bud. Describe an experiment that could be carried out in chicks to determine if indeed, FGFs are involved in limb formation
Implantation of FGF-soaked beads into the flank of chick embryos and observe if ectopic limbs develop
657
It was later found that ectopic FGF did in fact lead to the development of an ectopic limb in the chick embryo. What is the significance of the proximity of this ectopic signalling to either of the normal developing limb fields
Depending of the proximity of the FGF signalling (due to bead implantation) to either the hind or forelimb fields determined the ultimate identity of the ectopic limb. If it was closer to hindlimb field an ectopic leg would develop, similarly, if it was closer to forelimb field an ectopic wing would develop
658
The key genes involved in specification of the limbs were determined to be, FGF8 and 10, Retinoic acid, Hox genes and tbx transcription factors. Put these genes in the sequence in which they act to dictate limb field development
Hox genes --> retinoic acid --> tbx4/5 --> FGF10 --> FGF8
659
From medial to lateral, recall the mesodermal tissues in the developing chick embryo
Axial mesoderm, Paraxial mesoderm, intermediate mesoderm and lateral mesoderm
660
Which region of mesoderm gives rise to the urogenital structures such as the kidneys
Intermediate mesoderm
661
Which mesodermal region produces FGF10
Lateral mesoderm
662
Describe how FGF10 expression in the lateral mesoderm at the level of the limb is induced by the intermediate mesoderm
The intermediate mesoderm produces large amounts of FGF8 which induces the FGF10 expression by the lateral mesoderm
663
What signalling acts to restrict expression of FGF10 in specification of the limb fields
Wnt signalling
664
Explain how FGF10 signalling results in the induction of the AER
FGF10 from the lateral mesoderm acts to induce wnt3a expression in the overlying ectoderm which changes its identity to become thickened, producing the apical ectodermal ridge
665
After induction of the AER by FGF signalling from the lateral mesoderm and wnt3a expression, another set of FGF ligands are expressed at high levels. Which FGFs are these
FGF4 and 8
666
What the name of the structure that forms in the posterior limb bud that expresses the sonic hedgehog morphogen and plays a key role in patterning of the limb
Zone of Polarising Activity (ZPA)
667
What is meant by the progress zone and what is its role
The progress zone is a region of mesoderm where cells proliferate and that will ultimately give rise to cells of the limb
668
Outline the role of the AER in limb patterning
The AER is required and necessary for the growth and maintenance of the progress zone
669
As the limb grows outwards during development, the more proximal skeletal and muscle regions are specified, T or F
F – the more distal skeletal elements are specified as the limb grows outwards
670
As the limb grows its specifies the skeletal element that the cells adopt depending on how long they spend proliferating in the progress zone, T or F
T
671
Explain the temporal patterning of the proximal-distal axis of the vertebrate limb
There is a clock present in the progress zone of the limb bud that measures the number of cell divisions. Cells that have undergone a small number of cell cycles will form proximal skeletal elements, whereas those that have undergone more cycles will form distal skeletal elements
672
The temporal model for patterning the vertebrate limb was determined to be incorrect, T or F
T
673
Outline the two-signal model of proximal-distal limb patterning
The AER is only important in establishing the distal area of the limb. Another signal acts to establish the proximal regions. Its the interactions between proximal and distal elements that determines the intermediate regions
674
In the two-signal model of proximal-distal limb patterning, what are the signals involved
The distal signal from the AER are FGFs, whereas the proximal signal is retinoic acid
675
The molecules that act to specify proximal and distal regions of the vertebrate limb are secreted products that act as morphogens, T or F
T
676
Hox11, Hox 13 and Meis1/2 are all downstream genes upregulated in the limb bud that act to pattern the vertebrate limb. What type of genes are these
Transcription factors
677
Explain how interactions between the two secreted signals in vertebrate limb patterning act to form the stylopod
The stylopod is the most proximal region of the limb. This is the regions that’s is exposed to the highest levels of RA. Retinoic acid acts to induce the expression of Meis1 and 2
678
Explain how interactions between the two secreted signals in vertebrate limb patterning act to form the autopod
The autopod is the most distal region of the limb. This is the region that’s is exposed to the highest levels of FGF signalling. FGFs act to induce the expression of Hox13
679
Explain how interactions between the two secreted signals in vertebrate limb patterning act to form the zeugopod
The zeugopod is the intermediate region of the limb. This is the region that’s is exposed to intermediate levels of both RA and FGF signalling. A combination of this signalling acts to induce expression of Hox11
680
Distal-less (Dlx) is a gene found in Drosophila identified as playing a role in development of appendages, in particular, the leg. What is the significance of dlx in vertebrate limb development
Dlx is the Drosophila homologue of Hox13
681
Homothorax is the Drosophila homologue of Meis1/2. What is the importance of this gene in fruit fly development
Homothorax is important in controlling the patterning of the proximal wing
682
What is the ZPA and its role in limb development
The Zone of Polarising Activity (ZPA) is an area of mesenchyme in posterior limb bud that controls anterior-posterior limb patterning
683
Describe an experiment that could be carried out in chicks that would provide the evidence that the ZPA controls anterior-posterior patterning in the limb. What would you expect to see
Surgical removal of the ZPA from a donor chick embryo and implantation into the anterior limb bud of a donor embryo. This grafting would cause a mirror image duplication of distal limb structures
684
It was later found that ectopic grafting of the ZPA resulted in a mirror image duplication of autopod structures. What was seen in a similar experiment where grafting occurred earlier in limb development
There was also a duplication of the ulna
685
What molecule is secreted by the ZPA to control anterior-posterior patterning of the limb
Sonic hedgehog
686
Describe a similar experiment to ZPA grafting the proves its role in anterior-posterior patterning of the distal limb based on the knowledge of the molecules that it secretes
Implantation of sonic hedgehog-soaked beads into the anterior limb bud. This should also create a mirror image duplication of distal structures
687
Chick wings have three digits, describe the nomenclature of these digits
The chick wing has three digits named digit 2,3 and 4. There is no digit 1
688
How does the chick forelimb differ to its hindlimb
The chick wing only has three digits whereas the hindlimb or leg has 4. The leg has digits 1,2,3 and 4
689
How does sonic hedgehog dictate digit formation in the chick wing
Each threshold of sonic hedgehog correlates with the formation of a specific digit. At the lowest sonic hedgehog concentration digit 2 forms. Intermediate sonic hedgehog concentrations correspond to digit 3 with the highest sonic hedgehog concentration dictating digit 4
690
What experimentation methods could be employed to determine if sonic hedgehog is acting as a morphogen in anterior-posterior patterning of the distal limb
Carry out in situ hybridisation to look at where the sonic hedgehog mRNA is being transcribed in the limb bud. This would show a localisation of the mRNA in the cells of the ZPA. This could then be compared to immunohistochemistry experimentation which would stain for the sonic hedgehog protein using tagged antibodies. This would show a decreasing gradient of the protein from posterior to anterior throughout the limb bud and not localised to cells of the ZPA. This indicates that the sonic hedgehog protein is leaving the cells in which it is produced
691
Describe the phenotype of a sonic hedgehog mutant with regards to the effects on limb development
Sonic hedgehog knockout mice exhibit a complete loss of the distal most skeletal elements as well as a loss of identity of the zeugopods
692
Why does digit 1 often develop to some extent in the limbs of sonic hedgehog knockout mice
The anteriormost limb, or digit 1 forms independently of sonic hedgehog signalling. Ihh expression is seen in the most anterior region of the limb bud and acts independently of sonic hedgehog to dictate some autopod development. Ihh maintains the expression of sonic hedgehog target genes in the anterior region of the limb bud in the absence of sonic hedgehog hence leading to the digit development seen in sonic hedgehog -/- mice. Similarly Ihh -/- mice did not show development of the anteriormost digit
693
Which genes are mainily involved in proximo-distal patterning of the limb
RA and FGF
694
Which genes are mainly involved in anterior-posterior patterning of the limb
Sonic hedgehog (and Ihh)
695
Explain the coordination between the developing anterior-posterior and proximo-distal axis development
The AER releases FGF8 which plays an important role in maintaining sonic hedgehog expression by the ZPA. Sonic hedgehog then in turn plays an important role in maintaining FGF expression in the AER. This positive feedback loop allows for a mutual maintenance of both sonic hedgehog and FGF expression
696
What are the three genes involved in dorso-ventral axis formation in the developing limb
Wnt7a, engrailed1 and lmxb1
697
Which two genes show an exquisite complimentary expression pattern with one being uniquely expressed in the dorsal ectoderm whilst the other in the ventral ectoderm
Engrailed1 is only expressed in the dorsal ectoderm whilst wnt7a is expressed in the ventral ectoderm
698
Explain how the complimentary genes involved in d-v pattering of the vertebrate limb interact
Engrailed1 prevents the expression of wnt7a.
699
What is the effect of en1 knockout on vertebrate limb development
Engrailed knockout results in a dorsalisation of the limb as wnt7a expression isn’t inhibited. This is because wnt7a expression is driven throughout entire d-v axis ectoderm. En1 acts to prevent wnt7a expression from expanding ventrally
700
Where is lmx1b expression only seen in the vertebrate limb
In the dorsal mesenchyme
701
Explain the role of lmx1b in patterning of the vertebrate limb
Lmx1b acts to pattern the dorsal region of the developing vertebrate limb
702
What is the result of lmx1b knockout
Lmx1b knockout results in a ventralised limb with no dorsal mesenchymal development
703
Apterous is the invertebrate homologue of lmx1b. What is the significance of this in appendage development
Apterous is present in the Drosophila wing disc and controls dorsal fate by inducing wingless expression in Drosophila
704
Recall the pathway of interaction between the genes involved in the d-v patterning of the vertebrate limb
BMP --> En1 --| Wnt7a --> Lmx1b
705
How does the pathway of wing development in Drosophila differ from that of it vertebrate counterparts, particularly, in the way in which the Lmx1b homologue acts
Apterous acts upstream of the wnt7a homologue in Drosophila. Apterous acts to upregulate the expression of wingless leading to the adoption of a dorsal cell fate
706
The timing of heart development across the species is relatively conserved, when does this tend to occur
Immediately after gastrulation
707
Drosophila can be used to study the earlier development of the heart, however what prevents these models from being used to study the later stages of cardiogenesis
Drosophila have a linear circulatory system and don’t possess a multi-chambered heart
708
The first stage of cardiogenesis involves the formation of a structure from a mesodermal precursor. What is the name given to this early heart structure
Cardiac crescent
709
What is the name of the linear structures that form from the cardiac crescent and then fuse to form the looping heart
Heart tubes
710
What process takes place during heart remodelling
Duplication of chambers
711
Cardiac cells originate from a structure of the ventral lateral mesoderm, what is the name given to this region
Splachnopleura
712
What are the two different tissues contained within the region of the ventral lateral mesoderm that the cardiac cells originate from
Splachnic mesoderm and foregut endoderm
713
Heart precursor cells have two origins, the first and second heart fields, what structures are derived from the first heart field
Cardiac crescent, left and right ventricles, AV canal and the atria
714
What is the role of the second heart field and where is it located
The second heart field is located immediately behind the first heart field. It give rise to part of the right ventricles, inflow and outflow tracts as well as plating an important role in the growth of the heart
715
Lecture 22 Question 11 - See image
A – axial mesoderm, B – paraxial mesoderm, C – intermediate mesoderm, D – lateral mesoderm (splachnopleura)
716
Lecture 22 Question 12 - See image
A – notochord and prechordal mesoderm, B – somites, C – urogenital structure such as the kidneys, D – cardiac and circulatory system
717
Tinman was a gene determined to be involved in the development of the heart. What type of gene is tinman
Homeobox domain containing transcription factor
718
What is the precise role of tinman in heart development
Tinman is required to specify cells to a cardiac lineage
719
What signalling mechanism is responsible for the regulation of tinman expression in Drosophila. Also list the vertebrate homologue
Decapentaplegic (BMP in vertebrates)
720
Tinman expression is initially induced very broadly in the tract mesoderm, however what is the name of the structure that its expression becomes restricted to later in the development of the fruit fly heart
Dorsal vessel
721
Explain a loss of function approach used in Drosophila to investigate the role of tinman in cardiogenesis
Introduce deletions into the tinman gene and stain cells for a marker of cardiac cells
722
What marker can be used to indicate cells that have adopted a cardiac fate
Myosin heavy chain
723
What approach can be used to stain cells for the marker of a cardiac cell fate
Immunohistochemistry
724
Explain the results of tinman loss of function analyses on cardiac development and what this tells us about its function
Deletions inserted into Drosophila genome in the tinman gene results in mutations that show a loss of myosin heavy chain expression. This indicates that tinman is required for the formation of cardiac cells
725
What is the name of the family of tinman homologues found in vertebrates
NK-2 homeobox transcription factors
726
Which specific tinman vertebrate homologue is specifically expressed in the cardiac crescent
Nkx2.5
727
What is the result of knockout of this tinman vertebrate homologue specifically expressed in the cardiac crescent
Nkx2.5 mouse mutants do form hearts but show cardiac defects at the heart looping stage.
728
Why does knockout of Nkx2.5 not cause a failure of the heart to develop
Other members of the family carry some of the function and allow heart development to continue (heart ultimately fails to develop normally)
729
Which specific BMPs is decapentaplegic a invertebrate homologue of
BMP2 and BMP4
730
Explain a gain of function approach that can be used in mice to investigate the role of the dpp homologue on cardiogenesis
Implantation of a BMP2 soaked bead anterior to the primitive streak in early development.
731
What is the result of gain of function experimentation with BMPs involved in cardiogenesis
Bead soaked in BMP2 induces the expression of Nkx2.5 in cells that wouldn’t normally express it. This indicates that BMP2 is sufficient to induce expression of Nkx2.5 in cells that wouldn’t normally
732
What would be used as a control in a gain or loss of function experiment that involves bead implantation into chick/mouse embryos
Implant another bead that is soaked in PBS
733
A gain of function experiment was carried out to investigate the role of BMP2 by implantation of a BMP2 soaked bead into the posterior of the embryo. No Nkx2.5 expression was seen. Explain these results and what this indicates about BMP2 role in cardiogenesis
BMP2 needs to be acting in an environment that is favourable for its activity. Posterior implantation of BMP2 soaked beads wouldn’t result in a cardiac specification due to high concentration of BMP antagonists (Noggin, Chordin, Follistatin) released by the notochord. There needs to be a permissive environment to control cardiac specification by BMP2 signalling
734
Once the cells have been specified to a cardiac lineage, what happens in the stages leading to the formation of the heart tube
These cells migrate ventrally under the influence of signals coming from the foregut endoderm and fuse in the midline to form the heart tube
735
Once the heart tube has formed it contains two main tissues, the endocardium and the (epi)myocardium. What are the derivatives of these two tissues
Endocardium contains precursors of endothelial lining of heart and cushion cells that form valves. Myocardium contains myocytes of atria and ventricles, and Purkinje fibres
736
Endocardial tube in the inner part of the heart tube eventually contributes to the endothelial lining of the heart, T or F
T
737
What is the cardiac jelly
A structure that lies between the endocardial tube and the cushion cells that is rich in extracellular matrix proteins secreted by the myocardium
738
Dmef2 is present is all differentiated cardiac muscle cells, T or F
F – is it present in all differentiation muscle cells (skeletal and smooth muscle too)
739
What type of gene is Dmef2
Transcription factor
740
How is Dmef2 expression induced
By the expression of tinman
741
What are the results of mutations in Dmef2 in Drosophila
Mutations in Dmef2 result in no formation of the dorsal vessel in Drosophila
742
Like tinman, Dmef2 is also required to form the Drosophila heart, T or F
T
743
Dmef2 mutants still show expression of tinman. What can be inferred by this observation
Tinman acts upstream of Dmef2
744
What is the effects of Dmef2 mutations on cardiogenesis
In the absence of Dmef2 you can still form the precursors of cardiac myocytes. However, these cardiac myocytes are unable to differentiate to form cardiac myocytes
745
Dmef2 is also required to specify cells to a cardiac lineage, T or F
F – Dmef2 is required to differentiate cells into cardiac myocytes
746
What would be the effects of tinman mutations on Dmef2 expression
Tinman mutants would not show Dmef2 expression
747
What are the three homologues of Dmef2 in vertebrates
Mef2A, Mef2B and Mef2C
748
Which is the gene that is expressed earliest in the vertebrate heart
Mef2C
749
What is the result of a mutation in the earliest gene expressed in the vertebrate heart
No formation of the heart and no looping of the ventricles. Interestingly, upregulation of Mef2B is seen indicating a compensatory mechanism in vertebrates
750
What is the role of the GATA transcription factor family of gene in cardiogenesis
The GATA genes drives the fusion of the primordium into the early heart tube
751
What domain indicative of some transcription factors do the GATA proteins contain
Zinc finger domains
752
Which specific members of the GATA family are involved in cardiogenesis
GATA 4-6
753
What is the result of knockout for the GATA genes involved in cardiogenesis
Knockout of GATA4 in mice results in a failure of the heart tube primordia to migrate and fuse to form the heart tube. This is known as cardiac bifida
754
What cell adhesion molecule is controlled by GATA expression
N-cadherin
755
Following the formation of the heart tube, Drosophila cardiogenesis is complete as they possess a linear heart. What other embryo also has a neoheart tube with a single atrium and ventricle
Zebrafish embryo
756
What two processes take place in higher order animals after formation of the heart tube
Looping, chamber subdivision
757
What are the three hypothesised mechanisms of heart looping
Asymmetric cell division, asymmetric cell death or changes in cell shape triggered by asymmetric distribution of microtubules, actin bundles and cell adhesion molecules
758
A key factor in the looping of the mammalian heart is the breakage of left-right symmetry which occurs very early in embryogenesis immediately after gastrulation, T or F
T
759
Explain how left-right symmetry is broken in the chick embryo
In chicks, this symmetry is broken at the level of the (Hensen’s) node due to the asymmetric expression of sonic hedgehog in the left and right. Sonic hedgehog is inhibited by activin receptors on the right of the embryo leading to higher expression on the left of the embryo with low sonic hedgehog levels on the right
760
What two diffusible molecules of the TGF-? family are specifically expressed on the left side of the embryo
Lefty and Nodal
761
Which side does normal heart looping occur
To the right
762
Outline the role of the iv and inv genes in heart looping
Iv and inv involved in the development of motile cilia
763
What is the role of the iv gene
Inversus viscerum encodes a dynein protein involved in the movement of the cilia
764
What is the role of the inv gene
Inversion of embryonic turning encodes for Inversin, a protein containing ankyrin repeats found in cilia
765
What is the result of mutation in iv
Iv mutant mice have cilia that are immotile
766
How do inv and iv act to mediate heart looping despite being genes involved in cilia development
Inv and Iv are required for cilia movement, specifically their rotation. Cilia rotation establishes a preferential flow of Nodal and Lefty molecules on the lefts side of the embryo. This rotation prevents the lefty and nodal molecules from diffusing onto the right side of the embryo and allows looping to occur there
767
Formation of the septa in heart chamber formation involves the activity of the endocardia, T or F
T
768
The bHLH transcription factors dHand and Ehand are involved in the formation of the heart chambers. Outline their localisation
ehand and dHand show specific expression in right and left ventricules. Their restricted expression is complimentary with eHand restricted to the left ventricle and dHand restricted to the right ventricle
769
Whilst dHand knockout mice die at E10.5 with ventricule hypoplasia, eHand knockout mice die at E8.5 with placental defects. Why is this
The embryos don’t survive long enough to look at effects on heart development. dHand must be acting earlier in development in another vital process
770
When a gene has multiple vital functions as is common in development, how can we create mice models that specifically look at the later role of these genes if the embryos can't survive until that stage without it
Create a conditional knockout where the gene can be knocked out at a specific stage in development
771
What is seen in the eHand conditional knockout
Only knockout eHand in the cardiac myocytes. The mice show left ventricule defects, but survive until birth
772
Lecture 22 Question 27 - See image
A – BMPs, B – Nkx2.5, C – Mef2C, D – GATA4, E – inv and iv, F – eHand and dHand
773
Lecture 24 Question 1 - See table
c, a, b, d
774
Branching morphogenesis of the ureteric bud underlies the future nephrons, T or F
F – it underlies the prospective calyces
775
A remnant of the initial ureteric bud will from what structure in the adult
The ureter
776
What is seen at the level of the renal pelvis
Where the ureter ends and the where the branches of the major calyces begin
777
Kidney development is protracted, proceeding throughout prenatal and even into postnatal life, T or F
T
778
Stem cells of the ureteric bud and metanephric mesenchyme are retained into postnatal life, T or F
T
779
Older nephrons are found in the inner regions of the kidney and the newer ones are found at the periphery, T or F
T
780
The transcription factor Six2 can be used to identify stem cells of the nephron, what experimental evidence is there for this
Genetic lineage tracing has been used to determine that six2 positive cells give rise to cells of the nephron. These have involved the creation of transgenes with GFP fused to the six2 coding region and under the control of its promoter.
781
What is the result of a conditional knockout/loss of six2 expression
Depletion of the nephron stem cell pool
782
What is the results of overexpression of the six2 transcription factor
Prevention of differentiation
783
As well as six2 acting as a marker for the identification of nephron stem cells, what other role does it play
Six2 acts to maintain the nephron stem/progenitor cells by keeping them self-renewing
784
Six2 positive cells are nephron precursor cells that will undergo what type of change in cell behaviour
MET cell transition
785
As too much six2 expression prevents differentiation and too little leads to a depletion of the stem cell pool it is essential that only an intermediate level of its expression occurs to induce the creation of the two daughter cells required in nephrogenesis, T or F
T
786
What gene first expressed in the intermediate mesoderm prior to kidney development was initially discovered in a paediatric kidney cancer where kidney elements are incompletely differentiated and proliferate to form tumours
Wilms tumour 1 (Wt1)
787
Body attempts to build a kidney earlier in development at the cervical regions and then at the level of the developing lungs, T or F
T
788
The embryo attempts to form the adult kidney three times during development, producing the pronephros, metanephros and mesonephros. Put these in order in which they form
Pronephros, mesonephros and then metanephros
789
What structures in the adult does the mammalian pronephros give rise to
Although the pronephros gives rise to the pronephric duct and pronephric cord, the later degenerates with the anterior pronephric duct
790
What structures in the adult does the mammalian mesonephros give rise to
The mesonephros along with the most caudal pronephric duct (from the pronephros) give rise to the Wolffian duct
791
What structures in the adult does the metanephros give rise to
The metanephros gives rise to the kidney proper
792
From what two structures does the kidney develop from and which mesodermal tissue are these structures derived from
Nephrogenic cord and nephrogenic duct – derived from the intermediate mesoderm
793
Ultimately the kidney develops from the metanephric mesenchyme, from which structure is this tissue formed from
Nephrogenic cord
794
What type of cells are contained in the intermediate mesoderm
Mesenchymal cells
795
As the nephrogenic duct extends caudally, what structures does it induce the formation of in the nephrogenic cord
Nephric tubules
796
In mammals, the posterior nephric tubules degenerate and undergo apoptosis, T or F
F – In mammals, anterior structures degenerate, leaving only metanephric regions
797
The ureteric bud forms from the most causal region of the nephric duct, T or F
T
798
Which paired box transcription factor is expressed by the nephric duct
Pax2
799
Which structure expresses wt1 involved in kidney development
Nephric cord
800
Describe the anatomical relations of the nephric duct and nephric cord
Nephric duct lies dorsally to the nephric cord and elongates in a rostral caudal direction
801
Which three transcription factors are absolutely required for the formation of the nephric duct
Lim1, Pax8 and Pax2
802
Which cell adhesion molecules are identifiable markers of the nephric duct and can be observed to compare the effects of Pax2 and Pax8 knockout
Laminin and E-cadherin
803
What structure does the pronephric duct join with as it extends caudally
Cloaca
804
Outline the development of the mesonephros
As the pronephric tubules degenerate, the middle portion of the nephric duct induces a new set of kidney tubules in the mesenchyme. These new kidney tubules constitute the mesonephros or mesonephric kidney. Each mesonephric tubule attracts a blood supply from a branch of the nearby aorta which end in a capillary tuft – analogous to the glomerulus of the definitive nephron. Mesonephric tubule forms a capsule around this tuft, allowing for blood filtration
805
Discuss the ultimate fate of the mesonephros
The mesonephros largely generates, However, in males, some tubules retained and form vas deferens. But by now, the metanephric (definitive) kidney has been induced
806
Most likely different Hox code or signals from the cloaca mediate the formation of the kidney proper/metanephros, T or F
T
807
What is meant when we refer to early cells of the embryo as totipotent
They can give rise to any of the germ layers and subsequently any cell of the body
808
Most of the early derivatives of the totipotent cells will remain as adult or tissue specific stem cells, T or F
F – most will differentiate to form the somatic cells of the body
809
What is the name given to the set of cells set aside in an undifferentiated state that contribute to an individual during its lifetime
Tissue specific or adult stem cells
810
Tissue specific stem cells are maintained throughout life due to their ability to self-renew, T or F
F – they are gradually depleted over time
811
What are gonadal stem cells
Another type of specialised cells distinct from tissue specific stem cells that are set aside in an undifferentiated state for the next generation
812
In some animals as well as all plants, somatic cells can readily form new organisms, T or F
T – whereas in many animals there is an early division between somatic and germ cells
813
Generation of the germ cells is said to be a two-step process, what are the main stages in this process
The primordial germ cells (PGCs) are determined in a specific location just on the edge or outside of the developing embryo. Then PGCs migrate to the gonad and become the progenitor populations for eggs and sperm
814
A germ cell is a plastic/totipotent cell type capable of undergoing mitosis, T or F
F – they are capable of undergoing meiosis
815
C. elegans has given us a conceptual understanding of germ cell determination. What process of early cell division accounts for the differences in germ and somatic cells and is a process used to produce different daughters
Asymmetric cell division
816
What cell lineage in C. elegans is responsible for giving rise to the germ cells, discuss this lineage
The P-cell lineage in C.elegans gives rise to the germ line. The fertilisied egg goes onto to divide to give the AB cells and the P1 cell. The P1 cell then divides to give an EMS cell and the P2 cell. P2 divides asymmetrically to give the P3 and C cell fates
817
What causes the process used to create 2 different daughter cells during cell division
Asymmetric cell division is caused by the asymmetric localisation of cytoplasmic determinants within a cell
818
Describe how localisation of cytoplasmic determinants can both subsequently lead to both symmetrical and asymmetrical cell divisions
Once cytoplasmic determinants are localised within the mother cell, division along a meridian that intersects this localisation would result in the production of two identical daughters after division. However, cell division along the equator adjacent to the determinant localisation would generate two different daughter cells
819
What are the two daughters produced by asymmetric division in the formation of the germ line
One daughter cell will be maintained as a stem cell while the other will differentiation
820
Give an example of P-granule determinant that becomes localised during the generation of the P-cell lineage during C.elegans development
PIE1 – a transcription blocker that also subsequently prevents differentiation
821
Other than blocking transcription, what can other cytoplasmic determinants mediate in the P-cells
Translation blockers, promoters of stem fates, cause cells to undergo meiosis
822
P-granules become restricted to one cell, this cells will be the germ cell, T or F
T
823
What is the name of the region in the Drosophila embryo that is responsible for mediating germ line development
Germ plasm
824
Germ line determination occurs during what process in the Drosophila embryo
Cellularisation
825
Give an example of a specific gene product localised posteriorly in the Drosophila embryo
Germ cell-less
826
What is the result of loss of function mutations in some of the gene products involved in determining the germ line in Drosophila and the vertebrate homologues
Sterility
827
What transcriptional blocker homologue of gcl is found in Xenopus laevis and is expressed at the most vegetal pole of the bottom hemisphere of the frog embryo
Nanos
828
The frog cells that inherit the gcl homologue will become the primordial germ cells, T or F
T
829
What is different about the location where the PGCs are determined in mammalian embryos
In mammals, the primordial germ cells are determined outside of the main developing body axes at the junction of the epiblast/hypoblast
830
What sort of signals must the PCGs in mammalian embryos be prevented from receiving in order to maintain their stem capability
Signals that govern formation of the body axes such as wnts, FGFs, BMPs, Hedgehog and RA
831
In mammals, differentiation of PGCs is repressed by repressing gene expression until the rapid differentiation of the early embryo begins to decrease. What happens at this stage
The PCGs go inside the embryo and enter the hindgut at the posterior region. The germ cells then migrate to the specialised protective niche of the gonads.
832
What is meant when referring to the gonads as a protective niche
The gonad regions are a specialised protective microenvironment that prevent the PGCs from differentiating
833
Describe pole cell migration in the Drosophila embryo
Pole cells attach to the endoderm and move through the foregut. A combination of chemoattractive and repulsive cues drive the PGCs and gonad precursor cells together and to a specific destination (the gonad). The PGCs divide through the larval stage and differentiate at metamorphosis. In the ovaries, the cells attach to stromal cap whereas in the testes, the cells attach to hub cells
834
Describe how germ cells migrate in Xenopus embryos
Once convergent extension has happened, the primitive gut is formed from the archenteron. PGCs then migrate into the posterior part of the developing gut, before leaving the gut to migrate to the gonads. Roughly 30 PGCs reach gonads governed by a fibronectin pathway and a crucial contribution by the Sdf-1 chemoattractant
835
In mammals, the primordial germ cells stay out of the embryo (in the extraembryonic territory) while the major inductive events occur, T or F
T
836
Once they have migrated anteriorly through the foregut what happens to the PGCs
They leave the gut and move to the protective niche of the gonads via the dorsal mesentery, entering the genital ridges
837
What is the role of support cells in PGC migration in mammalian embryos
Support cells travel with the PGCs to maintain the undifferentiated stem cell phenotype by secreting stem cell factor (SCF)
838
Teratomas are tumours seen in some foetuses, what are these and how do they form
Teratomas are an embryonic cancer that contains over-proliferative cells derived from all three germ layers. They are caused by a failure of the PGCs to migrate to the protective niche/failure or to make SCF. This in turn causes the germ cells to differentiate without any organisation.
