Developmental Bio Flashcards
(220 cards)
1
Q
What is developmental biology?
A
Studying how cells acquire certain characteristics, behaviours, communications and organisations
From embryo formation to ante-, neo- and post-natal life
2
Q
Describe the evolution of developmental biology from Ancient Greece to ‘cell theory’ and ‘induction theory’.
A
Preformation (idea that everything already formed but gets bigger over time) vs epigenesis (new structures arose progressively). Cell theory (theory that organisms are composed of one or more cells) proved epigenesis correct. Induction theory is where one cell or tissue directs development of of another cell or tissue.
3
Q
What are some cellular and molecular processes underlying cell differentiation?
A
Cell division - can be symmetric or asymmetric (cytoplasmic determinants expressed asymmetrically causes this division)
Signal induction - e.g. growth factors causes signal transduction pathways resulting in differentiation changes
4
Q
What are the types of cell-cell communication involved in generating differences?
A
Paracrine - protein secreted and detected by nearby cells which activates a signalling cascade
Autocrine - protein secreted and detected by the same cell
Juxtacrine - factor is not diffused, instead is attached to cell and detected by neighbouring cells
5
Q
How does gene regulation support cell differentiation?
A
The gene content is identical in all cells but transcription and translation determine the protein content and therefore its behaviour. Can be controlled at different levels: by the production of mRNA, the processing/stability of mRNA, the production of proteins and the activity of proteins.
6
Q
What are two mechanisms which control the gene transcription?
A
Differential gene expression - where transcription factors can promote or repress expression of certain genes
Enhancer-mediated control of gene expression - enhancers promote gene expression
7
Q
What is the impact of developmental biology on biomedical science and medicine?
A
Stem cell therapy, cancer medicine, fertility understanding, congenital disease, degenerative disease, ageing, regenerative medicine
8
Q
Why is the concept that all cells contain the same DNA but express different genes important?
A
Mutations will only show up in cells where the mutated gene is expressed
E.g. Shh transcribed and translated in the developing limb and a mutation in this pathway will affect hand development
9
Q
What are 4 features of cell signalling?
A
Signal reception requires cells to be competent
Signals are instructive or permissive
Signals can act as morphogens
Require signal transduction cascade to reach nucleus
10
Q
What are the 3 germ layers (and which cells are the cells not derived from one of these)?
A
Ectoderm (external layer)
Mesoderm (middle layer)
Endoderm (internal layer)
Germ cells are the exception
11
Q
What will the ectoderm form?
A
Skin cells of epidermis
Neuron’s in brain
Pigment cells
12
Q
What will the mesoderm form?
A
Cardiac muscle Skeletal muscle Tubule cells of kidney RBCs Smooth muscle in gut
13
Q
What will the endoderm form?
A
Lung (alveolar) cells
Thyroid cells
Pancreatic cells
14
Q
What processes underlie embryonic development?
A
Pattern formation
Morphogenesis
Cell differentiation
Growth
15
Q
What is pattern formation?
A
The process by which cells are organised in space and time to produce a well-ordered structure within the embryo
16
Q
What is morphogenesis and what processes contribute to it?
A
Cell and tissue movement and changes in cell behaviour that give the developing organ it’s shape in 3D
Cell adhesion
Cell migration
Cell death
Cell shape
17
Q
What is cell differentiation?
A
The process where cells become different from each other over time and acquire specialised properties. (Governed by changes in gene expression which dictate protein synthesis)
18
Q
What are the steps involved in differentiation?
A
Egg/stem cell —> specification —> determination —> differentiation —> maturation
19
Q
What does the continuous growth process (increase in size) involve?
A
Cell proliferation (through mitotic divisions)
Cell enlargement
Accretion (of ECM tissues)
Also depends on age and organ
20
Q
What methods do we use to study changes in cell behaviour, cell-cell communication and gene expression which underpin developmental processes?
A
Embryology (observational biology and experimental manipulation) Developmental biology (study of genes and proteins) Animal models and use of genetics
21
Q
What makes animal models a good way to study developmental biology?
A
Early embryology is highly conserved therefore the foundations for all surviving organisms is very similar to that of humans
22
Q
What are experimental approaches to study gene expression?
A
RT-PCR In situ hybridisation Northern blot Reporter lines (transgenic) High throughput analyses (microarray, RNAseq)
23
Q
Describe how in situ hybridisation can be used to establish where and when a gene is expressed?
A
The target mRNA in a fixed embryo is recognised by a DIG-labelled probe with an antisense strand complementary to the mRNA sequence.
The DIG is recognised by an anti-DIG-AlkPhos antibody which is attached to alkaline phosphorylase that can cause a reaction that can be detected.
24
Q
Describe how a reporter line can be used to establish where and when a gene is expressed?
A
A reporter gene (e.g. betaGal or GFP) is added to the genome near the regulatory sequence of gene of interest.
The transgenic gene is then introduced into the animal model where it is expressed.
The reporter gene will give indication of when and where the gene of interest is expressed.
25
Describe how high throughput analysis (microarray) can be used to establish where and when a gene is expressed?
The RNA is isolated and cDNA is generated.
The probe is labelled with fluorescent tags and hybridised to array.
When imaged, the colours will show if the particular cDNA is attached.
26
Describe single-cell RNA Sequencing (scRNA-Seq)?
Individual cells from a tissue are separated and suspended in oil droplets with barcodes so that each transcript or can be sequenced.
Look at the individual genes in each cell and compare them.
27
Why is it important to study whether the protein is expressed with the same timing and place to the gene? What techniques could you use to investigate this?
Location gives an idea of function.
Want to find out spatial and temporal expression pattern.
Western blot
Immunohistochemistry
28
Describe how immuno-detection methods (immuno-histochemistry, immuno-fluorescence) can be used to investigate the distribution of proteins?
Each section of a sample will be incubated with a primary then secondary antibody.
The secondary anti-IgG is coupled to a fluorescent tag.
29
Describe how fusion protein construct can be used to investigate the distribution of proteins?
GFP is fused to the gene so when the protein is expressed, hybrid mRNA and a hybrid protein is produced.
30
How can you study whether a gene/protein is essential for development?
Genetics: gain- or loss-of-function
31
What is forward genetics and how do you study it?
Seeks to identify a gene whose mutation caused a particular phenotype.
Expose animal model to a mutagen and breed with WT for 3 generations.
Identify the gene which has mutated and caused the malfunction in this mutant.
(Phenotype —> genotype)
32
What is reverse genetics and how do you study it?
Seeks to characterise the phenotype of particular mutated gene.
Homologous recombination knock-in of target gene.
ES cell is transferred, selected and injected into blastocysts.
Chimeric mouse selected and bred to study phenotype.
(genotype —> phenotype)
33
How would you study how genes are regulated?
Embryology tissue manipulation (graft, ablation)
| Manipulating signalling pathways with drugs, transferring, electroporation or genetics
34
Describe how tissue graft can be used to demonstrate inductive function?
Inserting presumptive epidermis into a blastocoel of zebrafish on the opposite side to the primary invagination.
This will result in a phenotype e.g. a second head and will tell you the function of this tissue.
35
Describe how bead/cell implantation (e.g. signalling molecules or drugs) can be used to demonstrate inductive function?
They mimic signalling molecular instead of using an ectopic piece of tissue.
E.g. transplanting ZPA in ectopic location results in mirror image
36
What techniques can be used for studying fate mapping (i.e. tissues and organs derived from cells that express gene)?
Embryology (chick/quail chimera, labelling with dye)
| Genetics (labelling with retrovirus or GFP, brainbow)
37
How can cell/tissue transplantation be used to study fate maps?
Transplanting part of embryo from quail into chick.
| The quail tissue can be detected by antibodies and traced over time.
38
How can cell/tissue labelling genetically be used to study fate maps?
Different cells can be marked with different colours and traced over time.
39
What is a morphogen?
A soluble secreted molecule that acts at a distance to specify the fates of other cells.
It may specify more than one cell type by forming a concentration gradient.
40
How do morphogenesis gradients cause different fates?
Cell have certain thresholds of concentration. If it reaches the threshold for a certain fate then the cell will follow that fate.
41
Morphogens decay exponentially. What would happen if a morphogen is:
1. Increased
2. Reduced
3. Decays slower (‘long range’)
4. Ectopic (e.g. morphogen produced in two places)
5. Mutated
6. Uniformly expressed
1. More cells with highest response, same at lower concentrations
2. Loss of highest response
3. Less information as will not reach lower concentrations
4. Mirror image
5. No information so all one cell type
6. All one cell type
42
What is the difference between an instructive signal and a permissive signal?
Instructive provides information whereas permissive signals provide a switch (already had signal but waiting for timing cue) and is not a morphogen
43
How would you test whether a signal is instructive or permissive (2 methods)?
Adding an ectopic source with the the signal. An instructive signal will produce a mirror image whereas permissive will have no effect.
Introducing a signal at a uniform concentration will induce one cell fate if instructive and have no effect if permissive
44
Why is a “bucket brigade” mechanism not used by morphogens?
Morphogens act directly on cells. Bucket brigade involves induced cells producing their own signals.
45
How would you test whether a ligand is a morphogen or using the bucket brigade mechanism?
Using genetic engineering to make proposed morphogen juxtacrine so that it is on the membrane of the producing cell and only affects neighbours. If morphogen then other cells will not see signal and will not respond whereas bucket brigade will.
Make a cell lose receptor for original ligand. Bucket brigade will not be affected as does not respond to this whereas if morphogen cell without receptor will not respond.
46
What helps establish a morphogen gradient?
Restricted diffusion generates a steep gradient by having high concentrations of binding molecules (e.g. HSPGs) and receptors which bind molecules to the extracellular matrix
Rapid degradation of the signal can also help generate the gradient as well as planar transcytosis across cells
47
How is timing important in establishing morphogen gradients?
There must be a mechanism which blocks premature specification as the first cell would see all fates and if it continued to be produced then all cells would adopt the same fate. A ‘check point’ where the steady state of receptor activation is reached must be involved for cells to determine their fate.
48
What is the transcriptional read-out model?
Higher concentration of morphogen often results in a higher concentration of an activated transcription factor
49
How does enhancer affinity and genetic crosstalk result in reading the gradient?
Genes in a cell could have low or high affinity to the TF. If the concentration is lower then the high affinity gene is likely to be expressed. If the concentration is higher the both affinity enhancers will be activated. The low affinity enhancer will produce a repressor to inhibit expression of the high affinity enhancer (crosstalk).
50
How are strict thresholds achieved when the gradient is not steep?
Positive feedback - a transcriptional activator which binds to its own enhancer
51
What is hedgehog and what is its role in the drosophila embryo?
A segment polarity gene which acts in segmental patterning via a feedback loop with Wg/Wnt
52
How is the Hh ligand formed and released from membrane?
The protein is targeted and undergoes autoproteolyis and cholesterol modification. It then undergoes palmitoylation and as it is hydrophobic can move through membrane but is not easily diffused. The dispatched receptor and its ligand Scube are important in the diffusion of Hh where it interacts with (lipoprotein particles?, cytonemes? and) HSPG.
53
How is the Wnt ligand formed and released from the membrane?
The protein undergoes palmitoylation and palmitoleic acid modification. Wntless receptor helps membrane targeting, presentation and release as Wnt is very hydrophobic. Once released it interacts with (cytonemes?, lipoprotein particles? and) HSPG
54
What are cytonemes and how do they aid Wnt signalling?
Cellular protrusions which allow cells to reach out and contract other cells. Wnt becomes exposed to the tip of the cytoneme and as it grows out via anterograde transport it will reach a Wnt-receiving cell and change its fate.
55
What is the “handover” mechanism of WNT diffusion?
Dlp protein binds to Wnt. Dlp is coupled to the membrane via GP1 link so can be motile within the cell and laterally diffuse over membranes. Could also be possible that they can hand over Wnt molecules to other Dlp molecules on another cell.
Palmitate can block the binding pocket shielding the hydrophobic nature of Wnt
56
How is the Hh signal signal received?
When Hh binds to receptor Ptch, it no longer inhibits Smo. Smo relocates, accumulates and phosphorylates and increases the level of cholesterol on the inner leaflet.
57
What are the Hh downstream events?
When Hh binds to Ptch, Smo is no longer inhibited. Smo causes the Cos2-Fused-Ci complex and the SuFu-Ci complex to modify and become CiAct (activated TF). This is due to the CKI-PKA-GSK3 complex (which normally phosphorylates Cos2 complex) and Slimb (which usually is involved in ubiquitination of phosphorylates Cos2).
58
How does Hh signalling act on its own pathway?
Negatively by limiting activation levels of Ptch
| Positively by inducing Gli1 (acts similar to Ci) which cannot be proteolysed into a repressor
59
How is Hh involved in Drosophila wing patterning?
It’s expressed on the posterior side and diffuses across to induce a BMP like signalling molecule which acts to pattern the anterior-posterior plane
60
How is Hh involved in neural development?
It is produced in the floor plate and notochord. Cells in the neural tube differentiate into different types of Neuron’s depending on how long and how much Shh they receive
61
How is Shh involved in limb development?
It creates a ZPA which forms the anterior-posterior pattern.
| It also has inhibition pathways and a signalling loop to produce outgrowth.
62
What are two diseases caused by both a loss of Hh signalling or too much Hh signalling?
Loss: holoprosencephaly, cyclops
Gain: polydactyly, syndactyly
63
What could cause a gain of Hh signalling resulting in cancers such as basal cell carcinoma, medulloblastomas and rhabdomyosarcomas and Gorlin Syndrome come about?
Inactivation of Ptch1 or Sufu (tumour suppressor genes)
Activating mutations of Smo (proto oncogene)
Gorlin syndrome is due heterozygosity for Ptch1 therefore damage to other copy of Ptch1 e.g. by sunburn will result in activation of Hh signalling
64
How is Wnt received?
Ligand binds to both Arrow/LRP5&6 receptor and Frizzled receptor. This brings them together forming a receptor complex and it is likely this is what causes downstream activation of pathway
65
What are the downstream effects of Wnt binding?
Dsh phosphorylated and binds to Frizzled. Arrow is phosphorylated and binds to APC-Axin-CK1-GSK3 complex. This is phosphorylated and Slimb (ubiquitination complex) is lost. Beta-catenin can be released and displace the Grouch transcriptional repressor, activating expression of target gene.
66
What are the roles of Wnt in Drosophila and C elegans?
Segmentation and expressed in D/V boundaries in Drosophila
| Regulation of neuronal fate and migration in C elegans
67
What are some diseases related to loss or gain of Wnt signalling?
Loss of Wnt can cause a loss of stem cells in the gut
Gain of Wnt signalling results in colon cancers, breast, ovarian, uterine cancers, melanomas, prostate cancer, bone diseases, tooth agenesis
68
What is an example of non-canonical Hh signalling?
In myocytes and adipocytes Shh activates Smo which activates Ca2+ and Ampk signalling. This results in metabolic reprogramming towards aerobic glycolysis producing lactate and ATP.
Inhibitors of canonical Hh signalling activate the non-canonical, and could lead to too much Ca2+ and issues with muscles.
69
What is an example of non-canonical Wnt signalling?
Planar cell polarity/convergent extension pathway. Mediated by Wnt11, Wnt5, Frizzled and Dishevelled. These act to polarise cells within a sheet so affecting this pathway affects polarity (hairs on wings Drosophila and axis elongation zebrafish)
70
What are advantages and disadvantages of Drosophila as an animal model?
Advantages: accessible embryology and adult development stages, very low cost, fast, excellent genetics, no ethical concerns
Disadvantages: not a vertebrate, kept as live stocks
71
What are the main features of a Drosophila larvae?
3 thoracic segments and 8 abdominal segments. Mouth apparatus on thoracic end and posterior spiracles (for breathing) and anal pads on posterior end. Dentricles (lines of hair) on underside of larvae along abdominal segments.
72
What are techniques that use transgenic animals to either visualise, misexpress or reduce gene expression?
```
P-element transformation - transgenics
Enhancer trap - promoter trapping
Gal4/UAS - gene misexpression
FLP/FRT - ‘clonal’ mutant analysis
RNAi - both ex vivo and in vivo
‘Omic’ technologies - genome, transcriptome, proteome etc
```
73
Describe spermatogenesis in the Drosophila testes.
Hub cells in apical top of testes secrete factors e.g. Unpaired (JAK/STAT pathway ligand). The germline stem cells are located adjacent to the hub and cells further away will differentiate forming spermatogonial cells. Eventually will divide mitotically and meiotically into mature sperm.
74
Describe oognesis in the Drosophila ovarioles.
Stem cells at tip of ovariole maintained by JAK/STAT pathway. They divide until 32 cells (4 cytoblast mitotic divisions) but remain attached by bridges. One of the cells which has 4 attachments will become the egg, and undergo ‘meitotic’ recombination, and surrounding cells will become nurse cells and undergo endo-reduplication.
As it moves through the ovariole, follicle cells and nurse cells provide the oocyte with yolk, fats, lipids and maternally supplied proteins so it can grow and become fertilised.
75
How do nurse cells provide food and proteins etc to the developing oocyte?
Cytoplasmic dumping through ring canals
76
How do bicoid (anterior), oskar mRNA (posterior) and other mRNAs (dorsal) become localised in the oocyte?
Microtubule transport
Minus and plus ended motors
Glue anchors them in position
77
What happens in the first 14 divisions of a fertilised oocyte?
The nuclei of sperm and oocyte fuse and divide. By the 8th division most of the blastoderm nuclei are around the outside of the egg and this continues. At the 14th division there are vitellophages in the middle and pole cells at posterior as well. After the 14th division cellularisation occurs.
78
What are some common intracellular signals used Drosophila?
```
Hh
Wnt
Delta and Serrate
TGF-alpha and -beta
FGF
```
79
What is the role of the gap genes in Drosophila, what is an example and what would a mutation in this gene look like?
Gap genes are involved in development of a section of an organism. A mutation results in loss of a body segment e.g. knirps mutation sees loss of middle of the body
80
What is the role of pair rule genes in Drosophila, what is an example and what would a mutation in this gene look like?
Pair rule genes help define alternating segments. A mutation will result in loss of every other segment e.g. paired mutation will see every second segment pair lost therefore half the size.
81
What is the role of segment polarity genes in Drosophila, what is an example and what would a mutation in this gene look like?
Segment polarity genes help define the anterior and posterior polarities within each embryonic parasegment. A mutation will result in loss of naked cuticles e.g. gooseberry mutation will see all dentricle belts fused together.
82
What is the role of Bicoid and what does a Bicoid mutant look like? Can it be rescued?
Bicoid is a DNA binding transcriptional activator maternally loaded into a developing oocyte.
Mutant loses its anterior structures.
Yes can be partially rescued by transferring Bicoid WT cytoplasm to anterior part of body. Transferring to middle of body results in ectopic head structures and mirror image thoracic segments.
83
How does different Bicoid concentrations affect the segmentation pattern?
No Bicoid pushes everything anteriorly and top two segments missing.
Increased Bicoid pushes the pattern backwards.
84
How do enhancers on DNA help Bicoid form the French Flag Model?
Strong (high affinity) AND weak (low affinity) enhancers on a gene will be expressed at very low concentrations.
Just strong enhancers will express a gene at low concentrations.
Just weak enhancers will require higher concentrations to be expressed.
85
How do segmental genes form expression patterns?
From maternal genes gradients e.g. bicoid. Read these gradients to define blocks or domains of gene expression
86
Where is engrailed segment gene expressed in parasegment boundaries (and how does this compare to segment boundaries)?
On the posterior.
| Segment boundaries are not molecularly determined but instead define each segment so engrailed is expressed anteriorly.
87
Why do both Hh and Wg mutants result in loss of naked cuticle and so show lawn of dentricles?
They feedback on each other to maintain each other’s continued expression and refine segment borders. As Wg is needed to suppress hair growth and Hh maintains this, loss of either will result in loss of naked cuticle.
88
True or False?
| Expression of homeostatic genes along the D/V body axis occurs in the same order as the genes are within the genome.
False. They are the same as the A/P body axis.
89
Are Drosophila long or short germ band insects and what does this mean?
Long germ band insect. All 14 segments are defined at once. Embryogenesis is quick (24 hours) but complicated as maternal, gal, pair rule genes interact for every segment.
90
How do short germ band insects form?
Start with head and thoracic segments and add abdominal segments sequentially by posterior disc (proctodeum) budding off segments as it gets smaller. This is moderate complexity and not too slow.
91
How is Delta-Notch involved in segment addition (segmentation clock)?
Activation of Notch by Delta causes down regulation of Delta in that cell by Her1 expression. There is a time lag which causes oscillation between strong and weak signalling levels. This propagation of signal between cells causes wave of activation.
92
What is a blastula?
A mass of mostly unpatterned cells 3-4 hours after fertilisation
93
What are the two types of cytoplasm in a polarised unfertilised Xenopus egg and how do they differ?
Animal and vegetal. Animal is opaque and on the upper hemisphere. Vegetal is the lower hemisphere, yolky and contains cytoplasmic determinants e.g. mRNA and localised protein molecules.
94
How is the unfertilised egg polarised?
By gravity
95
How are the embryonic germ layers formed in Xenopus embryos?
Cells derived from the vegetal hemisphere signal to cells derived from the animal hemisphere. The animal cells closest to the vegetal hemisphere (in marginal zone) receive these signals and become mesoderm.
Animal hemisphere cells become ectoderm.
Vegetal hemisphere cells become endoderm.
96
What are the processes involved in vegetally-localised signalling?
VegT mRNA is tethered to structures in lower portion of vegetal hemisphere. VegT protein binds to gene promotors of genes encoding Nodal morphogen (Xnr genes) and activates their transcription.
97
What is the issue with the observing the presence of localised determinants that form mesoderm and endoderm?
It explains the induction but not the organisation and patterning within the embryo along A/P and D/V axes
99
What is the prefigured event that occurs at fertilisation leading to the formation of the Nieuwkoop centre in the blastula?
Symmetry-breaking event. The further dorsal side of the embryo will develop from a region opposite the site of sperm entry. A 30 degree rotation of cortical cytoplasm redistributes and activates maternal dorsalising factors.
100
What does the Nieuwkoop centre induce?
The Spemann-Mangold Organiser
101
What occurs within the Nieuwkoop centre?
Wnt11 mRNA and Dishevelled protein are transported along microtubules away from the vegetal pole towards the animal hemisphere. Beta-catenin accumulates in the nuclei of cells on the future dorsal side of the embryo causing transcription of Wnt-pathway target genes.
102
What are the 2 fundamentally different, temporally separated functions in the Xenopus embryo?
1. Specification of D/V axis in early embryo before gastrulation in induction of Nieuwkoop
2. Patterning of A/P axis in embryo after gastrulation for ventralisation and posteriorisation of the mesoderm
103
What does transcription of Spemann-Mangold Organiser-specific genes (chordin, noggin, goosecoid) require?
Activation of beta-catenin via Wnt to induce Siamois gene transcription alongside activation of Xnr proteins to induce Smad2 activation. Siamosis and Smad2 transcription factors are both uniquely present in S-M organiser and induce transcription of S-M genes.
104
What activates the gastrulation process?
Targets of organiser specific transcription factors (Goosecoid, Not1, Lim1) and Brachyury (which is present throughout the mesoderm including organiser) targets create the dorsal axial mesoderm which initiates gastrulation.
105
During gastrulation, how does the Spemann-Mangold organiser (first visible as the dorsal lip) pattern the embryos A/P and D/V axes?
It regulates involution, intercalation and migration of axial mesoderm across the blastocoel roof (“convergent extension”).
It also regulates the acquisition of distinct cell fates by mesodermal cells.
106
How does the Xenopus gastrulation compare to chick and human embryos?
Chick/human is a flattened multilayer disc rather than hollow multilayered sphere.
In chick gastrula the pharyngeal endoderm extends along the dorsal midline first, then prechordal mesoderm then notochord.
By day 12 the human embryo has separated into a top epiblast layer and bottom hypoblast layer —> very similar to Xenopus animal and vegetal hemispheres
—> conservation of mechanisms
107
What three embryonic tissues will the Spemann-Mangold organiser give rise to?
Pharyngeal endoderm
Prechordal mesoderm
Notochord
108
What is the result of a transplanting donor dorsal blastopore lip tissue into a blastocoel on the ventral side to the Spemann-Mangold organiser?
A secondary invagination resulting in induced secondary structures and a twinned embryo with mesodermal and neural tissues and organs
109
What evidence is there that the inductive properties of the Spemann-Mangold organiser changes during gastrulation?
If transplant early organiser then there is a complete second axis including head and trunk
If transplant is late then there will be a partial second axis comprising the trunk tissue only
110
What are the 4 functions of the Spemann-Mangold organiser?
1. It creates the A/P axis of the embryo
2. It induces neural tissue from the ectoderm
3. It patterns this neural tissue whilst creating the A/P axis of the embryo
4. It introduces A/P and D/V pattern into the mesoderm
111
How does early, mid and late organiser tissue induce formation of specific tissues?
Early organiser tissue expresses Wnt Antagonists and BMP antagonists which induces head and brain tissue.
Mid and late organiser tissue expresses only BMP antagonists which induces trunk and spinal cord
112
What are some BMP antagonists, where are they expressed and what is their role in gastrulation?
Noggin, Chordin and Cerberus
Expressed in the S-M organiser and its axial mesoderm derivatives.
They act on ectoderm to suppress epidermal fate and induce neural fate.
113
What are some Wnt antagonists, where are they expressed and what is their role in gastrulation?
Cerberus, Frzb and Dickkopf
Expressed in S-M organiser.
Induce anterior character with neural tissue, promoting brain development by antagonising the posteriorising and ventralising functions of wnt8 in the mesoderm. This preserves prechordal mesoderm identity.
114
What are the two gradients generated by the Spemann-Mangold organiser and by what signals does it do this?
A/P gradient of Wnt signalling activity in developing neural plate - lowest in anterior neural plate.
D/V gradient of BMP signalling activity throughout embryo - lowest in neural plate and dorsal axial mesoderm
115
What is the Activation-Transformation Model of neural tube A/P patterning?
Ectoderm becomes activated and neuralises/specifies the forebrain.
The prospective forebrain becomes transformed (caudalizes) in mid gastrula stages and onwards to induce posteriorised neural fates midbrain, hindbrain and spinal cord.
116
What signalling pathways does the Activation-Transformation pathway involve and where is each pathway active?
Wnt is inhibited in the early gastrula - gives rise to anterior features.
BMP is inhibited in the early, mid and late gastrula - gives rise to anterior character to anterior neural plate throughout A/P axis
Wnt, RA and FGF gradients exposed to posterior neural plate - posteriorises neural fates
117
How are the distinct domains of vertebrate spinal cord and and column defined?
Through specific combinations of Hox transcription factor expression induced by gradients e.g. RA concentration gradient
118
What are the three signalling systems involved in polarising the neural tube?
1. TGF-Beta ligands (mainly BMPs) secreted by the dorsal Roof Plate induce dorsal neuronal fates
2. Shh secreted by ventral Floor Plate (and notochord) induces ventral neuronal fates
3. Wnt signals are also secreted by the Roof Plate. Their functions include promoting expression of BMPs in roof plate and contributing to induction of neural crest
119
What happens when TGF-beta pathway is received for the target gene to be activated?
TGF-Beta binds to TGFBR2 and TGFBR1, causing dimerisation and phosphorylation of TGFBR1. This activates the SMAD pathway (SMAD2, 3 and 4) which forms a complex that gets phosphorylated. This binds to DNA-binding TF and activates target genes. The cell responses can also be induced by a non-Smad pathway.
120
What are the three branches of the TGF-Beta super family and what are examples of each?
BMP-like family e.g. BMPs, GDFs, AMH
GDNFs family e.g. GDNF, Artemin, Neurturin, Persephin
TGF-beta like family e.g. TGF-betas, Activins, Nodal
121
Why can implicated TGF-beta give rise to tumours?
Because it can act as a tumour suppressor or promotor due to its role in cell division, apoptosis, growth and migration
122
When is tissue organisation and axial patterning established?
During gastrulation
123
What is the canonical RTK activation pathway?
Ligand facilitates dimerisation of inactive RTKs. The kinase domains phosphorylate each other which increases activity, stabilises the receptor and creates docking site
124
What are 3 examples of proteins that bind to docking sites on RTKs?
1. Pl 3-kinase (inositol lipid pathway)
2. GTPase-activating protein (RAS/MAP kinase pathway)
3. PLC-gamma (inositol lipid pathway)
125
What enzymes are involved in converting the GTP-bound active RAS to inactive GDP-bound inactive RAS?
GTP-ase-activating proteins (GAPs)
| Guanine nucleotide exchange factors (GEFs)
126
How does docking lead to signal transduction in the RAS pathway?
GRB2 and Sos (GEF) couple the receptor to inactive Ras. Sos promotes dissociation of GDP from Ras, GTP binds Ras and then it dissociates from Sos.
127
What is the downstream signal transduction pathway of RTks?
Activated Ras activates MAP kinase kinase kinase (Raf). This activates MAP kinase kinase (Mek) which activates MAP kinase (Mapk). This then goes on to activate proteins and transcription regulators.
128
How many FGF ligands are in the family and what are the 3 different types?
22
Paracrine fgf
Intacrine fgf
Endocrine fgf
129
Which fgf plays an essential role in development?
Fgf8
130
Describe the structure of the FGF receptor.
Extracellularly there are 3 lg-like domains: D1, D2 and D3. The heparin-binding site is in D2 and the FGF ligand binds to D3. An acid box between D1 and D2 interacts with heparan sulfate binding site to prevent receptor activation in absence of fgf.
Intracellularly is the kinase domain split into two.
131
What are 5 features of Heparan sulphate proteoglycans (HSPGs)?
1. Protein core which can be transmembrane, tethered or secreted
2. Long chains of sugars called heparan
3. Each sugar can be modified e.g. by sulphation
4. Modification creates 'code' which creates binding sites and a sequence carrying information
5. Polyanionic (negative charge)
132
What is the role of HSPGs and how does affinity to these affect diffusion of ligands?
Fgfs can only bind and activate a receptor complex if HSPGs are present.
Paracrine Fgfs have a higher affinity to HSPGs and so will not diffuse far.
Endocrine Fgfs have a lower affinity to HSPGs and so diffuse into bloodstream and act long-distance.
133
What is the Ras signal transduction pathway of FGF receptors?
FGFR → FR52alpha → GRB2 →SOS→RAS→RAF→MAPKK→ MAPK→ FOS→ cell proliferation
134
What are pathways FGF signalling may trigger?
Cell proliferation
Cell survival
Cell motility
135
Where does mesoderm form during gastrulation?
At the primitive streak
136
What will the intermediate mesoderm go on to form?
Kidney
| Gonads
137
What will the axial mesoderm go on to form?
Notochord
| Pre-chordal mesoderm
138
What will the paraxial mesoderm go on to form?
```
Head
Somite (sclerotome, syndotome, myotome, endothelial cells, dermatome)
```
139
What will the lateral plate mesoderm go on to form?
Splanchnic (circulatory system)
Somatic (body cavity, pelvis, limb bones)
Extra-embryonic
140
What are somites?
Segmented paraxial mesoderm tissue
| They dictate the number of vertebrae
141
How does BMP signalling mediate medio-lateral patterning of the mesoderm?
It is high in the lateral plate mesoderm and low in pre-somitic mesoderm
142
What is responsible for the patterning of the A/P axis formation in the paraxial mesoderm?
Hox genes. They begin to be expressed during gastrulation in mesoderm cells. The anterior Hox genes are expressed first and a pattern is produced.
143
How do somites form?
In a continuous manner by budding off in pairs from the paraxial mesoderm
144
What is the 'Clock and Wavefront' model?
It predicts that a clock ticks in the posterior PSM and drives a molecular oscillator that dictates the periodicity of somites.
Where cells hit the travelling wavefront, an abrupt change of property occurs leading to the decision to form somites.
145
How is the oscillatory pattern of Hairy /Hes/Her mRNA formed?
Hes1 geneis activated by the Notch signalling pathway. They are expressed into Hes1 protein whichis very quickly degraded. It also acts as a transcriptional repressor meaning the level of Hes 1will oscillate.
146
How many cycles of oscillations does the chick presomitic mesoderm undergo before it starts forming somites?
12
147
How is the determination front formed and maintained?
A RA gradient from the somites opposes a posterior FGF8 gradient. These gradients regulate each other by negative feedback which maintains the level of the determination front.
148
What molecularly establishes the determination front?
FGF8 activates Tbx6. Tbx6 along with Notch signalling activates Mesp2 which is expressed at the determination front.
149
What effect will inducing Notch signalling inside a somite have?
It will introduce a new boundary
150
What is the overall model for the control of somite formation?
Molecular oscillation → c-hairy1 → lunatic fringe → delta1/notch1 → ephrins → cell adhesion changes → somite formation
151
What will occur as a result of absence of Notch?
Ossification centres do not align and will be defects in formation in axial skeleton
152
What are the main functions of skeletal muscles?
Motor function
Metabolism
Respiration
153
How is a muscle made from stem cells?
Stem cells undergo specification/determination and become muscle progenitor cells (myoblast).
This undergoes differentiation to become myotubes.
These mature into myofibres.
154
What happens when placing the gene for MyoD after a promoter and transfecting into a cell such as a fibroblast?
Within a few days the cell will convert to a myoblast and eventually myotube with muscle-specific proteins, receptors, membrane molecules and multiple nuclei.
155
Where do skeletal muscles originate?
Dermomyotome progenitor cells
156
What transcription factor is expressed in skeletal muscle progenitors?
Pax3
157
What are the MRFs expressed during embryonic development?
Myf5
MyoD
Myogenin
MRF4
158
What do knockout studies show about the role of MRFs?
Myf5 KO results in delayed myotome formation.
MyoD KO results slight delay in limb muscle development however Myf5 and MyoD can compensate for one another.
KO of both results in no myoblasts therefore they are required to generate myoblasts.
KO of Myogenin leads to reduced density of muscles so is required for muscle differentiation.
159
What pathways specify the epaxial muscle lineage?
Cooperation between shh and wnt signals to induce Myf5 and MyoD expression
160
What pathways specify the hypaxial muscle lineage?
Wnt signals induce Myf5 and MyoD in cells entering the lateral myotome.
BMP4 induces pax3 and represses Myf5 and MyoD in cells fated to migrate in the limb bud.
161
What pathways cause migration of muscle cells into the limb bud?
Mesenchymal cells from the limb bud secrete HGF/SF.
Pax3 induces the expression of c-met (receptor for HGF/SF) in time muscle progenitor cells in somites. Cells then migrate to the limb.
162
What are satellite cells and what stimulates their activation?
Precursors to skeletal muscle cells so quiescent stem cells. They sit under the basal lamina of muscle fibre.
Stimulated by injury, denervation, exercise.
163
How does muscle regeneration/growth by satellite cells occur?
1. Activated and induce Myf5 or MyoD
2. Expression of Myf5 and MyoD
3. Proliferation/self-renewal
4. Differentiation and fusion to existing fibres
164
What are the three tissues responsible for formation of bones?
Cranial neural crest (craniofacial skeleton)
Somites (axial skeleton)
Lateral mesoderm (limb skeleton)
165
What are the three steps leading to axial skeleton formation?
1. Sclerotome induction
2. Cartilage formation = chondrogenesis
3. Ossification of axial skeleton = osteogenesis
166
What are the steps in chondrogenesis?
Stem cells be come specified to become sclerotomal cells.
These undergo determination into chondroblasts.
These differentiate into chondrocytes.
These mature into hypertrophic chondrocytes.
167
What are the transcription factors expressed during sclerotome formation and whereabouts are they expressed?
Pax1 is expressed slightly more medially
| Pax9 is expressed slightly more laterally
168
How do Hox genes correlate to the axial skeleton formation?
Pattern of Hox genes correlates to the positional information of cells and also the time of expression.
I.e. Most anterior gene is expressed first and most anteriorly.
169
How are Shh and BMP4 involved in sclerotome development?
Shh expressed in notochord and induces sclerotome by inducing and maintaining Pax1 and 9.
BMP4 is produced in the lateral plate mesoderm and plays an important role in maintaining and allowing different domains of Pax1 and 9.
170
What processes are involved in the transformation of sclerotomal cell into chondroblast?
1. Migration of cells around notochord
2. Down-regulation of Pax1 and Pax9
3. Condensation of cells - extracellular matrix proteins
171
What processes are involved in the transformation of chondroblast into chondrocyte?
1. Proliferation induced by BMP2, BMP4, and BMP5
| 2. Production of cartilage matrix, requires Sox9
172
What are the two main modes of ossification?
```
Intramembranous ossification (mesenchymal cells —> nodules —> osteoblasts —> osteocytes —> bone. Mostly ossification of bones from skull)
Endochondral ossification (used for ossification of most bones I.e. limbs)
```
173
How does endochondral ossification form bones?
Chondrogenesis until chondraytes stop dividing. They die via apoptosis and blood vessels and osteoblasts enter this space (future bone marrow). Osteoblasts replace the cartilage and forms primary ossification centre. Blood vessels enter the epiphyses and secondary ossification centres form leaving cartilage plate between epiphysis and diaphysis.
174
How does Ihh and PTHrP control chondrocyte differentiation and maturation?
Ihh triggers PTHrP production which act in a negative feedback loop on the proliferation zone and zone of maturation to promote proliferation but not differentiation. Ihh is inhibited by FGFR3.
175
How does the 2D Drosophila wing imaginal disc become 3D?
It unfolds into adult shape by eversion.
176
Which transcription factor defines the posterior compartment of segments?
Engrailed.
| This is at the posterior of a physical segments, legs, wings etc even in adults
177
Where is the Hh pathway active in the Drosophila wing development and why?
In a stripe on the anterior side of wing. Engrailed suppresses Ci expression but drives Hh expression. Means Ci is only expressed in the absence of Engrailed. Patched is the Hh receptor and a pathway target gene so where Ci is expressed and Hh concentration is high is where the pathway will be active.
178
Where is dpp expressed in the Drosophila wing development and why?
The centre of the disc. As dpp is a Hh target gene it is expressed where Hh and Ci are present. It moves to both anterior and posterior as is difusible and so can cross compartment boundaries.
179
What is dpp and what is its downstream effects?
A secreted ligand of the TGF-beta superfamily.
Dpp → Tkv/Pnt (type I and type ll receptor heterodimer) → Mad (TF)
Mad inhibits brinker and promotes omb and sal
180
Which morphogen establishes the dorsal ventral boundary in the developing Drosophila wing?
Wg
181
How does Wg drive expression of targets such as Ac, DII, Vg?
In a concentration dependent manner
182
What are experimental techniques used to study development in Drosophila?
Reporter gene
Antibody staining
Confocal microscopy
Mutant clonal analysis altering patterning centres
183
What are the three regions the limb is organised into?
Stylopod (humerus)
Zeugopod (ulna and radius)
Autopod (carpals and digits)
184
What specifies the forelimb field in a developing embryo?
The anterior boundary of Hoxc6
185
What are the two T-box transcription factors that specify limb identity, what do they control and what does their misexpression cause?
Tbx5 expression correlates to wing development by driving EMT and Wnt2b.
Tbx4 expression correlates to the embryonic hindlimb and is activated by Pitx1 and drives Wnt8c.
Misexpression of either TF in their wrong position will result in leg-like structure in forelimb or wing-like structure 'in hindlimb.
186
What is the role of FGF10 in the lateral mesoderm?
Drives Tbx4/5 gene expression
187
What is the role of RA in the limb development and what restricts its activity?
Restricted by FGF8 to the fore-limb field. Activates Tbx5 which drives forelimb bud initiation.
188
What are the roles of Wnt2b, Fgf10, Fgf8 and Wnt3a in the forelimb bud initiation?
Wnt2b and drives Fgf10.
Fgf10 is important for later growth, drives mitosis, Wnt3a and also feeds back to Tbx5 and Wnt2b.
Wnt3a drives Fgf8 in the ectoderm.
Fgf8 feeds back to Fgf10.
189
What is the apical ectodermal ridge (AER)?
Structure that forms from ectoderm cells at the distal end of each limb and acts as a major signalling centre for development of the progress zone. Removal of the AER hinders development.
190
What is the progress zone model of proximo-distal patterning but what is the problem with this model?
Cells adopt a certain fate depending on how long they spend in the progress zone.
However progenitors for all elements found in the progress zone whereas model would suggest only proximal elements.
191
What is the two-signal model of proximo-distal patterning?
Two antagonising gradients (RA and FGF) work together to specify different regions within developing limb resulting in formation of different regions which are patterned to become distinct tissues.
192
Which morphogen is present in the ZPA and responsible for A/P patterning?
Shh
193
How does the A/P patterning coordinate with the P/D patterning?
Fgf8 from the AER feeds back to the ZPA to maintain shh signalling.
Shh from the ZPA maintains Fgf4 and 8 expression.
194
What is the proposed model for controlling dorso-ventral patterning?)
Bmpr → engrailed-1 (ventral pattern) —| wnt7a → Lmx 1b (dorsal pattern)
195
Why is branching mophogenesis essential for enabling physiological functions of different tissues?
It increases SA:V, maximising the efficient of gas, fluid or exchange, secretion or excretion.
196
What are some tissues that use branching morphogenesis?
```
Lung
Ureteric bud
Salivary gland
Prostate
Mammary gland
Pancreas
```
197
What is the organ responsible for respiratory gas exchange in Drosophila and how does it form?
Tracheal system.
Tracheal placode undergoes placodal invagination into tracheal sac. This develops protrusions via collective cell migration called tracheal tubules.
198
How does Branchless (Bnl) and Breathless (Btl) drive branching on tracheal epithelium and unicellular branching of tip cells?
Epidermal cells secrete Bnl which is detected by Btl receptor on Leader cells. When activated, these receptors induce branching. Only the Leader cells exposed to highest levels of Bnl can form the terminal unicellular branches.
199
What processes and proteins are involved in the collective cell migration and cell interacalation for branching in the Drosophila tracheal system?
Leader cells move towards Bnl pulling branches with it. Leader cells express Sprouty which inhibits tyrosine kinase signalling and therefore branching in neighbouring cells. Also Notch/Delta mediates lateral inhibition.
200
What are 5 features of receptor tyrosine kinases (RTKs)?
1. Mostly monomers (except insulin receptor)
2. Extracellular domains vary greatly (ligand binding activity)
3. Intracellular domains have kinase activity (enzyme linked receptors)
4. Single transmembrane domain (25-38 aA)
5. 20 subfamilies of RTKs
201
Where does the intermediate mesoderm lie and what will it give rise to?
Between somites and lateral plate.
| It will give rise to both ureteric bud epithelium and metanephrogenic mesenchyme of the kidney.
202
What are the three major phases of kidney development?
1. Pronephros formation (initial kidney)
2. Mesonesphros formation (transitional non-functional structure in mammals)
3. Metanephros formation (permanent kidney in amniotes, formed through inductive interactions)
203
How are metanephrogenic mesenchyme and the ureteric bud derived from pax2-expressing intermediate mesoderm in the embryo?
Anterior IM is exposed to higher Fgf9 and RA levels for longer and lower Wnt for less time --> forms ureteric bud epithelium
Posterior IM is exposed to lower levels of Fgf9 and RA for less time and higher levels of Wnt for longer --> forms metanephrogenic mesenchyme
204
How is branching morphogenesis of the ureteric bud regulated?
GDNF secreted from metamephrogenic mesenchyme --> causes proliferation and outgrowth in RET-receptor+ tip cells --> leading-edge tip cells arrest proliferation and bud flattens --> lateral tip cells continue to proliferate buldging laterally and forming a cleft --> RET-receptor+ ureteric bud cells secrete BMP7 and FGF2 to prevent apoptosis whilst cells in cleft undergo apoptosis --> lateral tip cells still surrounded by mesenchyme so branching repeats
205
What happens when branch formation of the ureteric bud has been induced?
The ureteric bud-associated mesenchyme aggregates, cavitates and epithelialises to form the renal tubules and glomeruli of the kidney. This is driven by Wnt signals produced by ureteric bud. Proximally the developing renal tubule will fuse with the developing collecting-duct and distally it will attract endothelial capillaries that form glomerulus.
206
What is the role of Wnt11 and Wnt9b in the developing kidney?
They convert aggregated mesenchyme cells into nephrons.
| Wnt9b is expressed in the UB stalk and Wnt11 in the mesenchyme-associated tips of branching UB.
207
How do the trachea, bronchi, bronchioles and alveoli arise from the respiratory diverticulum (a ventral bud of endoderm)?
The tip of the respiratory diverticulum undergoes branching morphogenesis to form lung buds. Lung buds then undergo further branching, surrounded by a sac of mesoderm.
208
What are the three periods of alveoli development?
```
Canalicular period (lung epithelium is cuboidal)
Terminal sac period (lung epithelial cells become Type 1 squamous cells, some interactions with capillaries)
Alveolar period (lung epithelial cells are Type 2, surfactant-producing squamous cells and directly interact with capillaries)
```
209
What are the roles of FGF, Shh and BMP signalling in branching morphogenesis of the lung (think mesenchymal-epithelial interactions)?
1. FGF10 expressed in mesenchyme up-regulates FGFR2B expression in nearby lung epithelium which stimulates bud outgrowth and induces Sprouty in tip
2. Sprouty suppresses proliferation and therefore FGF10 signalling via negative feedback
3. In response to high FGFR2B activation at the very tip cells, they express Shh and BMP4
4. Shh suppresses FGF10 expression locally on mesenchyme so domain splits into two and begin to grow laterally
5. BMP4 directly acts on tip cells to inhibit proliferation therefore non-BMP-expressing cells can grow out towards new FGF10 sources and next round of branching induced
210
How does the heart anatomy differ in model organisms such as Xenopus, Zebrafish and Drosophila?
Xenopus has a 3 chambered heart where blood appears to mix.
Zebrafish has a single circulatory system and therefore a 2-chambered heart.
Drosophila have a tubular heart.
Humans, mice and chicks all have a 4-chambered heart.
211
What are the key common morphological steps in heart development?
1. Bilateral populations of cells around the midline are the cardiac precursor cells
2. These migrate to the midline and fuse to form the heart tube
3. Heart looping occurs (an asymmetric bending morphogenesis)
4. Maturation resulting in formation of structres required for proper function e.g. valves, septa and trabeculae
212
What are the two populations of cardiac cells which are specified during early development and exhibit distinct spatiotemporal differentiation into the heart?
First heart field (FHF) - left ventricle and left and right atria
Second heart field (SHF) - right ventricle, left and right atria, outflow tract
213
What factors are involved in the differentiation of the cardiac mesoderm into the FHF and SHF progenitors?
The cardiac mesoderm is exposed to Non-canonical Wnt signalling.
BMP induces FHF progenitor formation by expression of Hcn4+, Tbx5+ and Nkx2.5+.
Wnt/beta-catenin and FGF induces SHF progenitor formation by expression of Isl1+, Nkx2.5+ and Flk1+.
214
What happens when the cardiac cells are specified from mesodermal tissue along the primitive streak?
They migrate anteriorly to form the primitive heart tube (FHF) and SHF in the adjacent mesoderm.
215
What needs to be patterned in the heart by functional regionalisation?
Chamber vs non-chamber
Atrial vs ventricular contractility (directional conduction)
Inflow (pacemaker/SA node), atrioventricular canal (valve/AV node), outflow (valves)
216
What signalling pathways are involved in the formation of Chamber and AVC?
AVC: BMP - Tbx2
Chamber : Notch - Tbx20
--> lots of other interactions between factors such as inhibition of Tbx2 in Chamber
217
What are the processes involved in heart morphogenesis and are they controlled intrinsically or extrinsically?
Changes in cell shape
Growth of the heart tube
Asymmetric cell movements at the poles of the heart
Regional changes in ECM composition (allows for shaping and maturation)
Lateralisatised cell signalling from the embryo
Mostly intrinsic as looping morphogenesis can begin to occur external to an embryo however lateralised cell signalling and growth are more extrinsic influences.
218
What helps promote looping morphogenesis?
Growth of the heart through SHF addition.
219
What are some congenital heart defects (including those associated with heterotaxia)?
Associated:
Situs inversus
Situs ambiguous
```
Not associated:
Ventral septal defect
Double Inlet Left Ventricle
Double Outlet Right Ventricle
Transposition of the Great Arteries
Persistent Truncus Arteriosus
Atrial Septal Defect
```
220
How is gene expression in the heart made asymmetric?
A transient cup-shaped organ forms at the posterior of the embryo and is lined with motile cilia which beat in a clockwise movement. This creates directional fluid flow and results in elevated Ca in the left side of the embryo. This facilitates transferal of nodal expression around the left lateral plate mesoderm and target genes are expressed in the left half of cardiac disc. This disc undergoes rotation and involution to form the tube.
221
Why is it important for the heart to be functional during development?
Cardiac contractility generates a mechanical force and blood flow generates sheer force. This is important for spatiotemporal gene expression e.g. forming valves and trabeculation. Genes such as klf2 are flow-responsive so respond to stress.
