BMS237 Advanced Developmental Biology Flashcards

Question

What is growth in development?

Answer 1

``` Continuous process (embryonic, fetal, post-natal, adult). Growth rate varies depending on age and organ. Cell proliferation, cell enlargement, accretion. ```

Answer 2

Accumulation of ECM around the cell in order to grow

Answer 3

Yes - Add specific transcription factors and it returns to a pluripotent form - Induced pluripotent stem cells

Answer 4

Funnel model - Wrong - That they are only useful in very early development - Decreases as development progresses Hourglass model - Right - Early stages can vary a lot between animals - The mid embryonic stages are the most useful as they share a lot of similarities with humans

Answer 5

- in situ hybridization, - northern blot, - RT-PCR, - micro-array - reporter lines (transgenic)

Answer 6

Study when and where gene expression occurs in an embryo

Answer 7

Generate a probe (nucleotide sequence) that mimics the gene of interest and label it The probe allows us to detect this compound using immunohistochemistry - Using an antibody that is coupled with an enzyme (alkaline phophostase) - Generates a chromogenic reaction where hybridisation of a probe to a specific transcript occurs Hybridisation is very stables so creates a striped pattern which can be seen in the developing embryo

Answer 8

Because the chromogenic reaction will continue to get stronger as the reaction is left - There is no correlation between amount of transcript present and the intensity of colour

Answer 9

Where and when a gene is expressed in a embryo

Answer 10

Information about regulatory sequences (promotors and enhancers) are required - If you have information regarding this then you use that molecular information to drive expression of another gene - Use green fluorescence protein - drive its expression - Fluorescence is observed

Answer 11

Qualitative

Answer 12

Global information of overall gene expression of a specific cell

Answer 13

RT PCR Microarrays RNA seq

Answer 14

Finding gene expression content

Answer 15

- Reverse transcriptase to produce cDNA - Label each cells cDNA with different fluorescent tags e.g.. cell A will all be red and B green - Hybridize on a life which have a collection of short sequences that are specific to every single gene in the genome of that species - If cell A was expressing a specific gene then a hybrid would form and red fluorescence would be seen , if B then green - Can use software that screens the dots and looks at intensity of fluorescence - tells us the amount of gene present

Answer 16

If cells A and B express different genes then red and green would be expressed - No co expression If genes expressed in both then would see yellow

Answer 17

Because proteins may not be expressed where it was transcribed In development there are mechanisms that may delay the protein production so that transcription occurs

Answer 18

Western blot | Immunohistochemistry

Answer 19

Requires the availability of antibody specific to the protein of interest Antibody specifically binds protein Detect this antibody by adding a second antibody specified for the immunoglobulin for the species that the original antibody is raised in

Answer 20

Western blot tells amount of protein in tissue but does not have as a high as a resolution

Answer 21

Gain of function and loss of function experiments

Answer 22

``` Mutation in a gene that disrupts the expression or the function of the protein product encoded by the mutated gene ```

Answer 23

Mutation in a gene that confers a gain in the activity of | the protein product encoded by the mutated gene.

Answer 24

Seeks to identify a gene whose mutation caused a | particular phenotype

Answer 25

Seeks to characterize the phenotype of particular | mutated gene

Answer 26

Forward genetics - Introducing random mutations in genomes and look for phenotype Reverse genetics - Disrupt function of specific genes

Answer 27

C elegans Drosophila Zebrafish Mice

Answer 28

ENU - N-ethyl N-nitrosourea

Answer 29

Time consuming - so usually done on animal models with short generation time Expensive

Answer 30

Mice, drosophila and zebrafish

Answer 31

Knowledge about the gene you're knocking out | - Its structure and location on chromosome

Answer 32

- Create a construct that is similar to the endogenous gene (structure) - Modify this construct with a gene (e.g. antibiotic resistant) that will disrupt function of endogenous gene - e.g. effecting start codon stopping transcription - Once made the construct introduce into embryonic stem cells from a mice line (of one coat colour) - Can select out embryonic stem cells that have incorporated your gene by growing the stem cells on antibiotics - only ones with the construct will survive - Triggers homologous recombination - in cell division cross over reactions occur in chromatids causing replacement of endogenous gene - Inject these cells into blastocyst and then surrogate mother - Breed mouse and eventually will have a conventional knockout mice where the construct is in every single cell of the mouse

Answer 33

By looking at the level of chimerism in coat colour as ES stem cells and blastocyst where from mice with different coat colours

Answer 34

The gene will have been knockout out from the earliest stage of development meaning that if that gene was required for normal embryonic development, it would be unlikely that the knockout mice would survive long enough to study the protein of interests function

Answer 35

Conditional knockout mice crossed with tissue - specific cre mouse

Answer 36

- Construct will contain targeting gene but will be surrounded by loxp sequences which does not disrupt endogenous gene function - Homologous recombination requires another mouse which expresses tissue specific cre recombinase - an enzyme that recognises loxp sites and triggers homologous recombination specifically in those tissues. The cre recombinase must be under the control of a promotor and enhancer - Cross the conditional mouse wth the tissue specific cre mouse then knockout will occur later in development

Answer 37

Variants of cre mice that are inducible that allows you to not only choose which tissue but also when the knockout occurs

Answer 38

- Embryology: tissue manipulation (graft, ablation) | - Manipulating signaling pathways: drugs, transfection/electroporation, genetics

Answer 39

Grafting different regions of developing embryo to different places and looked at the effect - lead to discovery of the organiser Similar experiment lead discovery of the zone of polarising activity in the limb bud

Answer 40

Fate mapping - Label small group of cells in early development before cells have become committed - Use antibody to detect the labelling later in development to see what has happened to the cell

Answer 41

Zebrafish Xenopus C.elegans

Answer 42

Injecting fluorescence dye | Adding a reporter gene

Answer 43

Using loxp and cre recombinase

Answer 44

A soluble secreted molecule that acts at a distance to specify the dates of cells. It specifies more than one cell type by forming a concentration gradient

Answer 45

Alan Turing 1951

Answer 46

Its not just making the morphogen that is important but also destruction of the morphogen at the other end of the field of cells

Answer 47

If morphogens from both ends and sink in the middle

Answer 48

- Induce different outputs at different concentrations | - Act directly at a distance

Answer 49

Morphogens are instructive not just permissive - ectopically implant the morphogen and if its is a real morphogen it will change the cell fate if not the the cell will goto original fate (permissive) - Can also make concentration of the morphogen the same in all of the cells then all cells would have the same fate

Answer 50

Get duplication of the digits | - Shh is acting as a morphogen

Answer 51

The morphogen must be able to diffuse all of the cells to reach the other end Signals that are morphogens do not diffuse through they trigger a new molecule to be produced at each cell and released - genetically engineer to make the morphogen juxtacrine - stopping it passing through the membrane of neighbouring cells - If its a morphogen then cells won't change fate - If not then still will change fate

Answer 52

The molecule doesn't diffuse trough its fuel of cells it binds to the membrane of the neighbouring cell and triggers a new molecule to be formed and released from that cell - This new molecule then triggers the same response

Answer 53

Remove the receptor for the morphogen on later cells I the field - If actual morphogen cell fates won't change in response to morphogen because won't be able to detect it - If bucket brigade then it won't be affected and cell fates will still be induced as long as the directly neighbouring cell still has a receptor for it

Answer 54

Binding to molecules in the extracellular matrix (e.g. heparan sulphate proteoglycans) and high concentrations of receptor can generate a steep gradient - called “restricted diffusion” Rapid degradation of the signal in the extracellular space may also generate a steep gradient

Answer 55

HSPGs are found in the extracellular matrix and are known to bind to many ligands. They are sometimes called co-receptors. They regulate morphogen diffusion by: - sequestration or slowing diffusion. eg BMP (TGFbeta) - facilitating diffusion (Hedgehog)

Answer 56

When the molecule inputs from one side of the cell and outputs from the other side - doesn't leave the vesicle

Answer 57

A pit forms in the cell membrane and engulfs the morphogen in a vesicle Repeated cycles of endocytosis and resecretion allows certain morphogens to travel through the cells in a tissue

Answer 58

Evidence for transcytosis in Dpp (TGFbeta) signalling: antibody staining shows that Dpp is found in vesicles AND mutations that block vesicle formation cause Dpp to act in a juxtacrine manner

Answer 59

All neighbouring cells will think thy are going to be a cell that requires little or no morphogen until the morphogen gradient is established and they received the instructions for their real fate - There is a checkpoint at which all cells differentiate but not understood why - As the gradient is established, gene expression is changing with time. There must be a mechanism to block premature specification. The cell probably waits for the steady state of receptor activation to be achieved - but the molecular mechanism for this "check point" is poorly understood.

Answer 60

How varying morphogen concentrations lead to different cell fates - Higher concentration of morphogen often results in a higher concentration of an activated transcription factor - In this model, receptor activation causes transcription factor to enter the nucleus and direct transcription. It is the same transcription factor in every cell - keep in mind these cells are initially identical. - It is the amount of transcription factor present that decided the cells fate

Answer 61

Binding sites for transcription factors that activated by high concentrations of morphogens have a lower affinity and those activated by low concentrations have higher affinity binding sites - Even though both sets of genes see the same levels of transcription factors, only the high affinity enhancers can bind enough transcription factors to activate gene expression. - Also presence of negative feedback - repressor that switches off low concentration activated genes when high concentration genes are activated

Answer 62

Positive feed back may help the cell commit to its fate. For example if one of the blue gene encodes a transcription factor that among other things activates its own expression

Answer 63

Because it allows us to see evolutionary conserved genes between them and humans

Answer 64

18 decrees C - If want to grow slowly with little maintenance 25 degrees C - If want to grow fast

Answer 65

10 days at 25 degrees C | 21 days at 18 degrees C

Answer 66

1910 - Morgan discovers a white eyed fly; white (w) 1913 - Sturtevant constructs first genetic map, genes are arranged in a linear order 1914/16- Bridges shows that chromosomes must contain genes. 1927 - Muller shows that X-rays cause mutations & chromosomal rearrangements 1979/1980 - Christiane Nüsslein-Volhard and Eric Wischaus undertake a saturation mutagenesis to identify genes involved in the development and patterning of the larval cuticle 1980s,90s - technical and methodological advances

Answer 67

``` 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 - transcriptome, proteome etc ```

Answer 68

24th March 2000

Answer 69

Can align genomes of different drosophila species and see which parts of the DNA have been conserved through time as the species have evolved over millions of years

Answer 70

Protein encoding exons are highly conserved | Most introns are not conserved

Answer 71

Because they have regulatory roles - May be binding sites that transcription factors bind to - Enhancer regions

Answer 72

Males are smaller with darker tip of abdomen | Females are larger and have distinct segments

Answer 73

It is genetically encoded, not learnt Very strong drive for reproduction Males face females and buzz wings and dance

Answer 74

Males have two testes and sperm tails At the tip there are germ line stem cells which are maintained by hub cells Hub cells produce a ligand that maintains stem cell fate of the cells surrounding them When the stem cells differentiate, one cell remains by the hub cell and the other grates away to differentiate into sperm cells

Answer 75

They produce JAK/STAT pathway ligand

Answer 76

String like structures which are present in drosophila ovaries Over 100 They bind together and are where eggs develop and mature

Answer 77

After conception, females store the sperm in seminal receptacles for 2-3 weeks and is used to fertilise her eggs

Answer 78

Stem cell niche keeps stem cells in stem cell fate They divide and one cell moves away The cells undergo cystoblast mitotic divisions Divide into 16 cells, two of the cells will have four connections and one of these cells will become the oocyte and the other 15 cells will becomes the nurse cells Oocytes grow at the expense of the nurse cells Egg activation occurs and undergoes meiosis

Answer 79

Large chromosomes in the drosophila | - Made of sub bands - over 5000 each 22kb

Answer 80

pronucleus and proteins are RNA | - Proteins and RNA are made in nurse cells and are transferred to the developing oocyte via ring canals

Answer 81

Microtubules - minus and plus ended motors

Answer 82

Pro nuclei from egg and sperm fuse 8 divisions of nuclei occur After the 14th division there will be thousands of nuclei Some remain in the centre to become the nuclei of yolk cells and other migrate to the top Membrane starts to upgrow and form around the nuclei forming cells

Answer 83

``` Hedgehog Wingless (Wnt) Delta Transforming growth factor Fibroblast growth factor ```

Answer 84

Morphogenesis Cell proliferation Differentiation Migration

Answer 85

Process by which cells become different from each other and acquire specialised properties Governed by changes in gene expression, which dictate the repertoire of protein synthesised Cells that become specialised lose potency

Answer 86

64-128 uniform cells - this is due to cell proliferation and occurs very quickly and occurs very little growth

Answer 87

``` Split into a top and a bottom hemisphere In xenopus - top hemisphere is animal - bottom is vegetal In chicks/humans - top is epiblast - bottom is hypoblast ```

Answer 88

At the 1 cell stage, before fertilisation, distribution of cytoplasmic components isn't even The oocyte is polarised and some of its components are only found in the vegetal half When division occurs (in the animal vegetal plane) the cells become fundamentally different to each other as the top dividing cells will not inherit vegetal cytoplasmic components but bottom cells will

Answer 89

Animal vegetal

Answer 90

The cytoplasmic components act on the DNA to regulate gene expression the vegetal hemisphere - As a result particular transcriptional factors are turned on in the vegetal hemisphere and not in the animal => differences

Answer 91

Blastocoel formation

Answer 92

The morula is much flatter in chicks and humans and may be depicted as two flat surfaces

Answer 93

Binds to the promotor region of Nodal gene up regulating it

Answer 94

It is exported out of the cell in which it was made (made in vegetal cells) and is transported up into animal hemisphere - Acts as a morphogen - Animal hemisphere cells that recieve high concentrations of nodal become endoderm - Lower levels of nodal become mesoderm - No nodal protein become ectoderm

Answer 95

The animal hemisphere | The vegetal hemisphere are yolky energy providing cells which eventually die

Answer 96

Nodal also causes the cells to migrate and redistribute as well as just forming the germ layers

Answer 97

All cells would have an ectoderm fate

Answer 98

Sperm always binds to the animal hemisphere of the oocyte - Opposite side to sperm binding activates Wnt signalling pathway which activating the transcription factor beta catenin - So on the opposite side of the embryo Wnt signalling is activated leading to the formation of quadrants - as horizontal axis from animal/vegetal and then vertical axis from beta catenin presence

Answer 99

The morula is split in half by animal/vegetal hemisphere it os then split in half vertically as beta catenin is present only in the side of the morula that the sperm doesn't bind - activating Wnt signalling

Answer 100

That is the quadrant with the most nodal signalling and the most Wnt signalling This nodal and beta catenin overlap is called the Nieuwkoop centre The future dorsal and posterior side

Answer 101

In the cells that lie just above the Neiukwoop centre as they receive the most nodal signalling acting a specialised sub set of genes

Answer 102

Two parts of the promotor for genes (such as Gsc) need to be activated in order to activate transcription Therefore requires the binding of SMAD2/4 (a downstream effector of nodal) and beta catenin (a downstream effector of Wnt)

Answer 103

Through in situ hybridisation - can see that different parts of mesoderm express different transcription factors and genes - Eg. Brachyury is induced in response to low levels of nodal, Gsc and Chordin - Xnot and Clim are induced in response to high levels of nodal and where Wnt signalling is activated

Answer 104

It undergoes convergent extension whereby the cells of the organiser form a long thin rod and internalise under the the ectoderm Froms axial mesoderm

Answer 105

Because the transcription factors expressed in the nucleus of cells of the organiser (e.g. siamois and goosecoid) begin to bind to promotors in an intrinsic manner to alter the cells behaviour - including changes to migration and differentiation

Answer 106

Prechordal mesoderm and notochord

Answer 107

Anterior end expresses Wnt and BMP antagonists Posterior end expressed Wnt, FGF and retanoic acid Antagonist actions from opposite ends creates a gradient forming the AP axis

Answer 108

Organiser produces secreted molecules - BMP antagonists such as Chordin and Noggin These diffuse into the ectoderm and interfere with BMP interacting with receptor on a responding ectoderm cell Causes the phosphorylation of SMAD Causes a cascade of transcriptional factors to cause a neural fate

Answer 109

BMP is able to bind to BMP receptors on the ectodermal cells | Activating transcription factors that induce a skin fate

Answer 110

Ectoderm Mesoderm Endoderm

Answer 111

Somites are formed

Answer 112

1920's: Spemann and Mangold - A donor organiser was grafted from a donor onto a host newt - found that a 'twinned' embryo developed with a secondary neural axis - Secondary neural tube was host derived showing it was induced from the ectoderm in response to signals from the organiser - The prechordal mesoderm ad notochord where donor derived showing they're formed from the organiser

Answer 113

Because its an amalgam of wingless (the Drosophila gene) and int, a vertebrate proto-oncogene that encodes a mouse homologue of wingless.

Answer 114

The early 70's

Answer 115

Wingless and Hedghog maintain each other so if you disrupt either of them it will lead to the same result - Will cause wingless flies and disrupted segment polarity of the embryo

Answer 116

Because vertebrates are more evolved so during evolution, duplications of the genes occurred Its an ancient gene that is found in all metazoic organisms

Answer 117

When the ligand enters the secretory pathway, the N terminus is cleaved off It becomes modified on two positions - (cys77 and ser209) by palmitoylation and palmitoleic acid modifications Causes two fatty acids to be covalently linked to the wnt protein

Answer 118

Wntless is required - Needs to either form multimers or be loaded onto a lipoprotein - Seen that HSPG is also important

Answer 119

Evidence in drosophila say that just cell contact is enough for normal function of Wnt but no evidence to say this is the same in other organisms - most likely requires diffusion

Answer 120

``` Frizzled Arrow or (LRP in humans) ```

Answer 121

Beta catenin

Answer 122

Frizzled - Made up of 7 transmembrane domains - LRP stands for LDL receptor related protein - LRP and Arrow are a single pass transmembrane protein which also makes contact with Wnt

Answer 123

It binds to the long N terminal extension of frizzled which is known as the cysteine rich domain (CRD)

Answer 124

Dickkopf1 (DKK1) | - Binds to LRP and Kremen promoting the internalisation of LRP

Answer 125

There is destruction complex made up of APC, Axis, CK1, GSK3, Slimb proteins Beta catenin binds to this complex and is phosphorylated CK1 and GSK3 This causes the Slimb protein to cause ubiquination of beta catenin leading to its degradation

Answer 126

It is not broken down - Wnt binds to Frizzled and Arrow causing them to be brought together leading to phosphorylation of Dsh - This recruits the destruction complex to move to the receptor casino further phosphorylation of arrow creating binding sites for destruction causing the loss of Slimb from the destruction complex - Beta catenin still binds to the complex and it is still phosphorylation but Slimb is not there to tag beta catenin with ubiquitin to target it for degradation - This means that beta catenin remains in the complex causing it to clog - Beta catenin can then leave the complex and enter the nucleus where it can activate transcription

Answer 127

Phosphorylation by both CK1alpha and GSK3 (kinases) is required for beta catenin recognition by an E3 Ubiquitin ligase complex (which contains β-TrCP/Slimb) to cause subsequent degradation by the proteosome.

Answer 128

CKI phosphorylates a site in the N-terminal tail of beta catenin at a specific recognition site Only after this occurs can GSK3 phosphorylate another recognition site. (GSK3 phosphorylates 3 times at 2 different sites) β-TrCP/Slimb can then bind to beta catenin once phosphorylation of the 3rd recognition site by GSK3 has occurred

Answer 129

β-TrCP/Slimb is the interacting site of an SCF E3 ubiquitin ligase complex - Contains a Roc component which binds to E2 - It contains an F box which is responsible for its interactions with Skp1 - It interacts with the substrate by a domain made up of WD40 repeats

Answer 130

When beta catenin is present - It binds to the promotor and activates acetyl transferases and BRC1 ( a chromatin remodelling enzyme) - Also activates transcription factors Pygo and Lgs - Allows transcription to occur

Answer 131

When beta catenin is not present - Groucho represses transcription by recruiting histone deacetylases wjocj causes acetyl groups to remove from histones causing tighter packing so it is less accessible for transcription

Answer 132

DKK1, DKK4 and Axin 2 are all produced during the Wnt signalling pathway but act negatively on the action of the pathway

Answer 133

Planar cell polarity and convergent extension - Involved Wnt11, Wnt3, Frizzled, Rho, Roc, Vang11/2 Axon guidance - Repulsion by RYK receptor tyrosine kinase

Answer 134

Wnt5a | - Via ror2

Answer 135

To make all cells point in one direction | eg. hairs on rats, bristles in flies legs, hair cells in ear

Answer 136

Convergent extension does not work successfully | - Vang12 plays a role in the Wnt signalling pathway

Answer 137

Segmentation, also expressed at D/V boundary of the wing required for patterning and outgrowth - The first wingless gene

Answer 138

Doesn't have a vital role but is used to regulate neuronal fate - If Wnt5 is active then the QL.d neurone will move backwards instead of forwards - Useful investigating the roles of Wnts and the strength of the signals

Answer 139

Wnt11 induces a dorsal fate due to its activation of beta catenin Wnt8/3 induces ventral/posterior fates in the late zygotic stage Loss of Axin1 due to Wnt activation leads to posteriorisation of the anterior brain causing the loss of eyes

Answer 140

Required to maintain intestinal stem cells in stem cell fate - If there is a loss of TCF4 or an over expression of dkk4 (and therefore loss of Wnt signalling) then you can see that there is a loss of these stem cells - Can be investigated using in situ hybridisation

Answer 141

Ectopic wnt signalling can occur by the loss of APC gene (in destruction complex) If the other copy of APC gene on the other chromosome is lost to (eg. due to mutation) then it can cause familial adenomatous polyposis as stem cells will just continue to divide uncontrollably without differentiation - cancer

Answer 142

``` Familial adenomatous polyposis Hepatocellular carcinoma Breast cancer Ovarian and uterine cancers Melanomas Prostate cancer ```

Answer 143

Wnt3-tetra-amelia - Loss of function in Wnt3 - Babies born without limbs Bone diseases - LRP5 point munition cause insensitivity to Dkk causing an increase in bone density - Axin2 mutations can cause severe tooth agenesis (oligodontia) - multiple missing teeth

Answer 144

Undertook a saturation mutagenesis to identify genes involved in the development and patterning of the larval cuticle

Answer 145

They found 580 genes that didn't cause lethality Put them into complementation groups to check if they were on the same chromosome Found that there were in 139 different genes in this experiment

Answer 146

They crossed each gene with each of the other genes | If a phenotype was shown then it failed to complement, if no phenotype was shown then complemented

Answer 147

Lots including Knirps - causes a loss middle chunk of segments Paired - only has alternate segments Gooseberry - lose half of each segment - segment polarity gene

Answer 148

Maternal genes Gap genes Pair rule genes Segment polarity genes

Answer 149

A DNA binding transcriptional activator that acts as a morphogen It is maternally loaded into the developing oocyte (by nurse cells)

Answer 150

Loss of anterior structures

Answer 151

Implant anterior tissue from wild type oocyte into the bicoid mutant Get the development of some anterior structures

Answer 152

If you take anterior tissue from wild type oocyte and implant in the middle of a bicoid mutant then you get bilaterally symmetry as thoracic segments all face the middle ectopic head

Answer 153

If there are 0 copies then results in an oocyte with only 5 segments that are spread throughout the whole embryo (posterior) If 1 gene (normal bicoid levels) then there are 7 segments and the original five or more posterior If 4 genes (too much bicoid) then it compresses the segments posteriorly

Answer 154

High affinity binding site: activated at lower threshold concentrations of bicoid Low affinity biding cites need high concs of bicoid to be activated

Answer 155

Controlled by gap genes on a stripe by stripe basis Kruppel and giant are repressors and bicoid and hunchback are activators Each stripe is activated dependant on the interaction of positively and negatively acting transcriptional regulators

Answer 156

At this stage there is 14 segments but they need to be put together and show polarity within the segment (hairy cuticle and naked cuticle)

Answer 157

Hh and Wg feedback onto each other to maintain each others expression and refine segment borders

Answer 158

Hh maintains Wg expression which suppresses denticle development but an asymmetric pattern is formed because hedgehog causes degradation of wingless in posterior cells but not in the anterior cells creating a steep gradient on the posterior side

Answer 159

Provide ‘who am I’ information to each segment. Expression of homeotic genes along the a/p body axis occurs in the same order as the genes are within the genome. Controlled by a combination of gap and pair-rule genes Homeobox containing, DNA binding, transcription factors

Answer 160

Two clusters | Patterned from anterior to posterior

Answer 161

Mutation in drosophila hox genes that causes the antenna to turn to legs Antennapedia gene is expressed in head as well as thoracic segments

Answer 162

All 14 segments are defined at once | Meaning embryogenesis is complete in just 24 hours

Answer 163

Hedgehog (Hh)

Answer 164

``` Sonic Hedgehog (Shh) Indian Hedgehog (Ihh) Desert Hedgehog (Dhh) ```

Answer 165

The genomes of vertebrates have undergone a number of genome duplications early in their evolution. As a result for several genes that are present in drosophila have more than one homolog.

Answer 166

- Hedgehog genes are transcribed - N-terminal region targets them the secretory pathway. - The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein (this c terminal part has no other role than promoting this cleavage). - N-terminal part is coupled to a cholesterol molecule - A palmitoyl group is added to the N terminus by the skinny hedgehog gene in drosophila and Hhat in vertebrates - Both cholesterol and the palmitate are strongly hydrophobic and will render hedgehog quite insoluble in water and target it to membranes.

Answer 167

The hydrophobicity of the signaling component would make it impossible for the molecule to leave the cell membrane so it therefore requires the Dispatched protein - a 12 transmembrane protien - requires Scube glycoproteins - Mechanism not understood - likely to create multimeric hedgehog signaling particles that have their hydrophobic tail buried inside the particle or help load hedgehog molecules on lipoprotein particles

Answer 168

``` Drosophila - Patched - Smoothened Vertebrates - Patched 1 and 2 - Smoothened - Hedgehog interacting protein (Hhip) ```

Answer 169

Patched continuously inhibits smoothened by stopping it from reaching the membrane by sending it for degradation When Hh is present, it relieves the inhibition allowing smoothened to be active

Answer 170

Not 1:1 | one patched can inhibit many smoothened

Answer 171

It takes place in the cilia | - discovered as cilia mutants had the same phenotype as hedgehog mutants

Answer 172

It acts as a pump

Answer 173

- Prokaryotic RND (resistance-nodulation division) permeases confer multi-drug resistance by pumping out toxins - NPC1 (Niemann-Pick protein C1), which promotes the movement of cholesterol-laden vesicles along microtubules and can transfer some molecules across membranes

Answer 174

Ptc is also related to NPC1, the gene mutated in Niemann-Pick type C1 disease. Niemann-Pick disease (NP) refers to a group of inherited metabolic disorders known as the leukodystrophies or lipid storage diseases in which harmful quantities of a fatty substance (lipids) accumulate in the spleen, liver, lungs, bone marrow, and the brain

Answer 175

Hedgehog interacting protein is a vertebrate specific molecule that most likely acts to “mop up” free hedgehog thereby preventing it from reaching the patched receptor and thus keeping the signal down

Answer 176

CDO BOC and and the verebrate specific gene Gas1 most likely act as a co-receptor promoting the binding of hedgehog signal to patched and thereby promoting signaling.

Answer 177

In the absence of hedgehog, patched inhibits the activity of smoothened and two complexes exist that keep the transcription facor Ci reponsible for the effects of hh signaling out of the nucleus - One containing costal2 (a kinesin like molecule that acts a scaffold protein) and fused a serine threonine kinase - The other contains the Ci and supressor of fused gene (sufu) a gene without clear domains - Under the influence of the first complex that is bound to Smo three other genes can act on Ci. They form complex consisting of casein kinase I Protein kinase A and Glycogen synthase kinase 3 beta. - The transcription factor Ci which as a full length gene is a transcritional activator is processed under the influence of these genes to a shorter form via Slimb. - The short form acts as a transcriptional repressor and is called CiR. In this way hedgehog target genes are actively repressed.

Answer 178

When low concentrations of hedgehog are present it is thought that the PKA/GSK3/CKI complex dissociates from the complex containing Ci and as a final result, active repression by CiR is lost

Answer 179

- At high concentrations, hedgehog acts through both complexes to release a fulllength Ci that will actively promote transcription of target genes - This activation could involve phosphorylation but the exact nature if this phosphorylation is unknown - it is thought that phosphorylation of sufu by fused promotes formation of the active form of Ci.

Answer 180

PKA phosphorylates Ci first, priming phosphorylation by GSK3 and CK1. Mutation of any of these sites reduces processing to Ci 75, and reduces Slimb binding Slimb is an F box protein, a component of the SCF ubiquitin E3 ligase complex

Answer 181

Negative - One of the best known targets of Hh signaling is Patched1/Patched - limit level of activation and reduces the range of movement of the Hh signal - CDO/BOC/GAS-1 (positive regulators) downregulated Postive - Gli1 (not Ci), which is always an activator of transcription, is induced

Answer 182

In myocytes/adipocytes - Ca2+ acts on smoothened and activating AMPK - Causes cellular metabolism stimulating aerobic glycolysis, where glucose is used as an energy source but without fully “burning” it in the mitochondria - leads to the production of lactate, which leaves the cell and will acidify the extracellular medium. In drosophila embryo - Wg and Hh maintain each others expression in an autoregulatory loop In wing patterning - Diffuses to anterior side and produced app at the boundary between cells Neural development - Different cell types are induced depending on the amount and duration of the hedgehog signal.

Answer 183

Hedgehog loss of function - holoprosencephaly, caused by a loss of ventral brain structures - cyclopamine polydactyly (extra digits) and syndactyly (webbed digits)

Answer 184

Gain of Hh signaling -Basal cell carcinoma (BCC), medulloblastoma rhabdomyosarcoma -Inactivation of Ptc1 or Sufu: tumor suppressor genes -Activating mutations of Smo: Smo is a proto oncogene BCC most common form of cancer

Answer 185

In other cancers, Hh acts as a growth factor, but the pathway is not mutated - small cell lung carcinoma (SCLC) - pancreatic cancer - metastatic prostate cancer

Answer 186

By signals released from the niewkoop centre | This activates BMP inhibitors

Answer 187

- BMP binds to BMP receptor and a secondary signal (Smad) is phosphorylated - Smad can then up regulate transcription factors like Msx1, GATA1 which leads to epidermal differentiation

Answer 188

- BMP inhibited - No Smad phosphorylated so different transcription factors are up regulated (xlpou2, soxd) - Neural differentiation pathway

Answer 189

Somite formation

Answer 190

When the neural plate rolls up to form the neural tube

Answer 191

Establishes in response to diffusible molecules | - BMPs released from surface ectoderm (dorsally) and Shh released from mesoderm ventrally

Answer 192

Intermediate levels of BMP signalling triggering transcription factors (msx)

Answer 193

- It was thought that they act as morphogens | - But recent work shows that roof plates may express many different BMP's which induce a particular dorsal cell type

Answer 194

- Notochord secretes shh - shh diffuses through spinal cord - conc gradient (morphogen) - It induces different patterns of transcription factors - High concs of shh cause floor plate cells to develop which occupy ventral midline of neural tube - activates shh - shh mRNA in both notochord and floor plate - shh diffuses into neighbouring cells causing ventral fate

Answer 195

- The epiblast cells converge towards the primitive streak and involute down the streak - Epithelial cells transform to mesenchyme which is important in the ability of cells to migrate - Endoderm cells go deeper and mesoderm stays between endoderm and epiblast cells - Formation of mesoderm

Answer 196

It is posterior to Hensen's node and forms the somites

Answer 197

Found laterally to the axial mesoderm | Responsible for limb and heart formation

Answer 198

Contributes towards formation of urogenital system - kidney and gonads

Answer 199

The notochord and precordal mesoderm

Answer 200

Perfectly symmetrical small cells on either side of the spinal cord - Show evidence of segmentation in invertebrates - Segmented paraxial mesoderm

Answer 201

Somite number dictates number of spinal vertebrae - Humans have 33 vertebrae at adulthood - Human embryo has 38-44 somites

Answer 202

When somites no longer form - number of somites is fixed for a given species

Answer 203

Although it is not segmented, it pre-figures the | future segmentation of somites

Answer 204

From by budding off pre somatic mesoderm - the paraxial mesoderm, becomes positioned in the tail of the pre-somatic mesoderm - form in pairs

Answer 205

The clock and wavefront model (Cooke and Zeeman, 1976)

Answer 206

It predicts that a ‘clock’ ticks in the posterior pre somatic mesoderm 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

Answer 207

- Cooke and Zeeman looked at the C hairy expression in the pre somatic mesoderm and saw that embryos have the same age all have very different expression patterns - They counted number of somites to see the developmental stage of bisected embryos - Concluded that expression of C hairy oscillates over 90 minutes which is the exact time it takes for a somite pair to form

Answer 208

Implanting a somite boundary into a non boundary region - It creates a new boundary - Meaning boundary cells can instruct cells that are anterior to form a boundary Implanting lunatic fringe in anterior of somite - Represses notch in anterior causing a boundary Over expressing notch in posterior of somite - Activates notch in posterior causing a boundary

Answer 209

Formed at the interface between notch expressing and non notch expressing areas - Anterior somite expresses high levels of notch signalling - Posterior somite expresses Mesp2 which activates lunatic fringe, this inhibits notch signalling creating a boundary

Answer 210

Lunatic fringe eg. Glycosyltransferase | - Notch signalling activates mesp2 which activates lunatic fringe

Answer 211

The interface of two opposing gradients of retanoic acid and FGF8 There is a high level of retanoic acid in already formed somites (anteriorly) decreasing towards presomatic mesoderm FGF is expressed posteriorly so is high in mesoderm and low in somites

Answer 212

Raldh2 is required for retanoic acid synthesis and is only produced in somites forming its gradient FGF8 drives the expression of cyp26 which is an inhibitor of retanoic acid synthesis causing low retanoic acid where FGF8 is present

Answer 213

- FGF8 causes the expression of Tbx6 - Tbx6 and notch signalling drive the expression of Mesp2 which is maintained by a negative feedback loop involving Ripply2 causing its expression to oscillate - Mesp2 leads to the suppression of delta and therefore decrease in notch activity - It is responsible for the formation of somite boundaries

Answer 214

Somites do not align | In humans, this causes spondylocostal dysplasia. This is called Jarcho Lewin syndrome

Answer 215

- Movement and posture - Communication: speech, expression & writing - Maintain body temperature: heat released through muscle contraction participates in control of body temperature. - Respiration: importance of diaphragm

Answer 216

- Injuries - Ageing - Muscle-degenerating disease = Dystrophy (Duchenne and Becker)

Answer 217

Stem cell undergoes specification/determination to form a myoblast The myoblast then undergoes differentiation o form myotubules - Myotubules then mature into myofibres

Answer 218

Weintraub, 1987 - Fibroblasts could be driven to myoblasts if treated with a demythalating agent 5Aza (epigenetic changes) - He isolated the mRNA from both treated and untreated fibroblasts and converted it to cDNA - He hybridised these two cDNA together - Genes that are only found in fibroblasts or myoblasts will not be able to find a counter part to hybridise with so will remain single stranded - Found enriched cDNAs that were muscle specific - Isolated a number of genes through this including MyoD

Answer 219

A master regulatory gene that is sufficient to drive muscle differentiation

Answer 220

The cell will convert to a myoblast

Answer 221

Myf5, myogenin, Mrf4

Answer 222

They are transcriptional activators They from heterodimers with E12 or E47 Binds to E box sequence in promotors of genes - Abundant in promotors of genes which are involved in muscle cells

Answer 223

Has a basic domain which is a binding site for DNA | Has a helix loop helix domain which allows or dimerisation with E12 and E47

Answer 224

Dorsal part of the somites remain as epithelial cells and form dermomyotome

Answer 225

Where skeletal muscles originate from - contains progenitor cells for skeletal muscles of the trunk and limbs

Answer 226

The cells undergo a reverse process of epithelial to mesenchymal transition The mesenchymal cells populate ventral pr too the somite and are responsible for formation of bonds

Answer 227

Myf5 - straight after somite formation

Answer 228

No obvious defects at birth | A slight delay in myotome formation until onset of MyoD expression

Answer 229

No obvious defects at birth | Increased Myf4 expression in somites

Answer 230

Because only Myf5 or MyoD is required for the generation of myoblasts Have the capability to compensate for each other - functional redundancy

Answer 231

Mice die shortly after birth due to a diaphragm defect Reduced density of myofibres so are replaced by myoblasts Myogenin is required for muscle differential

Answer 232

Myf5, MyoD or MRF4

Answer 233

``` Epaxial - Neural tube and notochord secrete Shh - Wnts released from ectoderm - Both required to drive Myf5 and MyoD expression Hypaxial - BMP from lateral plate mesoderm - Wnt from ectoderm - BMP4 drives Pax3 expression which represses Myf5 and MyoD expression. This is to ensure that the mytotome doesn't specifiy until it reaches its site of differentiation ```

Answer 234

Mesenchymal cells from limb bud expresses HGF/SF BMP4 induced Pax3 in cells fates to become limb muscle Pax3 induces C-met (receptor for HGF/SF) After C-met activation, cells migrate to limb bud Splotch mouse - Deletion of Pax3 gene which drives expression of C-met - If Pax3 is not present, migration does not occur - Without this migration, MyoD and Myf5 will not be activated

Answer 235

Adult muscle stem cells that are responsible for repair

Answer 236

Pax7 specifies satellite cells | - In its absence, repair will not occur and cells will die

Answer 237

Satellite cells are dormant until required Activated by release of reactive oxygen species when muscle is damaged or after exercise Reduced expression of Myf5 and MyoD which triggers repair - Proliferate and differentiate activating myogenin - Fuse with each other or existing fibre to repair muscle

Answer 238

During embryogenesis - stage 12

Answer 239

A wing disc grows from about 30 columnar epithelial cells to about 50000 by the end of the third larval instar

Answer 240

If make mutant in one original cell (of the 30) would have a huge impact on the overall wing eg. Mutant fasIII clones produced a mosaic - normal structure but some cells are mutant and some are wild type

Answer 241

They have no eyes

Answer 242

Heterozygous - small eyes | Homozygous - do not survive - embryonic lethal

Answer 243

Homozygous - don't survive | Heterozygous - aniridia (loss of iris)

Answer 244

A homeobox transcription factor homologous to vertebrate pax6 A key regulator of eye expression

Answer 245

- Found that Pax6 could rescue eye development and dorm new eyes so is sufficient - human pax6 expressed in flies - Also found to be necessary

Answer 246

They have two large eyes on the side of their head | Three small eyes on the top called ocelli

Answer 247

Used for orientation

Answer 248

Consists of 800 ommatidia per eye

Answer 249

Hexagon shaped Curved so that the top ones face up and the bottom ones face down Have 8 photoreceptors, 4 cone cells and 2 primary, 6 secondary and 3 tertiary pigment cells Has a lens and a bristle

Answer 250

Secrete the overlying lens

Answer 251

They lattice optically insulates each until eye (stops light from dazzling)

Answer 252

Cluster of photoreceptors called rhabdomeres - Photoreceptors stretch across the length of the ommatidia - Small central R7 photoreceptors surrounded by larger R1-R6 - R8 lies directly underneath of R7

Answer 253

They have a line symmetry where the ommatidia face opposite ways - Separates the dorsal and the ventral eye fields - Though to face a role in orientation

Answer 254

Retina, lamina, medulla, lobula and lobule | 1/3 of the brain - shows the importance of vision

Answer 255

Project axons into the lamina

Answer 256

Project into the medulla

Answer 257

Each rhabodmere are slightly angled meaning that light hits in the side focuses on the edges Means any single object is seen by multiple rhabodmeres because they all overlap slightly but they activate different photoreceptors within those rhobodmeres This process is the way a fly can increase the resolution of its sight - detect movement

Answer 258

Develop during the third install stage Moves from anterior to posterior to fill the whole area with photoreceptors - No photoreceptors in the morphemic furrow

Answer 259

An indentation in the eye disc It moves across the imaginal disc from posterior to anterior in response to a wave of signals that initiate development of ommatidia It recruits each cell in the cluster and each cluster has a distinct morphology

Answer 260

R8 is the first photoreceptor to be recruited by notch signalling R8, R2 and R5 secrete EGF pathway ligand spitz which recruited R3 and R4 and then R1 and R6 R7 is the last photoreceptor to be recruited Then cone cells are recruited

Answer 261

Single cell fate specification

Answer 262

R7 not present but all other photoreceptors present | Shows R7 is not required for other photoreceptors

Answer 263

They cannot see ultra violet light | Th wild type favours UV light

Answer 264

When R7 is not present, R8 is always a mutant | When R7 is present, R8 is always a wild type

Answer 265

Shows that R8 requires boss to produce R7 | Boss is a ligand in the ERF pathway which interacts with the seven less receptor - activates transcription pathway

Answer 266

Anterior/posterior Left/right Dorsal/Ventral

Answer 267

Easiest way to identify | - Associated with specific muscles and nerves

Answer 268

Form where limb will from | Appear first as protrusions from the flank at precise positions along the AP axis of the embryo

Answer 269

If transplant to an area that is not normally a limb bud | - ectopic limb bud formation

Answer 270

Before limb formation, there is molecular characteristics present in the mesoderm to form a limb

Answer 271

T-box transcription factors - Tbx 5 - wing - Tbx 4- leg - Tbx5 can restrict expression go Tbx4

Answer 272

you get a leg

Answer 273

Expression of hox provides a permissive environment that allows synthesis of retanoic acid in mesoderm Induces expression of tbx genes

Answer 274

Limb formation wherever the beads are paced Depending on where the bead is placed, depends on whether a wing or leg is formed If implanted closer to the forelimb then a wing will develop (because it is close to tbx5)

Answer 275

Hox proteins allows the synthesis of retanoic acid Retanoic acid induced Tbx transcription factors Tbx causes the cascade activation of FGF10 in the mesoderm FGF10 induces Wnt3a activating FGF8 in the ectoderm Positive feedback loop between these causes limb outgrowth

Answer 276

``` The apical ectoderm ridge - Expresses FGF8 Zone of polarising activity - Expresses Shh Progress zone - Area of mesenchyme that lies under the AER ```

Answer 277

John sanders used microsurgery - took embryos form different stages and removed their AER and then allowed to continue to develop - Looked at there skeletal - If AER was removed at 3 days then proximal bones were developed but not distal - If removed at 3.5 days then proximal fully developed but no digits - If removed at 4 days then Neal full development but digits were truncated - He concluded that the AER plays a role in proximal distal development and maintaining cells in the progress zone - Concluded that progress zone cells are specified by the more time spent there: the more time in the progress zone, the more distal the cells

Answer 278

Disproved in early 2000's when they used technique of cell fate mapping - The fate of each cell didn't match the predictions made from this model

Answer 279

Early in development, there are precursor cells for all p/d elements but they only develop into these cells based on what signals they receive - The signal for proximal formation is retanoic acid and is released from the paraxial mesoderm - The signal for distal formation is FGF and is released from the tip of the limb bud - They act antagonistically to each other

Answer 280

``` Meis (a form of HOX) - retanoic acid production - Causes expression of proximal structures Combination of Hox11 an Hox13 - FGF production - Distal structures ```

Answer 281

Meis = Hth | Hox11 and 13 = Dll

Answer 282

Creates a mirror image duplication of distal digits

Answer 283

Mirror image duplication of digits | - Same is if you graft ZPA

Answer 284

Suggested that ZPA secretes Shh which acts as a morphogen High concentrations would specify digit 4 Low concentrations would specify digit 2

Answer 285

Complete loss of most skeletal elements - Small portion of digit present which is a remnant of digit 1 - Because cells that are most anterior are outside of ZPA range so cells for digit 1 develop independently of Shh

Answer 286

Negative feedback loop between Shh and Fgf8 | - ensures coordinated development of limb bud along the axes

Answer 287

In the neural plate: - Under the influence of signals that define the anterior-posterior axis, an early territory is established that expressed distinct transcription factors: Rx, Six3, Pax6. This is the early eye-field - Slightly later, Shh from the prechordal mesoderm induces floor-plate like structure in the middle which splits the eye field - It does this by suppressing Pax6/Rx expression in the centre of the embryo, which is splits the field into two

Answer 288

Unable to suppress Pax6/Rx in the centre of the embryo Eye field is not separated Results in cyclopia - single eye

Answer 289

Anterior neural plate

Answer 290

1. The eye field grows sideways and contacts the ectoderm 2. The ectoderm then thickens forming the optic placodes 3. Once induced, the placode signals back to the optic vesicle, causing it to undergo a mophological transformation, invaginating and forming a 2-layered cup-like structure 4. This then pitches off to form a vesicle

Answer 291

Six3 and Rx transcription factors which control proliferation and migration

Answer 292

Must be transparent

Answer 293

- The lens placode is induced by contact between the optic vesicle and the overlying ectoderm - The lens placode is then induced, by the inner layer of the optic vesicle, to invaginate and pinches off to form a hollow lens vesicle - These cells are ‘stem-like’ – capable of ‘self-renewing’ to give more of themselves, or differentiating to lens fibre cells - Lens fibre cells are long, fibre-like highly specialised cells that fill the inside of the lens. They immediately make lots of a protein called crystallin – a transparent protein - Nucleus undergoes deterioration because so much crystallin produced - Stem like cells remain on outside of the lens

Answer 294

They have to lose their nuclei because the lens must be transparent However, this means that these cells do not live very long so have to have a maintained population of stem cells to renew them

Answer 295

Stem cell populations can deplete in older people | Can lead to formation of cataracts

Answer 296

Forms the retina

Answer 297

Consists of the outer retinal pigment epithelium and the inner neural epithelium

Answer 298

Retinal progenitor cells in the outer layer form pigmented cells, giving the eye its colour

Answer 299

Retinal progenitor cells in the inner layer give rise to the ganglion cells, bipolar cells amacrine cells, photoreceptors and Muller glia.

Answer 300

The astrocytes of the optic nerve

Answer 301

- The optic vesicle infolds, forming a bilayered optic cup - The inner wall of the optic cup becomes the neural retina, while the outer wall becomes the pigment epithelium - The cells of the outer layer produce melanin pigment (one of the few tissues other than the neural crest cells that can form melanin) and ultimately becomes the retinal pigment epithelium - Inner layer cells contain a stem cell-like population, which can either self-renew or can differentiate, via progenitors that differentiate to the diverse ganglion cells, interneurons, light-sensitive photoreceptor cells in the neural retina.

Answer 302

Six3, Rx1 and Pax6

Answer 303

if the mouse Pax6 gene is inserted into the Drosophila genome and activated randomly, Drosophila eyes form from Pax6+ cells

Answer 304

Pax6 knockouts lack eyes, and heterozygotes have small eyes

Answer 305

Neuronal stem cell Radial glia can divide asymmetrically, giving rise to one daughter which is like its mother – ie a radial glia (stem cell), and a 2nd daugher that will differentiate to a neuron. This daughter uses the scaffold provided by its sister to migrate away from the ventricular zone

Answer 306

Initially the neural tube is a single layered neuroepithelium. During early stages of neural tube development, some cells continue to ‘span’ the width of the neural tube. Their nuclei migrate back and forth at diferent stages of the mitotic cycle: - In G1 and S phase of the cell cycle, the nucleus is away from the lumen. - At M phase and cytokinesis, the nucleus is close to the lumen. - At cytokinesis, the lateral attachment is lost, then reforms These cells, which derive from the stem-like neuroepithelium, are called ‘radial glia’ and are believed to be the later ‘neural stem cells’

Answer 307

Neuorblasts sort into functional layers during development to form: Rods and cones Bipolar neurones Ganglion cells Axons of the ganglion cells join to form the optic nerve

Answer 308

Rat retina: - A virus containing a functional β-galactosidase gene is injected into the back of the eye of a newborn rat to infect some of the retinal precursor cells - After a month to 6 weeks, the eye is removed and the retina is stained for the presence of β-galactosidase - Stained cells forming a strip across the neural retina, including five rod, a bipolar neurone, a rod termina, and a Müller glial cell

Answer 309

Chx10 promotes bipolar cells | Prox1 is involved in horizontal cell fate

Answer 310

To collect urine and void it into the bladder

Answer 311

Induce different cell types (nephron vs collecting ducts/calyces/ureter) Induce these in a certain place in the body (lower back, bilateral) Proliferation Form branches (branching morphogenesis) Form many nephrons Attract blood capillaries to one end of nephron Form tubes Connect tubes (nephron and collecting ducts

Answer 312

Out of 929 known human disease genes, 548 have homologous genes in drosophila Major signalling genes were discovered in fly: Hh, Notch, Wg, Dpp etc

Answer 313

Immediately after gastrulation

Answer 314

The ventral lateral mesoderm called splanchnopleura

Answer 315

Dorsal: Somatopleura - Splanchnic mesoderm and ectoderm Ventral: Splanchnopleura - Splanchnic mesoderm and foregut endoderm

Answer 316

First heart field - First origin developed - It has a low proliferative capacity so doesn't contribute much to the structure of the heart - It lays down the scaffold for the heart and is the exclusive source of the left ventricle Second heart field - High proliferation so can contribute to the structure of the heart - It forms the right ventricle and outflow tract

Answer 317

Precursor cells lie anterior part of embryo in the chick - angiogenetic cell clusters

Answer 318

The homeobox gene Tinman - As the drosophila develops, Tinman expression becomes restricted to the dorsal vessel - It is originally expressed everywhere

Answer 319

In the wild type - Myosin is stained In drosophila - Cant see myosin (a muscle specific protein) - Therefore, Tintin is required for muscle formation

Answer 320

The Nk-2 family of homeobox transcription factors - Specifically Nkx2.5 (expressed in the cardiac crescent) - Responsible for the specification of mesodermal precursor cells

Answer 321

They, unlike tinman mutants, form a heart but show cardiac defects at the looping stage Suggests that there are some compensatory mechanisms to allow heart formation

Answer 322

BMP (app homologue)

Answer 323

Gain of function experiments - Take a bead soaked in BMP2 and a control bead and implant them either side of the anterior primitive streak - Allow the chick to develop and then carry out in situ hybridisation for Nkx2.5 - Saw more expression in lateral mesoderm by BMP2.5 bead - In presence of BMP2.5, Nkx2.5 is ectopically expressed in paraxial mesoderm (a tissue that doesn't usually express it). BMP therefore has the capacity to induce Nkx2.5 expression

Answer 324

Angiogenetic cell clusters come together and the mesoderm thickens around it forming bilateral tubes Migration occurs, bilateral tubes fuse to form a single endocardial tube - Gives rise to endothelial lining of the heart and cushion cells that form valves

Answer 325

Dmef2 | Responsible for differentiation of all muscle including skeletal and smooth

Answer 326

Differentiation of all muscle does not occur | Tinman is still present meaning the initial specification stages aren't affected

Answer 327

No Dmef2 present suggesting tinman is required for its induction, placing up stream genetically to Dmef2

Answer 328

Mef2A, Mef2B, Mef2C - Mef2c is the earliest gene expressed - Responsible for differentiation of cardiac muscle cells in vertebrates

Answer 329

There is no heart looping or right ventricle | There is upregulation of Mef2B suggestion part of a compensation mechanism

Answer 330

GATA transcription factors - Zinc finger proteins - GATA4 plays a role in controlling expression of genes involved in migration - Fore gut endoderm also important

Answer 331

2 tubes remain in bilateral positions - they fail to migrate - formation of heart tibe does not occur

Answer 332

Looping is the first physical sign of a break in left and right symmetry

Answer 333

Can be seen molecularly - Lefty and Nodal, two TGFβ-related molecules, are specifically expressed on the left side of the embryo. - First asymmetry established when signalling through Activin receptor IIa inhibits Shh expression on the right side of the embryo.

Answer 334

- iv (inversus viscerum) encodes a dynein, protein involved in the movement of cilia - inv (inversion of embryonic turning) encodes for Inversin, a protein containing ankyrin repeats found in cilia - Involved in heart looping - Inv and Iv are required for cilia movement - rotation. (In inv, cilia are smaller, creating a slower flow) - Cilia rotation establishes a preferential flow of Nodal and Lefty molecules on the left

Answer 335

Mutations in inv or iv genes cause random (iv) or complete reversal (inv) of heart looping Nodal and Lefty are expressed on the right, but not the left of embryo

Answer 336

dHand and eHand, two bHLH transcription factors, with specific expression in right and left ventricles Initially expressed everywhere but then becomes restricted

Answer 337

Die early in development | Right ventricle hypoplasia

Answer 338

Died very early due to placental defects as it also has a role in tissue formation A conditional knockout showed left ventricle defect but mice did survive until birth

Answer 339

Metanephros

Answer 340

Definitive kidney arises as a result of reciprocal interactions between 2 structures, a small outpocketing of an intermediate mesodermal structure called the ureteric duct (outpocket is called the ureteric bud), and the adjacent mesenchyme, called metanephric mesenchyme

Answer 341

The definitive nephric tubules

Answer 342

- The interaction between the ureteric bud epithelium and nephrogenic mesenchyme is an inductive interaction - The ureteric bud induces the nephrogenic mesenchyme to condense around the bud and undergo a mesenchymal-to-epithelial cell transition and form renal epithelium, then renal vesicles, in response to Wnt4 - Renal vesicle proliferation is governed by signalling pathways forming an s shaped body - S body becomes polarised as the distal and proximal end have different properties - The proximal end secretes chemoattractants and angiogenic molecules to rearrange local endothelial cells and form new capillaries - The distal end fuses with the collecting duct by selective apoptosis

Answer 343

Because the ureteric bud undergoes branching morphogenesis | - Therefore forms millions reba vesicles

Answer 344

Governed by signals from the mesenchyme - The mesenchyme releases the signal GDNF which is reviewed by receptor tyrosine kinase (Ret) on the bud - Cells at the tip of the ureteric bud undergo proliferation and outgrowth - There is an arrest in the proliferation of the leading edge tip cell on the bud causing the bud to become flattened - The lateral tip cells continue to proliferate forming a cleft and two tips - This is repeated again and again to form multiple branches

Answer 345

Initially identified as a neurotrophic factor and acts to promote proliferation, migration and outgrowth

Answer 346

Studied the process in real time using transgenic animals, organ cultures and sophisticated imaging - fluorescence

Answer 347

Branching morphogenesis of the ureteric bud underlies future calyces - In humans, at 6 weeks there are 16 branches and at week 7 minor calyces form - Major calyces are the remnants of the first branches

Answer 348

The initial ureteric bud

Answer 349

Kidney development is protracted, proceeding throughout prenatal and even into postnatal life

Answer 350

Relies on stem like cells in both the ureteric bud and the metanephric mesenchyme

Answer 351

Six2 - Activates genes that from the nephron and all the different cells within it - Six2 is not just a marker but regulates the events that keep the nephron stem cell in a self-renewing state

Answer 352

Depletion in nephron stem cell pool

Answer 353

Prevents differentiation of nephron stem cells

Answer 354

The first attempt of building the kidney in development is called pronephros and is at the top of the back - fails to form functional kidney The second attempt is called mesonephros and is in the middle of the back - fails The final attempt is metanephros at the lower back and successfully builds functional kidneys - Evolutionary development

Answer 355

The intermediate mesoderm is a line of cells that extends along the whole of the posterior body It undergoes a mesenchymal to epithelial cell transition to form two structures, called the nephric cord and nephric duct The duct develops into a lumen Duct will eventually give rise to ureteric bud The nephric cord then undergoes another epithelial to mesenchyme transition which will give rise to metanephric mesenchyme

Answer 356

- Each mesonephric tubule receives a blood supply from a branch of the aorta, ending in a capillary tuft analogous to the glomerulus of the definitive nephron - The mesonephric tubule forms a capsule around the capillary tuft, allowing for filtration of blood - This filtrate flows through the mesonephric tubule and is drained into the continuation of the pronephric duct, now called the mesonephric duct or Wolffian duct.

Answer 357

Provides huge breakthroughs in understanding kidney diseases? - WT1 was originally identified as a gene involved in Wilms tumor, a pediatric cancer in which kidney elements are incompletely differentiated and instead proliferate to form tumours

Answer 358

Endoderm - Will give rise to the epithelial lining go the trachea, larynx, bronchi, alveoli, through branching morphogenesis Mesoderm - Will give rise to cartilage, muscle and connective tissue

Answer 359

The bud that is responsible for the formation of trachea, bronchi, bronchioles and alveoli

Answer 360

The most ventral tube in the embryo is the foregut The top part of the tube (the oesophagus) starts to form a bud which is separated and sealed called the respiratory diverticulum which will form the trachea It then undergoes branching morphogenesis

Answer 361

.Its medial edge is the visceral pleura and its outer edge is the parietal pleura. The space in between is the pleural cavity

Answer 362

1st bud - 4 weeks Bronchi buds - 6 weeks Developed lungs - 10 weeks (still very small)

Answer 363

Drosophila - Discovered through analysis of drosophila tracheal development - discovered the role of FGF10 and its inhibitor sporty

Answer 364

- Endothelial epithelial cells that express FGF receptor respond to secretion of FGF from nearby mesenchyme by bud formation/extension toward the FGF source - Exposure of tip cells to high concentrations of FGF induces the expression of genes that code for signals eg. BMP4, Shh and cyclins (cell proliferation) - BMP4 is expressed at highest levels in the ‘leading edge’ tip cells and inhibits epithelial cell proliferation causing flattering of the bud - At the same time, Shh expressed by the tip cells diffuses to the mesenchyme, and inhibit FGF10 expression in the mesenchyme nearest the tip. This splits FGF10 expression promoting the next round of branching. - Sprouty 2 is then induced which inhibits FGF signalling so that branching is restricted to the tip of the bud

Answer 365

FGF10 induces expression of genes BMP4 and Shh which increase proliferation and growth However, FGF10 then induces sprouty2, a molecule which inhibits FGF signalling and stops the proliferation and migration of distal tip cells - This is negative feedback because a signal (FGF10) induces its own inhibitor (sprouty2) limiting its own action

Answer 366

They up regulate VegF, a signalling factor which promotes angiogenesis, allowing for the formation of new capillaries This forms an extensive capillary network around each alveoli

Answer 367

1/6 of adult alveoli

Answer 368

In vivo approaches - Conditional knockouts Organ culture - Can grow tissues in vitro and easily manipulate them Light microscopy - Using sophisticated microscopy provides high resolution of living tissues

Answer 369

Because this process extends into postnatal life | Constantly growing makes them more sensitive to mutations which can lead to cancer

Answer 370

Adult stem cells - In an undifferentiated from to contribute to the individual over its left time: to grow, restore, repair Germ cells - Undifferentiated state to be used for the next generation

Answer 371

Primordial germ cells (PGCs) are determined I a specific location just 'on the edge' or 'outside' of the developing embryo PGCs migrate to the gonad and become the progenitor population for eggs and sperm

Answer 372

A plastic cell type (totipotent) | A cell capable of undergoing meiosis

Answer 373

C elegans | - The first division produces a specialised cell at the posterior side of the embryo - the P lineage cell

Answer 374

Non autonomous divison - Cell division plane is often a crucial determining factor in the fate of two daughter cells - For example division down the meridian may produce two identical cells but down the equator may separate cytoplasmic components and produce two different daughter cells

Answer 375

Acts as a pre-germ cell - Through asymmetric division, P cells inherit specialised P-granules (a mix of proteins and RNAs) that can be in the cytoplasm and get into the nucleus

Answer 376

- Bind to DNA of the P cell and block almost all transcription and therefore differentiation - Block translation in the cytoplasm - Promote stem cell fate and cause cells to undergo meiosis

Answer 377

Always at the posterior of the embryo, even after each cell division

Answer 378

Epigenetic silencing mechanisms | - Alter DNA stability through DNA methylation and histone modifications

Answer 379

Drosophila - Germ cells enter the body from pole cells by attachment to endoderm and migration through the gut into the gonads - A combination of chemoattractive and repulsive cues drive the primordial germ cells and gonad precursor cells together and to specific destination

Answer 380

A small localised area that protects the germ cells from differentiating A type of stem cell niche

Answer 381

In males - Primordial germ cells end up in niche and attach to hub cells where they are maintained In females - In ovaries, the germ cells end up in the niche and attach to stromal cap where they are maintained

Answer 382

After convergent extension in vertebrates, the formation of the primitive gut occurs. Once this is formed, the primordial germ cells migrate into the posterior of the developing gut, before leaving the gut to migrate to the gonads

Answer 383

Stay in the extra embryonic territory - While the major inductive events occur, then migrate through the posterior gut, then leave the gut to move laterally to the gonads

Answer 384

A failure to migrate to the protective niche causes the germ cells to differentiate

Answer 385

Support cells travel with primordial germ cells to maintain the undifferentiated stem cell phenotype

Answer 386

Stem cell factor (SCF)

Answer 387

They follow a fibronectin train | Chemoattractant, Sdf-1 is also required

Answer 388

A cell that are endlessly capable of self renewing and/or differentiate to multiple lineages and cell fates

Answer 389

A stem cell that can give rise to multiple lineages

Answer 390

A stem cell that cannot give rise to multiple lineages but can give rise to multiple cell types within in the same lineage

Answer 391

A stem cell are endlessly capable of self renewing and/or differentiate to multiple lineages and cell fates A progenitor cell has a limited ability to self renew (only certain number of divisions) can give rise to a small number of specialised cells but not multiple lineages

Answer 392

- Self renewal is needed because if the stem cells didn’t copy themselves, you would quickly run out. It is important for the body to maintain a pool of stem cells to use throughout your life - Differentiation is important because specialized cells are used up, damaged or die all the time during your life. Specialized cells cannot divide and make copies of themselves, but they need to be replaced for your body to carry on working.

Answer 393

Red blood cells - Don't have a nucleus so only survive about 6 weeks - Need to be produced more in women when pregnant - release of oestrogen activates haematopoietic stem cells

Answer 394

Adult stem cells

Answer 395

The same thing - When in the embryo they are called inner mass cells - When taken out of embryo (about 32 cell stage) and cultured in a lab they are called embryonic stem cells

Answer 396

They are pluripotent - If add the correct proteins in the specific order that mimics those that occur in vivo, you can drive the stem cells to a specific fate and produce specific tissues - Grow organs for transplantation - Understand how diseases develop - Test drugs in the laboratory

Answer 397

- Have to use IVF to gain the embryos - Unethical - The stem cells aren't completely identical so differentiation is more complex then first appeared

Answer 398

Bone marrow Liver Gut Skin

Answer 399

``` Bone marrow Liver Gut Skin Brain Muscle ```

Answer 400

An adult stem cell is an undifferentiated (or partially-differentiated) cell found in tissues and organs that can self-renew and differentiate to become most or all of the specialized cell types within their specific tissue lineage. They are used to - Maintain cell populations - Help you heal - Play a role in ageing

Answer 401

The constant or periodic generation of new cells to replace old, damaged, and dying cells, or the addition of new cells as needed Adult stem cells fill this role

Answer 402

- Hematopoietic stem cells: blood and immune system - Mesenchymal stem cells: bone, cartilage, fat, muscle, tendon/ligament - Epithelial stem cells: skin, gut, other linings - Muscle stem cells Neural stem cells: neurons, glial cells, retinal cells

Answer 403

In stem cell niches - Complex microenviornments around the stem cells, made up of many cells that interact with the environment and the stem cell to decide whether to activate it - Very vascularised - to carry information in the circulatory system

Answer 404

Give rise to all the blood cell types: - Myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) - Lymphoid (T-cells, B-cells, NK-cells) Found in the bone marrow from very early on in development, as well as in umbilical cord blood and placental tissue

Answer 405

in its dormant form - not dividing

Answer 406

- Stem cells cycle between a quiescent and an active form; that - Many other adjacent cells in the niche provide factors that regulate stem cell activity and the decision as to whether to remain quiescent, or become active, and then divide to give another stem cell or a daughter that will differentiate; - That stem cells respond to physiological signals – inlouding blood-borne factors (eg hormones). So niches include endothelial capillary cells.

Answer 407

These stem cells will differentiate into: - cartilage cells (chondrocytes) - muscle cells (myocytes) - fat cells (adipocytes) - tendons, ligaments, and - connective tissue (epithelial cells including osteoblasts) These cells are found in bone marrow, fat, and blood

Answer 408

- Give rise to epithelial cells which constitute 60 percent of the differentiated cells in the body. - Responsible for covering the internal (i.e. intestinal lining) and external surfaces (i.e. skin) of the body, including the lining of vessels, glands, and other cavities. - Epithelial stem cells are also found in the bulge region of the hair follicle

Answer 409

Neural stem cells (also called neural precursor cells) give rise to neural progenitors and then to neurons, oligodendrocytes, and astrocytes

Answer 410

- Subventricular zone lining the lateral ventricles, where they give rise to newly-born neurons that migrate to the olfactory bulb via the rostral migratory stream - Subgranular zone, part of the dentate gyrus of the hippocampus - Hypothalamus lining the 3rd ventricle, where they contribute new neurons to energy/feeding circuits

Answer 411

Quiescent - dormant Active Self renew or differentiate

Answer 412

Stem cells rebuilt patients immune system using stem cells harvested from their own blood - helped multiple patients

Answer 413

Haematopoietic stem cells are easier to access then other types as they can be collected from blood and bone marrow

Answer 414

Patients travel to other countries with fewer restrictions to receive stem cell therapies

Answer 415

Stem cells are self renewing | - If put in the body uncontrolled then it could lead to cancer

Answer 416

Because it is surrounded by support cells It also looks the same as the support cells so can't be identified In bone marrow, the cells are isolated and look different so easier to identify

Answer 417

A stem cell niche - The crypt is a tube of cells with stem-like cells in a niche at the distal end and differentiating/ed cells at the proximal end - 1000's of them in the body as each villus has its own crypt

Answer 418

Support cells - Paneth cells

Answer 419

They are amplified by transit amplifying cells (TA) - These are progenitor cells which go through a number of rapid rounds of cell cycle to amplify the progenitor pool before differentiation

Answer 420

Wnt and notch are expressed highly by the panath cells Stem cells express frizzled and other components of the wnt signalling pathway in order to respond to wnt - It causes the transcription of genes such as Lgr5 and axin which promote stemness

Answer 421

The stem cell is pushed up out of the crypt away from proliferating signals (Wnt) and towards differentiating signals (BMP) that are expressed in the villus - When the cell encounters BMP, it causes the phosphorylation of SMAD and the inhibition of stemness causing differentiation

Answer 422

Stem cells and support cells look the same in the gut - Wnt (from support cells) binds to the promotor of Lgr5 in the stem cell - You would therefore make a Lgr5-EGFP transgenic mouse by taking the promotor of Lgr5 and fusing it upstream to GFP - Put it back into the mouse and normal development will occur but with the addition of the fluorescent gene - Wherever Lgr5 is being expressed will fluoresce when lit with a fluorescent light so will be able to recognise the stem cells in vivo - You can now dissociate epithelium and pass down a FACS ,machine which sorts out cells that show fluorescence from those that don't

Answer 423

Cancer treatment - Cut out the cancer leaving a wound bed which can be filled from the stem cells Diagnostic tool - Make mice have a disease and use to study Experimental tool

Answer 424

Cell proliferation - Increases the number of cells by mitotic cell division Cell enlargement - Individual cells increasing their mass and getting bigger - Skeletal and cardiac muscle never divide once differentiated they just increase in size Accretion - Increase in the volume of extracellular space which is achieved by secretion of large quantities of extracellular matrix by cells

Answer 425

G1: Cells grow in size S: DNA synthesise G2: Gap Phase M: Mitosis

Answer 426

The timing of the events in the cell cycle are controlled by cyclins. Cyclins control the passage through key transition points in the cell cycle They act by forming complexes with cyclin dependant kinases (Cdks), activating them. These kinases phosphorylate proteins that trigger the events of each phase, such as SNA replication in S phase or mitosis in M phase

Answer 427

Intrinsic control - Drosophila initially develop as a syncytium, this is a single cell with multiple nuclei. These nuclei go through very rapid synchronous cell cycles consisting only of an S and an M phase - At the 14th cycle, the cycle slows and a G2 phase is introduced and undergo cellularisation - Now depending on is precise position in the A/P and D/V coordinate system, each cell will acquire its own cell division rate - Controlled by a protein called String, a phosphatase that activates cyclin dependent kinases - During the first 13 divisions, uniformly distributed maternally-supplied string, allows rapid and synchronous divisions. After the 13th division zygotically expressed string is produced under the direct control of the patterning genes that set up the A/P and D/V coordinates

Answer 428

There is a protein call tribble, an inhibitor of string - The mesoderm needs to invaginate and formation of this ventral furrow can be inhibited by cell division, thus tribble functions to promote invagination by preventing cell division at an inappropriate time

Answer 429

If you implant a limb bud from a larger species of newt to an embryo of a smaller species of newt, a larger limb develops. This suggests that once the limb bud has formed, limb growth is controlled intrinsically – not by other factors from the body

Answer 430

If additional thymus glands in an embryo are transplanted all organs maintain their size and multiple amounts of tissue will be present, suggesting intrinsic control. In the case of the spleen, this is not true and both spleen will only grow to half the size such that the total amount of tissue remains the same suggesting, systemic control

Answer 431

May be determined by morphogens and the steepness of a morphogen gradient rather than by the cell size or number

Answer 432

TOR pathway - Increases cell size Hippo pathway - Limits organ size

Answer 433

The transcription factor Yki is in the nucleus activating gene expression involved in stimulating growth and survival of cells

Answer 434

YAP and Taz are vertebrate homologs

Answer 435

- Hippo, a kinase phosphorylates Yki stopping it from entering the nucleus - It therefore limits growth - Hippo integrates various signals to create a ‘stop growing’ signal. For example, overcrowding of cells

Answer 436

- Vertebrate homolog of hippo is Mst1/2

Answer 437

- In a hippo mutant, there is less growth restriction - A knockout mice results in a large liver but not enlarged everywhere suggesting the hippo pathway is only important in some tissues

Answer 438

The size of the adult is determined by the size of the larva which is determined by insulin signalling which affects both duration and rate of larval growth

Answer 439

In a drosophila where insulin has been ablated, the larva and resultant adult is much smaller

Answer 440

Insulin-like growth factors 1 and 2 (IGF1 and IGF2) are important for both embryonic and adult growth Post embryonic growth is controlled by Growth Hormone. Many of the effects of GH are mediated via IGFs. The production of growth hormone in the pituitary is stimulated by growth hormone releasing hormone and inhibited by somatostatin both from the hypothalamus. GH feeds back on its own production, it promotes production of somatostatin and inhibits GHRH IGF is mainly produced in the liver but can also be produced locally

Answer 441

Barker and Clark 1997 - Found a correlation between low birth weight, due to mother’s malnutrition, and an increased risk of CHD Dutch famine - A short caloric restriction of pregnant women resulted in normal weight babies but still with an increased risk of obesity and diabetes in later life

Answer 442

The most common tissues to become cancerous are those that continue to divide throughout life as they are already proliferative and are constantly dividing so may lead to copying errors 85% of cancers occur in epithelia! (3% leukemia)

Answer 443

Require activation to cause cancer | E.g. Ras, Raf, EGRF

Answer 444

If these genes become inactivated then it can lead to cancer E.g. Retinoblastoma, p53, patched If a person is heterozygous for a tumour suppressor gene, e.g. retinoblastoma, then if they lose the other functional copy during their life time, due to mutation, then it can lead to cancer

Answer 445

It is thought that these lead to mutations that again interfere with normal growth control, often involving developmental pathways eg. BRCA2

Answer 446

- Activated Wnt - Colon cancer, Hepatocellular cancer - Activated Hedgehog - Basal cell carcinoma Medulloblastoma - Activated Nodal - Melanoma - Activated Notch - Leukemia - Activated EGF - Lung cancer, Breast cancer

Answer 447

- Molting is initiated by activation of stretch receptors in the cuticle this leads to release of protothoracicotropic hormone which in turn leads to release of ecdyson from the protothoracic gland - Under the influence of ecdyson molting occurs - First the cuticle separates from the epidermis. While the original cuticle is still present the freed epidermal cells proliferate, secrete a fluid that forms a barrier, and start secreting new cuticle. Only after this stage the old cuticle will be shed

Answer 448

It was thought that regeneration ability was directly linked to the complexity of the organism where the lower the complexity, the better the regeneration. However, this is not correct as some very simple organisms have little or no ability to regenerate e.g. C. elegans

Answer 449

Morphallaxis - There is little new cell division and growth, and regeneration occurs mainly by re-patterning of existing tissue and the reestablishment of boundaries Epimorphosis - This type of regeneration is completely dependent on the growth of completely new, correctly patterned structures

Answer 450

Regeneration of the hydra

Answer 451

Hydra are very simple organisms formed by two germ layers, the endoderm and ectoderm It consists of simple body column and a mouth region, called the hypostome, surrounded by tentacles They grow continuously but are a fixed size. Cells, therefore, must be lost to maintain its size. This occurs at the tip of the tentacles, at the basal disc and by asexual budding, leading to the formation a new individual

Answer 452

Experiments have led to a model that states that two opposing morphogen gradients are responsible for this regeneration - The first gradient is one in positional value, meaning that the location of the cell determines the head inducing ability and level of resistance to the head inhibitor - The second gradient is produced in the head itself and is the head inhibitor

Answer 453

- Implantation of head tissue to the body of a different hydra doesn’t cause the formation of an ectopic head However, if the head of the hydra is removed before the implantation then an ectopic head does form - The head produces a head inhibitor stopping the formation of another head even when head tissue is implanted - If the original head of the hydra is removed then there is no inhibition stopping the formation of the ectopic head - If head tissue is implanted at the bottom of the hydra, even with the original head still present, an ectopic head still forms - Suggests that the head inhibitor forms a gradient with the top of the hydra receiving high levels of the inhibitor and the bottom of the hydra receiving low levels

Answer 454

The Wnt signaling pathway is thought to be involved in head formation Activation of the Wnt signaling pathway with lithium lead to all regions of the hydra acquiring characteristics of the head organiser

Answer 455

Regeneration in urodele amphibians

Answer 456

They are capable of regeneration of almost anything, including their limbs, retina, lens, jaw and their dorsal crest

Answer 457

In the iris

Answer 458

After amputation, the epithelial cells migrate over wound surface, this is essential for regeneration to occur. This is known as epidermal cell migration Once the epithelium has covered the wound, the cells below dedifferentiate and form a blastema This then restores the damaged limb

Answer 459

The blastema is derived from the dermis but also from dedifferentiating muscle and cartilage

Answer 460

The fact that the muscle can also participate is quite surprising since muscle cells are multinucleate. It has been found that multinucleate muscle cells can revert to mononucleate cells in cell culture under the influence of thrombin Dedifferentiation of muscle cells involves expression of msx, a homeobox transcription factor, and inactivation of the Rb gene, which inhibits proliferation of muscle under normal circumstances The presence of thrombin is crucial for dedifferentiation and may have wider significance, lens regeneration also requires local activation of thrombin

Answer 461

No, it is thought that there is very little dedifferentiation occurring Usually, the cells used in regeneration stay true to their fate and only dedifferentiate enough to allow the cells to enter the cell cycle

Answer 462

This is shown in a GFP experiment. Different cell types were labelled before regeneration and their lineage was traced. This found that most cells stay true to their fate, e.g. muscle cells restore muscle with the exception of dermis, which has the ability to generate cartilage as well. This indicates the blastema provides only a limited differentiation environment

Answer 463

Limb regeneration always occurs distally to the wound It occurs according to the positional value of the site of the cut It does not just try and replace missing parts

Answer 464

If the distal limb of a newt is amputated and the stump is inserted into the flank (to establish vascular connections) and the limb is subsequently cut again, the anterior limb will regenerate the missing distal parts. Importantly, the remaining stump will also regenerate a distal limb, and not a proximal humorous The wound blastema “reads” the local positional value and generates more distal positional values, it does not determine what is missing. As a result of this, a limb with two radii and ulnae is formed

Answer 465

If a distal blastema is transplanted to a proximal wound an entire limb will form. This shows that the site of the wound can sense a discontinuity in positional values between the distal blastema and the cut site. The remaining tissue will be formed by intercalary growth

Answer 466

If a distal is combined with a proximal blastema the proximal blastema will start to engulf the distal blastema this can be interpreted as a sign the distal cells stick more tightly to one another and that the proximal cells stick more to the distal cells than to themselves. Differential adhesive properties might also help to explain why distally transplanted cells do not mix with the proximal blastema cells that will form because of the intercalary regeneration

Answer 467

Retinoic acid has been found to be capable of resetting this positional value to a more proximal value. The effect of retinoic acid is dose dependent. The higher the level the more proximal structures will be regenerated. Proximalisation by retinoic acid may occur via upregulation meis homeobox genes and/or prod 1. Prod1 is a protein that is expressed at the highest level in the proximal regenerating blastema, and is induced by Retanoic acid treatment

Answer 468

Regeneration is usually dependant on innervation. If before the limb is amputated it is denerved then no regeneration occurs. However, if limbs are produced that have never had nerves present, regeneration of the limb does occur after its amputation

Answer 469

Newt anterior gradient (nAG) was discovered which can rescue regeneration of a denerved limb. nAG binds to Prod1 but the mechanism is unknown

Answer 470

nAG becomes expressed in the nerve sheath in response to wounding, driving regeneration. nAG is also expressed in the embryonic epidermis of the limb but is switched off when the nerves form. This explains why regeneration can occur in a limb that has never had innervation: nAG will still be expressed in the epidermis

Answer 471

Regeneration in mammals is very limited but does exist - Young children and mice have the ability to regenerate the tips of their digits - Regeneration of axons can occur in the peripheral nervous system. However, if the neurone is lost then no regeneration can occur - There is very little regeneration in the CNS. This is thought to be due to oligodendrocytes providing a non-permissive environment - The liver is able to regenerate fully after losing 2/3s of its tissue - As long as the membrane surrounding the ribs remain, they will also regenerate

Answer 472

After resection of the heart it will bleed but soon a blood clot will form preventing the circulation from coming to a complete standstill Very soon after injury the endocard (inside lining of the heart) will be activated and start expressing certain genes The epicardium (outside lining of the heart) will expand rapidly and start to cover the wound. Newly forming muscle expresses FGF and epicardial cells respond to this signal by invading the regenerate and reform blood vessel that will help to restore a functional ventricle Very recently it was reported that the epicard expresses an EGF like factor, Neuregulin, which can stimulate cardiomyocyte cell division

Answer 473

No The necrotic tissue that forms after infarction is replaced by scar tissue. In addition, there is a hypertophic response of the remaining muscle cells, which is thought to be maladaptive, and may in fact increase the risk of further infarction.

Answer 474

A recent paper correlated this loss of regeneration in mice with reduction in erbb2 expression which is required for Neuregulin signal transduction. To put this to the test a transgenic mouse that had a doxycycline inducible- dominant active form of erbb2 was created. This transgene was then induced for some time after an infarct was created in these mice. They were able to induce cardiomyocyte proliferation a significant improvement in function, and could show a smaller scar area

Answer 475

The age-related decline in vital physiological functions

Answer 476

Wear and tear - In C. elegans, the muscles that are required for feeding degenerate over time because of their use - In elephants, it is known that they die of starvation at old age as their teeth wear off Genetics - Salmon die soon after laying their eggs

Answer 477

Natural selection tunes life history of the organism so that sufficient resources are invested in maintaining the repair mechanisms that prevent aging, at least until that organism has reproduced and cared for its young As soon as an individual cannot increase number, or the chance of survival of its offspring any further, there is no natural selection against decline/aging in that individual. This theory was stated by Haldane

Answer 478

Metabolism Reactive oxygen species (ROS) DNA damage

Answer 479

Rate of living theory - This theory explains the link between rate of metabolism and age. This is especially clear in cold blooded animals like drosophila where low temperatures slow metabolism and increase life span

Answer 480

High metabolism leads to the formation of reactive oxygen species and oxidative damage. - Manipulation of ROS by various methods have produced contradictory results. In worms, paraquat or juglone treatment can increase lifespan, suggesting their are superoxide dependent - Glucose restriction extends C. elegans life span by inducing mitochondrial respiration and increasing oxidative stress - Resistance to oxidative stress is induced by longevity genes

Answer 481

Progeria syndromes cause premature aging and are caused by mutations in genes involved in maintenance of the genome

Answer 482

Progeria syndromes cause premature aging and are caused by mutations in genes involved in maintenance of the genome. This suggests that aging is related to DNA damage However, it is also clear that it is DNA damage rather than mutations that will lead to aging. This is shown by the fact that there are mouse models that have an increased mutation rate but these animals do not age more rapidly

Answer 483

The DNA damage theory of suggests that it is unrepaired damage in non-replicating cells that causes ageing. - In non-replicating cells unprepared/able DNA damage may accumulate and cause aging (in dividing cells it will lead to mutations) - It is suggested that NAD depletion by DNA damage induced PARP activation that might be to blame. PARP is an enzyme that is essential for certain DNA repair processes

Answer 484

Signals from the somatic gonad Environmental stresses Dietary restriction

Answer 485

- Restricting the diet of the organisms leads to longer lifespans in all animal models - This is not due to a lower nutrient metabolism that causes damage

Answer 486

E.g. Heat, ROS generators ROS generators have also been shown to increase life span disagreeing with the ROS theory of aging One explanation for this has been that a small insult can unleash a disproportionate protective response a process called Hormesis, reducing overall ROS impact. However, another theory says that ROS are simply not/barely relevant for aging, but that ROS are used as a molecular reporter for stress leading to activation of anti-ageing mechanisms

Answer 487

- There is strong evidence in flies and c elegans that the gonads produce factors that increase life span which come from the gonad not the germ line - Removing the entire gonad does not extend life span, but if only the germ cells are removed it does. It appears the germ cells inhibit a longevity signal that is produced by the soma. Daf12 and its ligand, dafachronic acid, are involved in this. However, giving dafachronic acid to normal worms does not lengthen their lifespan

Answer 488

By finding a mutant that has a modified life span, cloning the gene and then see what it does Identification of short lived mutants is likely to be very successful as many mutations can lead to the death of an organism by removing an esential gene, but this does not directly mean the gene is involved in aging Long lived mutants are more relavent to this research. C Elegans and Drosophila are often used because they have appropriate genetic tools and a short life span

Answer 489

IGF signaling inhibits the transcription factor DAF16 aka FOXO. The FOXO transcription regulates genes that increase resistance to various stressors This has also been seen in drosophila as mutations in the IGF Pathway have been found to double its lifespan and loss IGF signaling has been linked to resistance to oxidative stress

Answer 490

C. elegans have a normal life of 25 days but under adverse conditions it can enter a dauer state which can last up to 60 days. This will not affect the life span after the dauer stage so truly extends the lifespan Genetic screens have led to the identification of mutants that promote longevity and many of these were found to be in components of the insulin signaling pathway, leading to a block in insulin signaling

Answer 491

This signaling pathway is also involved in extension of the mammalian lifespan. Female mice with a mutation in IGF1R (IGF1 and 2 receptor) live 33% longer FOXO1 and FOXO3A, AKT and IGF1 receptor variants have been linked in Human longevity in multiple cohort studies

Answer 492

- FOXO downregulates Insulin-like genes meaning that it can exert its action outside of that cell - Genes affected include antioxidant genes (oxidative damage is a potential cause of aging), metabolic genes, chaperones, and antibacterial genes.

Answer 493

The TOR kinase is a major amino-acid and nutrient sensor that stimulates growth and blocks salvage pathways such as autophagy when food is plentiful Under stress this pathway is inactivated. It activates TSC, TSC blocks TOR, this then activates 4E-BP1 and blocks S6K1. This pathway inhibits protein synthesis and cell growth and activates a salvage pathway Growth factor, however, blocks Tsc, causing TOR to be active. This blocks 4E-BP and activates S6K 1. This pathway activates protein synthesis and growth – ‘wasteful’ pathways

Answer 494

Rapamycin, a blocker of TOR extends lifespan In C elegans another Fox gene, FoxA is required for TOR dependent lifespan extension Blockage of S6 kinase extends lifespan in yeast, worms, flies and mice along with overexpression of 4E-BP in drosophila

Answer 495

Sirtuins are NAD+ dependant protein deacetylases

Answer 496

They have been put forward as being able to prevent aging. They are thought to be the target of resveratrol, the presumed anti-aging compound from red wine. There are conflicting reports on their role in flies and c elegans. Most recently SIRT6 was put forward as the true anti aging gene and it was shown that it downregulates IGF1 levels

Answer 497

Through a protective mitochondrial response called the mitochondrial unfolded protein response which by an unknown mechanism protects against aging

Answer 498

Sirtuins (which require NAD) lead to activation daf16 and a protective mitochondrial response called the mitochondrial unfolded protein response Mitochondrial mutations have also been found to extend lifespand. A theory for this is that, similar to activation of sirtuin, this will lead to the mitochondrial unfolded protein response

Answer 499

NAD+ can also protect against aging, and it is thought that supplementing this compound will boost Sirtuin activity and possibly production of mitochondria, leading to an imbalance between mitochondrially produced and nuclearly produced mitochodrial proteins thus again leading to the activation of UPR-mt and ROS If NAD is central, it provides a potential link with DNA damage (which is thought to be at the basis of many human progeria syndromes) as DNA damage will lead to activation of PARP genes. PARPs are NAD consuming enzymes and can reduce the level of NAD

Answer 500

Adult stem cells that have been reprogrammed back to a pluripotent state

Answer 501

Can be used to grow human organoid that are being used to understand the development of organs and disease

Answer 502

A measure of how many types pf specialised cells a stem cell can make

Answer 503

John Gurdon in 2012

Answer 504

John Gurdon and Shinya Yamanaka

Answer 505

- He took a xenopus egg cell and eliminated the nucleus - He took a skin fibroblast, which are fully differentiated cells, and removed the nucleus and implanted it into the xenopus egg - This diploid status of the egg allows the first stages of mitotic division to occur - Found that a full new tadpole developed meaning that the fibroblast nucleus must be reprogrammed to a pluripotent state - This must be in response to a factor in the egg cytoplasm - Cloning

Answer 506

Because they are very large and easily manipulated

Answer 507

He hypothesised that they reprogrammed by activation of transcription factors in the differentiated cell that sit at the top of a hierarchy of de-differentiation programmes and return to pluripotency

Answer 508

- He guessed four transcription factors that induce pluripotency by taking an educated guess based on developmental biology studies which had identified transcription factors expressed in pluripotent cells which were then subsequently shut down when the pluripotent cells differentiate - When he transferred these transcription factors into cells taken from the skin, they were reprogrammed into pluripotent stem cells that could develop into all cell types of an adult mouse - He named these cells induced pluripotent stem cells

Answer 509

Any animal

Answer 510

First step is genetic reprogramming of a differentiated cell | The second step is differentiation into a desired cell type using specific transcription factors

Answer 511

- There is no need for embryonic stem cell research (more ethical issues) - Can also get individual specific pluripotent cells which will stop the problems of rejection - An example of personalised medicine

Answer 512

- Ability to differentiate into many cell types - Vastly renewable - Easily renewable - Individual specific – personalised medicine

Answer 513

- One of the transcription factors was first described as an oncogene (cancer causing) - Viral vectors for gene delivery that carry the risk of insertional mutagenesis - Low efficiency and slow kinetics

Answer 514

- Lung cells are being cultured on sophisticated tissue engineered substrates ‘lung on chip’. Can see how lungs respond to mechanical ventilation in vivo - Brain organoids can be made from IPS cells and can help us understand the brain development and its response to diseases

Answer 515

Parazoa (simplest) Diploblasts (two germ layers) Bilaterians (three germ layers)

Answer 516

Deuterostomes which are a branch of bilaterians

Answer 517

Being larger has advantages – the predator rather than prey Cells could sacrifice themselves to provide food for ultimately one last surviving cell, this could explain the evolution of an egg

Answer 518

The evolution of gastrulation could have resulted from the advantage of having a specialized surface/cavity that could aid in digesting large chunks of food Primitive multicellular organism

Answer 519

Trichoplax represents this. When it encounters a food source it forms a digestive cavity around the nutrient particle and digesting it externally

Answer 520

Has inverted over time An idealised arthropod has the circulatory system on the dorsal side and the nerve chord on the ventral side An idealised vertebrate has the circulatory system on ventral and nerve chord on the dorsal

Answer 521

In drosophila, an arthropod, dpp patterns the dorsal side and is counteracted by the protein sog. Dpp is the drosophila homolog of the BMP proteins whereas sog is the homolog of chordin. BMP induces ventral fates and chordin counteracts BMP on the dorsal side it seems that the body plan in metazoans has evolved and an inversion has occurred

Answer 522

The stage where vertebrate embryos are most similar

Answer 523

There is a stage called the phylotypic stage. At this stage vertebrate embryos are most similar. The stages before that are changed by reproductive adaptation, e.g. egg size in mammals vs birds and reptiles, the stages afterwards show more and more species specific adaptation.

Answer 524

The genome sequence, cell types and signalling pathways are all conserved throughout animals

Answer 525

Change in the promotors and enhancer of genes changing their expression - Usually modification of existing mechanisms

Answer 526

Duplication and divergence - Big driving force for evolution - Genome duplications have led to knew functions of proteins - These changes are rarer then promotor changes Genome duplication - There has been instances where the entire genome has been duplicated - Leads to changes in function because there is still another function protein that will allow the organism to survive

Answer 527

Through duplication and divergence - Drosophila have one set of Hox genes, mammals have 4 sets and fish have seven - Primitive chordates like amphioxus only have a single Hox cluster meaning that ancestral vertebrate underwent 2 rounds of genome duplication - This fits well with the fact that the fishes, the most species-rich group within the vertebrates probably duplicated their genome one further time

Answer 528

Different types of vertebrae are correlated with expression boundaries of Hox genes. For instance, the number of neck vertebrae can vary in vertebrates but it is always correlated with the Hoxc6 expression boundary

Answer 529

They have completely lost their limbs which are usually formed at the anterior expression boundary of Hoxc6. In the python loss of limbs is correlated with an anterior expansion of both hoxc6 and hoxc8, expanding the non-limb bearing flank territory all the way anterior

Answer 530

In flies, 12 genes important for wing development and 6 are repressed by Ubx in the haltere In the butterfly, due to changes in the upstream regions of some of these genes they are not repressed and can thus act to form a second wing

Answer 531

Different finches have evolved to have different types of beaks depending on the diet they were specialising on

Answer 532

- The differences in beak size were shown to correlate with onset and level of BMP4 expression. Importantly, it was also shown that misexpression of BMP4 in similar staged chick embryos could change the shape of the beak of a chick embryo in a similar manner. This provides important indication that it is not just correlation but BMP4 may cause this change. Thus, changes in level and duration of bmp4 expression are at the basis of evolution of beak size and shape

Answer 533

From the fossil record, insect and vertebrate wings do not derive from a common ancestor as wings have evolved twice in different lineages However, many genes involved appear to be similar. This is because the patterning mechanism used to set up an axis is conserved

Answer 534

Fins | - Fins had humorous, radius and ulna but no digits

Answer 535

There is a correlation between Hox expression and evolution of digits In mice an enhancer drives distal expression of HoxA and HoxD clusters When the same region from fish was tested in mice it only drove proximal expression However, when the same region was tested in a coelacanth, an ancient lobe-finned fish and the closest living ancestor of the tetrapods, expression was driven more distally

Answer 536

Cave fish have lost their eyes as they are useless in the dark. However, the eyes are still present in the developing embryo because genes that drive eye expression in cave fish have other roles in the embryo so eyes still form as it is difficult to get rid of those genes

Answer 537

Heterochrony is the change in the duration of growth

Answer 538

An example of this can be seen in dogs. The face of a bulldog is much smaller than the face of a dog meaning it stops growing sooner Another example is the evolution of horses as the increased duration of growth of the central digit may have caused loss of contact of the lateral digits which in turn favored further reduction of their development. This has resulted in the single digit (hoof) which we can recognize in the present-day horse

Answer 539

This describes a situation where the animal reaches sexual maturity in the larval stage

Answer 540

An example of this is in the axolotl. It reaches sexual maturity and remains in its larval form It can still be induced to undergo metamorphosis by injection of thyroxin, changing it to its lost adult form of the species

Answer 541

Long germ band - All segments are defined at once meaning embryogenesis is quick but segmentation process is complicated - e.g. drosophila Short germ band - Segments are added progressively overtime - eg. centipede

Answer 542

Short germ band - Segments are formed progressively by the budding off of the proctodeum - Works through notch and delta signalling - Notch activates HER protein which is a negative regulator of delta, therefore decreasing notch signalling - negative feedback - Her protein is unstable so is degraded quickly allowing notch signalling to increase - This causes oscillations in a time dependant manner which allows for the formation of segments

Answer 543

Beatle | - Head and thoracic segments are formed together and he abdominal segments form progressively

Answer 544

They use short germ band insects showing our evolutionary path

BMS237 Advanced Developmental Biology Flashcards

(584 cards)