Devlopmental Flashcards
What are the two opposing theories for development?
Epigenesis: Progressive process where new structures are added slowly leading to the development of the whole organism.
Preformationism: organisms develop from miniature versions of themselves.
What is the germ plasm determinants theory?
Weismann: theory that ‘determinants’ get split up into cells unevenly as they divide. it is only the germ cels that contain all of the genome, and these self differentiate and give rise to somatic cells.
Roux experiment initially supported this mosaic determining of cell fate at cleavage, when ablate one of the two cells of the xenopus with a hot needle and still developed into a well formed half larva.
What is the induction theory?
One cell or tissue directs the development of another. Dreisch if divide blastomas at the two cell stage, and even 4 both will form a full sea urchin, just smaller.
Also Spemann and Mangold, grafting the dorsal lip of blastopore and inducing a second axis=organiser.
When cells are close vs when far apart?
Condensed vs dispersed
What does factor x do?
Negative feedback to inhibit proliferation.
Balance of stem cells is important why?
Too few: ageing and degeneration
Too much uncontrolled growth=cancer
Cell changes that cause morphogenesis?
Cell adhesion
Cell migration
Cell death e.g digits
Cell shape
Steps (single words) from Egg/stem cell to its final fate?
- Specification- early comittment, if transplant will likely change fate.
- Determination-more stable comittment, now if transplant will likely keep same fate.
- Differentiation
- Maturation- e.g. muscle fibres fast or slow depends on level of innervation they recieve.
Two opposing theories for embryo development between animals?
Haeckel’s Funnel theory: wrong- early all develop very simialrly then gives variety.
Von Baer’s hourglass model: Early v different e.g. gastrulation, then similar in middle section, before differentiating a lot more.
Instructive vs permissive?
Instructive: Instructs the fate e.g. tells the cell what action to do.
Permissive: Allows something to happen, e.g. already had some info e.g. a HOX gene, that is turned on to alter fate. (gives permission)
What is a morphogen?
Soluble secreted molecule acting at long distances to specify the fates of cells. Can form a concentration gradient.
Is a morphogen instructive or permissive?
Instructive.
How can test if the signal is a morphogen or not by working out if its instructive or permissive? (2)
Ectopic expression: Provide a second source of signal. If permissive the same patterning, if morphogen different fate, creating a mirror image for example.
e.g. Shh in chick wing, mirror digits.
Or make expression uniformly high, see if changes fates.
How can test if the signal is a morphogen or not by working out if it acts directly at distance? (2)
- Genetically engineer the signal to ahve a TMD so can only work juxtacrine signalling to the next cell. If put the morphogen in the first cell in a line, if bucket brigade this will work as normal, however if morphogen cant diffuse to other cells, no patterning of further down cells.
- Make one cell in the centre lack a receptor for the signal. If morphogen this one cell will not be patterned, if bucket brigade then won’t matter.
What is meant by a morphogen working directly at distance?
A gradient is created away from the source, which impacts the cells. If not may work in a Bucket brigade (chinese whispers) like way, signalling to the next cell.
As well as passive diffusion what else helps to establish the gradient of morphogens?
Binding to molecules in the ECM e.g. heparin sulfate proteoglycan, and if want a steep gradient, degradation over distance, and high conc of receptors to amplify the signal.
How can heparin sulfate proteoglycan regulate morphogen diffusion? (2)
Binds many ligands e.g. Sequestering and slowing BMP, or facillitating the diffusion of Hedgehog.
How can morphogens travel through cells?
Planar transcytosis.
Cell can endocytose into a pit coated vesicle
Evidence that morphogens travel through cells?
If block vesicle formation caused Dpp to ask juxtacrinely.
How does timing play a part in morphogen induction?
Poorly understood, but likely the receptors respond to the signal when it gets to a steady state.
Transcriptional read out model?
Higher conc of morphogen, leads to higher conc of activated transcription factor (moving it into the nucleus).
How else is the expression of genes controlled as well as concentration of morphogen (transcriptional read out model) lead to differential differentiation intrinsically and autonomously?
Affinity of enhancers
e.g. if receptors see only low levels of transcription factors and the enhancers have a low affinity definitely no transcription will happen, whereas if both high lots of transcription will occur. So two cells could see the same concentration of transcription factor but one could have the genes expressed if high affinity, whereas other not if has low affinity enhancers.
Name an example of a signal that is both a morphogen and a transcription factor?
Bicoid mRNA is localised at the anterior of the egg and is translated into protein during early embryogenesis. Bicoid protein then diffuses through the cytoplasm and accumulates in nuclei of the syncytial blastoderm generating a concentration gradient.
How else is the expression of genes controlled as well as concentration of morphogen (transcriptional read out model) lead to differential differentiation non-autonomously?
Negative feedback crosstalk
if one cells genes are activated, it can also code for a repressor and repress neighbouring cells, to create differences.
How are strict thresholds achieved when the gradient is not steep?
Positive feedback, once weakly activated the gene may code for a transcription factor which feeds back to activate its own expression, thus amplifying it.
Epiblast and hypoblast equivalent to … and .. in the xenopus
Epiblast: Animal
Hypoblast: Vegetal
How is the egg already polarised in xenopus before fertilisation?
due to gravity, heavy yolk sinks down, whereas the pigment cortex is more animal.
First cleavage in the xenopus?
Asymmetric. All of the cytoplasmic components sink to the vegetal (bottom) cell.
hypoblast or epiblast goes on to form the ICM?
epiblast
What do the vegetal cytoplasmic components of the xenopus cell do?
Act on the DNA to regulate gene expression e.g. some are DNA binding proteins. As a result certain transcription factors are turned on in vegetal hemisphere cells.
In the xenopus what causes the formation of the blastocoel?
Changes in the osmolality cause an influx of water creating the fluid filled blastocoel.
After the sperm enters what is the next event to happen?
30degree cortical rotation.
Sperm entry into ventral cortex causes the cortex to become loose from the cytoplasm so it can move. Dorsalising factors are activated and through the actin filaments in the microtubules causes relocation of maternal proteins.
What are the dorsalising factors in the xenopus during cortical rotation?
wnt and dishevelled, Wnt binds to frizzled receptors in cells, which leads to activated Beta Catenin, leading to upregulation of these genes.
What forms opposite the sperm entry of the xenopus embyro?
differentiation of the organiser.
From the dorsal part of the xenopus embryo what factor is secreted? leading to?
Wnt, which binds to Frizzled, and eventually causes the accumulation of Beta-catenin turnin gon Wnt genes in the dorsal quadrant.
The dorsal of the embryo will become future…
posterior
Relevence of the dorsal region in development stage?
The dorsal lip/ blastopore folds inwards in gastrulation.
How do transcription factors work?
gene is transcribed as mRNA in the cytoplasm and then translated into a protein which can be secreted or autonomously migrated back to the cells nucleus to bind to promoters and enhancers of genes to alter transcription.
…….. accumulates dorsally in the xenopus embryo, whereas ….. vegetally
Beta- catenin
TGF B signal (Nodal or Vg1)
Vegetally ……. located in vegetal nuclei transcriptionally activates …….. in the xenopus.
VegT (to Veg1)
Nodal
Nodal is a what? Function?
secretory Morphogen, which sets a gradient across the animal hemisphere for gastrulation.
What is Nodal’s mode of action?
Cells that contain receptors for Nodal respond by the Receptor Serine Threonine Kinase Type 1 and 2’s dimerizing and phosphorylating Smad 2/4, which translocates into the nucleus and leads to the induction of target genes such as Nodal, Lefty, the antagonist of nodal Cerberus, to cause differentiation of the 3 germ layers.
Cells that see high Nodal will become….
Low Nodal….
No Nodal…
In a gradient Vegetal to animal, so those most vegetal in the animal hemisphere (or epiblast) become Endoderm. Low levels become Mesoderm and no Nodal cells become Ectoderm.
Why is the hypoblast so important in gastrulation?
It is the hypoblast that signals to the epiblast via morphogens such as Nodal, to differentiate into the 3 germ layers.
From a coronal view of the epiblast what is happening during gastrulation?
The cells of the mesoderm undergo an epithelial to mesenchymal transition and invaginate and drop down into the middle layer causing the formation of the primitive streak.
As well as low levels of Nodal what other transcription factor is expressed in mesoderm that can show its mesoderm?
Brachyury
Where both ….. dorsally and ……vegetally overlap what develops?
Beta- catenin
Nodal
The Nieuwkoop centre.
How is the Nieuwkoop centre induced?
Where both high Nodal signalling (P Smad 2/4 effector) and B-catenin (Wnt effector) vindn to the promoter elements, zygotic genes such as Siamois and later Goosecoid are activated. These are characteristic of the Nieuwkoop centre.
What does Siamois do in the Nieuwkoop centre?
T.F. which causes the upregulation of the gene goosecoid (Gse) by binding to the promoter region, not only in the Nieuwkoop centre itself but also just animal to in the dorsal mesoderm. Causing the formation of the organiser.
The high Nodal and Wnt signals the organiser to express what transcription factors?
Siamois, Goosecoid, Chordin, Xlim, Xnot etc.
What does the organiser/node do?
secretes BMP inhibitors e.g. Chordin, Noggin to Dorsallises the neighbouring mesoderm and neuralise the overlaying ectoderm.
Under the transcription factors of …. and ….., the node self-differentiates into? How?
Siamoid and Goosecoid
Axial mesoderm
These TF go back into the nucleus, binding to promoters causing the differentiation. Under slightly different concentrations of, the A/P axis is established.
What movements does the node do when self-differentiating?
Migration by convergent extension into a long thin rod underlaying the midline of the ectoderm. The Pre-cordal mesoderm migrates first anteriorly, and then the notocord posteriorly.
What transcription factors are expressed in a gradient along the A/P axis of the axial mesoderm?
Wnt and Retonoic acid are highest posteriorly, giving the posterior characteristics of the hindbrain, as differrent HOX genes are turned on.
intermediate mesoderm (in terms of dorsal to ventral), induced by BMP inhibition from node, goes on to form..?
somites and heart
Ventral mesoderm forms?
Blood and kidney
Dorsal mesoderm forms?
Notocord (self differentiated node)
What is the structure that runs anterior to posterior thats formed during gastrulation?
The primitive streak, with hensons node which progreses posteriorly.
What happens along the primitive streak during gastrulation?
The mesoderm undergoes an epithelial to mesenchymal transition and migrates down (ventrally) and laterally under the overlaying ectoderm.
After gastrulation what happens to the node?
It self differentiates into axial mesoderm, made of notocord and precordal mesoderm.
what are the three types of mesoderm found in a coronal cross section through the primitive streak?
Paraxial mesoderm, most medial, either side of the neural tube.
Intermediate mesoderm, ventral to and slightly lateral
Lateral mesoderm, furthest lateral in a C shape.
What does paraxial mesoderm go on to form?
Head and somites from.
what does the intermediate mesoderm go on to form?
Urogenital system- kidney nd gonads.
What does the lateral mesoderm go on to form?
Circulatory system e.g lungs and somatic cells. and extraembryonic tissues.
What 5 structures come from the somites?
Sclerotome (cartilage) Syndotome (tendons) (people with a syndrome tend to..) Myotome (skeletal muscles) Endothelial cells Dermatome (Dermis)
Paraxial mesoderm is split into 2 why?
The anterior and posterior ahve different fates, the posterior segments into somites, whereas the anterior is unsegmented.
What are somites?
Segmented paraxial mesoderm.
What is expressed in both invertebrate and vertebrate somites?
Engrailed1
The number of somites dictate the number of…
vertebrae.
In humans some vertebrae fuse- from 33 at birth to 24 in adulthood.
Do somite numbers vary between species? Inviduals?
Yes, but not individuals. Humans: 38-44 chicks: 55 Mouse: 65 Zebrafish:33
Somites form in … from …… to …… as the ……. migrates
Pairs
Anterior
Posterior
Node
In chicks somites bud off every ……whereas a mouse is every….?
90minutes,
2 hours.
How is the periodicity of somite formation controlled?
Clock Wave- front model. A molecular oscillator, where cells hit the travelling wavefront and have an abrupt chnage in property and become somites.
How was the Clock wavefront model first found in somites?
The periodicity of C-Hairy every 90mins, correlated with the addition of a new somite in chicks. Cooke et al 1976
How are the boundaries formed in the oscillations of the clock-wavefront model? (2)
Negative feedback mechanisms
Short half life
How is the most anterior region protected from the clock wave-front model?
Retinoic acid from the anterior anatagonises so the presomatic region continously getting longer.
Where is the pre-somatic region found?
Between the node and the last formed somites cells, before the paraxial meso is converted to somites
What is the determination front?
Position of the last somite-2
If take a boundary cell and transplant into a non boundary location, instructs cells anterior to becomes a boundary, why?
Notch family genes are expressed and at a somite boundary these are inhibited by lunatic fringe Glycosyltransferase enzyme.
What cell movements govern somite fomation?
Mesenchymal to epithelilal transition.
What are the two oposing gradients in the clock wavefront model?
Anterior: Retinoic acid gradient
Posterior: FGF/wnt
When in the clock-wavefront model do the somites actually form?
when FGF/Wnt levels drop below a threshold level, then gene expression is turned on, to cause somatogenesis
How does lunatic fringe work?
Glycosylation affects the ability for the extracellular domain of Notch to bind to the ligand delta, so creates a negative feedback loop.
Therefore causes the formation of a boundary between somites.
4 stages of muscle differentiation?
Stem cell, myoblast, myotube, myofibre
Stem cell- specification/determination, loss of pluripotency-> Muscle progenitor cell myoblast- differentiation-> Myotubes-maturation->myofibres
From a stem cell to a muscle progenitor myoblast what happens?
Specification/determination, muscle specific genes turned on such as MyoD, Myf5, MRF4.
What is 5Aza?
A demethylating agent that can drive stem cells to differentiate into myoblasts.
By removing the methylation of histones, which repress the muscle specific genes being expressed.
How can 5Aza be proved experimentally?
- Treat a fibroblast (already differentiated cell) with 5Aza.
- extract the mRNA, and reverse transcriptase into cDNA.
- Screen the cDNA for muscle specific genes using myoblast specific probes.
- compare results with a control.
What evidences that MyoD is a muscle promoting gene?
If treat already differentiated cells such as nerve cells, fat, fibroblasts etc, with MyoD along with an active viral promoter, they get a myotube fate.
With muscle specific receptors and proteins, and become multinucleate.
What is the structure of MyoD family transcription factors?
They are basic helix- loop helix proteins.
They have a basic domain- which binds DNA to, and a helix-loop-helix domain which causes dimerisation with E12 or E47 proteins, whcih act as a co-factor for transcription activation.
If MyoD is ablated what happens?
Nothing, it is compensated by Myf5 and Mrf4, in which it acts redunantly with.
Where does MyoD bind to?
it is a transcription activator which binds to the E-box sequence of enhancers to drive transcription of genes e.g. muscle specific genes have this specific e-box in.
Forms dimer with E12 or E47 too.
CANNTG sequence.
The dermomyotome contains progenitors of the ……. These express ….
skeletal muscles
Pax3
The dermomyotome comes from the …… mesoderm
Paraxial
What causes the differentiation of the paraxial mesoderm? e.g…
Signals from adjacent tissues,
e.g. Wnts from the doral enural tube and ectoderm and BMP4 from the lateral mesoderm cause formation of the dermomyotome.
Whereas Shh from the notocord and floorplate cause the formation of the sclerotome.
How does the sclerotome form?
Shh Signals from the notocord and floorplate, which causes the sclerotome to express PAX1 and undergo a mesenchymal to epithelial transition and migrate laterally.
The ribs are formed from what?
Sclerotome
How does the dermomyotome form?
BMP4 from lateral mesoderm signals and Wnts from the dorsal neural tube and ectoderm.
Dermomyotome forms a flat sheet- dermis, while other cells migrate away MET to become the myotome.
The somite myotome forms two tissues which are? Signals that specify?
Epaxial- more medial, higher Shh and Wnt
Hypaxial- more lateral, high Wnt and BMP4.
Hypaxial mesoderm forms the ….. Mutant?
Abdominal and limbs
Splotch mutant lacks Pax3 and lacks limb muscles.
(normally high in Pax3, which induces c-Met expression
What is the first embyonic muscle to form??
Dermomyotome.
What are expressed in myoblasts during embryogenesis that regulate myogenesis?
Myogenic regulatory factors- can tell by doing a blue insitu hybridisaion ane expression correlates to skeletal muscle.
What does the epaxial muscle go on to form?
deep back muscles (more dorsal)
Epaxial tissue down regulates…
Pax3
Pax1 present
At 8 days the first muscle gene …… is expressed in epaxial tissue until 12 days.
Myf5
Myf5 knockout?
Redundancy with MyoD so little difference, just delay in myotome formation.
Double knockout of Myf5 and MyoD?
No skeletal muscle present, and no myoblasts.
Myogenin knockout?
Mice unable to take their first breath so die shortly after birth, as there is no diaphragm.
In epaxial mesoderm both …… and ……. signals are needed for …… expression
Wnt and Shh
myf5
Hepaxial mesoderm is regulated by ….. signalling as well as ….. which leads to the expression of both … and …. but …… induces …… which represses in the limb bud those.
Wnt and BMP4
MyoD and Myf5
BMP4 induces Pax3, and represses Myf5 and MyoD in cells fated to migrate
in the limb bud.
MyoD is expressed later in the epaxial and hypaxial at what time?
9.5days
How does hepaxial tissue migrate to the limbs?
It expresses Pax3, which drives C-met receptor expression, which is a RTK to Scatter factor (or hepatocyte growth factor HGF) in the limb mesenchyme, which acts as a chemoattractant for the somite cells to the limb.
How do myoblasts differentiate into myotubules?
Fusion requires integrin B on the cell membrane, and structural reorganisation of Multinucleate myotubules.
Myogenin also expressed.
Growth factors inhibit, as promote myoblast proliferation, and not differentiation.
Without, it withdraws from the cell cycle under P21 (myogenin and MyoD activate transcription of)
WHat happens to the hypaxial tissue after its migrated to the limb bud?
it then differentiates, Myf5 and MyoD determination.
Pax 7 knockout?
Lose ability to repair muscles in adulthood.
Order of the 5 ‘-tion’ s of limb myogensis?
Delamination, Migration, Proliferation, determination, differentiation
Satellite cells originating from the somites sit where?
under basal lamina in the extracellular matrix of all muscle fibres.
How does the maturation from myotubules to muscle specific protein happen?
Myf4
Maturation decides whether short or fast fibres, and the muscle expresses proteins such as actin, tropomyosin, creatine phosphate etc.
How do cells intiate a determination front?
Where the retinoic acid gradient from the anterior meets the FGF and Wnt gradient from the posterior = the level of the determination front.
This is when there is an arrest in oscillations of Wnt and FGF8.
How do Retinoic acid anteriorly and Wnt and FGF8 posteiorly inhibit each other?
By Mesp2, RA activates expression of, which inhibits FGF8. But also are morphogens which just gradually diminish over distance.
What is Jarcho Lewin syndrome?
Absense of Notch signalling.Spondylocostal dysplasia, ossificaion centres of vertebrae dont align due to the misalignment of somite formation.
Molecular cascade from C-hairy to somite formation? Explain?
C-Hairy1 -> Lunatic fringe -> Notch1 -> ephrins -> cell adhesion changes
Genes that encoding C-Hairy are periodicially activated then inactivated in the posterior mesoderm. When high levels are detected this activates Lunatic fringe, which works to inhibit Notch
(mesenchymal to epithelial cells condense together)
How do cells know to initiate a boundary after somite formation?
Oscillation slows down as cells mature
Acquisition of anterior and posterior identity:
Notch1 and Dl1 in post.
Notch2 and Dl3 in ant
Structure of C-Hairy?
bHLH transription factor
What is the determination front?
It creates a boundary between RA anterior and Wnt and FGF8 posterior which initiates somite formation. Cells posterior to the determination front are maintained in a non-determined state by FGF activity, whereas those anterior initiate the formation.
Determination front and length of the somite?
Theory holds that the size of each somite is defined by the number of mesodermal cells that pass the determination front between these two opposing signalling domains during one cycle of the segmentation clock.
Mesp2 expression in the somite?
Mesp2 is only expressed in the anterior half of the somite,anterior to the determination front, as Notch from the anterior activates Mesp2 via TbX-6.
Whereas Mesp2 is low posterior to the determination front, as Mesp2 is inhibited by FGF and Ripply2.
What does Mesp2 do in the somite?
Induces boundary formation, by activating Ephrin and E cadherin signalling, causing epithelialisation and the formation of a furrow.
Anterior posterior, proximal distal, ventral dorsal axis of the limb?
Proximal near shoulder, distal at phalanges, anterior thumb, posterior little finger. Dorsal back of hand, whereas palm is ventral
What do the limbs form from?
A limb bud.
Two …… transcription factors specify limb bud formation These are?
T-box
Tbx5 specifies the forelimb in mouse, or wing the case of drosophila (induced by Pitx1)
Tbx4- hind limb- leg
Evidence for T-box genes?
If force transcription factor expression of other limb, that limb will form- sufficient.
What induces the expression of the T-box genes in the limbs?
Differential HOX genes along the anterior to posterior axis, which cause differential gradient of Retinoic acid (highest anteriorly) which leads to the different T-box genes being activated.
What experiment proves that FGF is involved in limb bud formation?
If ectopically put an FGF bead in a flank region not destined for limb formation early, an ectopic limb appears.
Depending on proximity to which limb, this could be either a forelimb or a hindlimb.
PITX1?
expressed in the hindlimb, and is thought to determine the differences in hindlimb and forelimb
Mutations in PITX1, TBX5?
TBX5- mutation in humans causes Holt-Oram syndrome defects in upper limbs and heart
PITX1- mutation associated with lower limbs required for TBX4 expression
What do the T-BOX transcription factors do?
control local production of FGFs that initiate limb developement, FGF10 is the ligand.
FGF10 K/O?
Mice lack limbs
FGF is involved in establishing and maintaining which two regions? What do these do?
Apical Ectodermal Ridge- essential for limb bud formation (FGF8)
Zone of polarizing activity-determines pattern along the anterior posterior of the limb.
…… induces the apical ectodermal ridge to express ….. which is also acts back to maintain …. in the …….
FGF10
FGF8 in the overlying ectoderm
FGF10 in the proximal mesoderm
FGF8 induces …. in the overlying ectoderm also
Wnt
Removal of the AER causes?
Truncation in growth of the limbs, the earlier its removed the bigger the impact. Reduced proliferation in this area and more cell death.
3days- develop only the proximal bones
3.5days- proximal and distal but no digits
4days- only truncated digits
The longer the cells spend in the progress zone the more proximal/distal their fate in the progress zone model
Distal
the progress zone moves ditally as the limb grows
What are the two models of proximo-distal patterning of the limbs?
Progress zone model- amount of time in determines fate
Two-signal model- Proximal signal from paraxial meso antagonises the distal gradient from AER giving differential expression,
What are the two models of proximo-distal patterning of the limbs? Which is correct?
Progress zone model- amount of time in determines fate
Two-signal model- Proximal signal from paraxial meso antagonises the distal gradient from AER giving differential expression, (correct)
How does the two-signal model work?
Retinoic acid from the proximal paraxial mesoderm, and FGF and Wnt from the AER.
Retinoic acid drives expression of Meis homodomain genes which is required for proximal fate.
And FGFs drive expression of HOX11 and 13 (most distal), which give distal fate.
Meis expression in the distal region of limb?
Limb abnormalilites as normally in proximal
The anterior/posterior axis of the limb is specified how?
By the Zone of polarising activity which acts as a source of the Shh morphogen gradient from the posterior.
If graft the ZPA or Shh beads anterior what happens?
Mirror image duplications of digits forms e.g. in chick 4,3,2 -> 4,3,2,2,3,4
K/O of Shh in limb?
Complete loss of distal skeletal elements and single bone of ulna and radius(zeugopods), as this sets the anterior to posterior axis.
Pod names for the humerus, ulna and radius and digits?
Humerus: stylopod
Ulna and radius: zeugopods
Digits: autopod
Cardiovascular disease accounts for how many deaths a year? (fraction), cost to NHS?
1/4 of UK deaths a year, 1 person has a myocardial infarction every 3 mins in UK.
Costs NHS £11billion a year
Congenital heart problems account for how many deaths a year and fraction of babies?
800
1/3 of babies deaths before 1
Ratio of human disease genes that have a drosophila homologue?
Over half, 548/929 - 550/930
When does cardiogenesis happen in vertebrates vs drosophila?
Vertebrates it happens immediately after gastrulation e.g. human and mouse at around 6-12 hours.
Drosophila later, delay for mesoderm to form.
