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.
Cardiac cells originate from what tissue?
The Ventro-lateral mesoderm, called the Splanchnopleura (the Ventral bottom part of the lateral C shaped mesoderm) or Splanchnic lateral plate mesoder,
The structure that forms and lays down the scaffold for the heart?
First heart field (cardiac crescent)
First heart field characteristics?
Makes the open cuved tube heart scaffold, low proliferative capactiy,
Forms two layers of cells: the inner endocardium and the outer myocardium.
After the first heart field cardiac tube starts pumping blood what happens?
The tube elongates and becomes divided transversely nto the atrial and ventricular chamber. Epicardium forms to surround the myocardium.
Unlike in the drosophila heart which stops at the first heart field what happens in higher vertebrates?
Looping into a multichambered heart.
Difference in the structures formed by the heart fields?
first heart field earlier: cardiac muscle cells of the primitive left ventricle, and myocardial cells of the outflow tract.
Second heart field: elongation of the early heart tube causing looping, and formation of the separate chambers later.
Where are the second heart field dcells located?
The second heart field myocardial cells lie medially and posterior to the first heart field cells.
Characteristics of the second heart field?
Develops slightly later but has a high proliferative capacity.
3 very broad stems in heart formation?
- Angiogenic cell clusters- on the heart plate.
- These angiogenic cell clusters coalesce to form right and left endocardial tubes at around day 21.
- Heart formation
4 steps in the Drosophila heart formation?
- Mesodermal precursor (splanchnic mesoderm) specification by Dpp-> causing expression of the transcription factor Tinman.
- Cardioblast differentiates under DMef2 into a cardiomyocyte.
- Expression of Pannier is upregulated creating a linear heart tube as the two endocardial tubes fuse together
- Linear heart tube further differentiates into the dorsa;l vessel.
5 Steps in vertebrate heart formation?
- Mesodermal precursor (splanchnic mesoderm)is specified as BMP induces the NKX2.5 transcription factor
- A cardioblast is formed, which under MEF2C, which causes muscle differentiation, differentiates into a cardiomyocyte
- Under GATA4 the cardiomyocyte (endocardial tubes) migrate ventrally and fuse into a linear heart tube.
- inv and iv expression causes the looping of the heart tube.
- dHand and eHand causes the final multichambered heart formation
Tinman is initially expressed throughout the …… but the expression is limited until only expressed in the ….
mesoderm
Dorsal vessel heart tube.
Tinman vertebrate homologue is…?
NK-2 family, with Nkx2.5 specifically found in the cardiac crescent.
Nkx2.5 mutants?
Do form a heart (compensatory mechanism?) but the looping is often defected.
(human congenital heart malformations
What induces Nkx2.5 expression?
BMP
How can it be proven experimentally that BMP induces Nkx2.5 expression?
Electroporate BMP genes ectopically in Chick, or bead soaked in BMP2 anterior to the primitive streak.
Nkx2.5 induced in the paraxial mesoderm,
Why would ectopic expression of BMP to induce Nkx2.5 only work anterior to the primitive streak?
The tissue posterior isnt permissive to the signal, and posteriorly BMP inhibitors are secreted from the posterior notocord. Also anterior neural plate secretes Wnt inhibitors (dkk and Ceberus) which stops the Wnt inhibiting the BMP like in the posterior.
What do the angiogenic cell clusters form?
Angiogenic cell clusters go on to form bilateral endocardial tubes and dorsal aortas with epimyocardial cells around
What happens to the bilateral endocardial tubes?
As they migrate medially epimyocardium surrounds them. They then migrate ventrally below the foregut, and fuse into a single endocardium, surrounded by epimyocardium.
Tinman induces..? required?
DMef2 (drosophila Mef2)
Is required, as no tinman no Mef2
Endocardium heart precursors to?
Endocardium contains precursors of endothelial lining of heart and cushion cells that form valves.
Myocardium precursors to?
Myocardium contains myocytes of atria and ventricles, and Purkinje fibers
Mef2 structure?
Mef2 proteins contain both MADS-box and Mef2 DNA-binding domains. DNA binding causes the dimerisation
Function of dMef2 in the heart? mutation?
It causes muscle differentiation, so a mutation causes the loss of differentiation,
What is the earliest gene expressed int he vertebrate heart? Knockout for?
Mef2c
-/- no heart looping, no right ventricle, upregulation of Mef2B as compensation.
GATA transcription factor structure? Function?
Zinc finger domain.
GATA1-3 role in hematopoiesis
GATA 4-6- Cardiogenesis (GATA 4 earliest expressed)
GATA4 -/- ?
Fail to form the heart tube, as the endocardial tubes dont fuse. PLays a role in driving ventral migration CARDIA BIFIDA
BMP is a what?
Growth factor. Bone morphogenetic protein.
Shh is what?
a morphogen.
The neural tube has two concentrations of what?
BMP Dorsal (roofplate and ectoderm), and Shh ventrally (floorplate and axial mesoderm). This is converted into GliR dorsally and GliA ventrally.
What transcription factors are upregulated in the roofplate of the neural tube?
Pax3,6 and especially important Pax7 and Lim1 which lead to formation of Dorsal progenitors fate of dorsal cells.
The most anterior endoderm of the axial mesoderm will form?
Pharyngeal
What was previously thought and now been disputed about BMP in the neural tube?
Previously thought the BMP acted as a morphogen but now evidence suggests that many different types of BP’s diffuse varying differences giving different transcriptional profiles.
Shh ventrally in the neural tube how?
It is made in the notocord and floor plate and secreted in a gradient away MORPHOGEN. -> GliA
How does the axial mesoderm migrate?
Covergent extension following a fibronectin rich pathway that binds to integrins expressed by organiser cells.
The Ventral neural tube expresses what?
GliR represses Nkx 6.2, NKX2.2 and Nkx 6.1, but ventrally this is converted to GliA, so these are disinhibited immediately dorsal to the floorplate
FoxA2 is expressed in the floorpalte.
What transcription factors does the early eye field express?
RX, srx3, Pax6.
What splits the eye field in two? Induced by?
A Shh expressing flooplate like structure, which is induced by Shh from the precordal mesoderm.
What does the Shh expressing floorplate cause to happen in the embryo?
Division of the eyefield in two. The Shh supresses Pax6, RX expression in the centre of the embryo eyefield.
Mutation of Shh?
Cyclopia or holoprosencephaly- failure for the ventral tube to close
Where is the eyefield set up?
In Anterior neural plate
How do the eyes form in vertebrae?
- Eyefield grows sideways and contacts surface ectoderm laterally forming a T like shape with two optic vesicles from the neural ectoderm diencephalon.
- Optic vesicle induces the lens placode in the surface ecoderm overlaying.
- Ectoderm thickens (e.g. optic placode) and the membrane starts to invaginate in.
- The optic vesicle invaginates around the optic placode which is also invaginating inwards.
- Eyefield forms a double layered cup, with the inner forming the neural retina, and outer the pigmented epithelium.
- Lens vesicle dettatches from the surface ectoderm as it invaginates in making a tube with the cornea over.
AT what stage does the evagination of the neural tube eithelium elongate out into the optic vesicles?
5 vesicle stage- e.g telencephalon, diencephalon, mesencephalon, metencephalon, myelincephalon
22 days.
How are the optic vesicles connected to the diencephalon?
By the optic stalk which will become the optic nerve.
Where do the structures of the vertebrate eye originate from?
Neural ectoderm/ Neuroepithelium: Retina and Retinal pigment epithelium
Surface ectoderm: lens, Cornea
Migrating cells: Sclera, A. chamber (fluid filled space between the iris and cornea)
How is the lens placode induced?
As optic vesicle grows out it contacts the surface ectoderm and induces thickening.into the optic placode.
Chain of events of induction in the vertebrate eye formation?
Neural to optic vesicle-> epidermis to lens placode/optic-> induces involution and invagination of optic vesicle into 2 layer cup -> signals to lens placode to pinch off into lens and diffentiate -> Optic vesicle becomes neural and pigmented retina -> lens vesicle induces overlaying ecto to become the cornea.
Characteristics of the lens ealry?
Pinches off to form a hollow ball of cells that is stem-like, capable of self renewal anteriorly or fibre-like cells that elongate from the posterior.
They make lots of transparent crystallin and get rid of their nucleus
2 layers of the optic vesicle form?
outer: Retinal pigment epitheliium- produce melanin give colour
inner: neural epithelium, stem like pop, ganglion cells, bipolar, photoreceptors etc
What does the optic stalk give rise to?
astrocyte cells, bt turns into the optic nerve.
Ectopic activation of PAX6?
Necessary for eyeformation, can cause ectopic eye growth
Evidence that PAX6 is highly conserved?
Insert PAX6 from mouse in Drosophila and normal drosophila eye forms.
PAX6 throughtout eye formation?
Early: in eyefield
Maintained in forming retina and play a role in stem cell maintainance and differentiation
Structure of the neural tube early?
1 cell wide neuroepithelium, when these proliferate they migrate away from the ventricle.
Proliferation of neural tube neuroepithelium
1 cell wide initially. Initially these are symmetrical division, then radial glial like. Asymmetric divison, the neuron uses the radial glial cell to migrate away from the ventricle.
Formation of the neurons in the vertebrate eye?
From 5 weeks the outer neuroblastic layer signals to the inner neuroblastic layer to form at 6-7 , then to innermost at 7-8weeks.
Outer neuroblastic- rods and cones
Inner- bipolar
Innermost- ganglion
with synapses between
How do we know that neuroblasts are stem/ progenitors?
Virus containing a functional Bgalactosidase gene injected intothe back of a newborn rat to infect some of the retinal precursor cells.
After a month-6weeks, the eye is removed and retina stained for B-galactosidase and found.
Which Transcription factors cause different eye neuron fates?
bHLH TF’s:
Chx10 promotes bipolar cells( inner neuroblastic layer), whereas Prox1 is involved in horizontal cell fates.
Diseases of the eye link to age?
Age-related macula degeneration.
Neuones in the eye can degenerate and stem cel numbers decline over age
NEw research for macula degeneration?
transplant stem cells into patients and see if regenerate.
optic cup inner part contains?
A stem- cell like population, which can either self renew or differentiate into ganglion, interneurons, photoreceptors etc
Cell movements to produce the vertebrate eye?
Changes in cell fate: neural to optic vesicle
Invagination- of the optic cup, cells need to change shape to go from a columnar rectangular shape to a triangular.
Involution- optic vesicle to optic cup, growing larger than area to take up.
Pinching off- lens vesicle from epithelium placode. ETM
Migration- lens moving inwards
Branching of the kidney?
ureter to renal pelvis, to major calyces to minor- nephrons
Increasing surface area
What are the two separate structures and orgins of these that go on to form a nephron to the collecting duct?
Nephric duct- bud
Nephric cord- mesenchyme
From what do the kidneys form?
Intermediate mesoderm running bilaterally down the posterior of the body
What are the 3 places that the kidneys try to form called?
Pronephros-24days near heart
Mesenephros-25days midway down
Metanephros-26 days
Formation of the kidneys nephrons?
- Intermediate mesoderm undergo a MET to form bilateral nephric Ducts (Anterior) and cord (posteriorly)
- The duct forms a lumen and expresses PAX2.
- The duct Tries to bud at 24days (pronephros), 25days (mesenephros) and 26 days (metanephros) =uroteric bud.
- A uroteric bud grows towards and induces the nephrogenic mesenchyme to condense (was cord earlier)
- Mesenchymal to epithelial transition to renal epithelium then renal vesicle, then proliferates into S-Shaped body towards to uroteric bud (collecting duct)
- Defined apoptosis and enables the two to fuse, as the S-shaped body elongates into a long tube (by proliferation and differentiation)
- Local signals for angiogenesis via chemoattractants and angiogenic factors, so capillaries form around, making the glomerulus
- The nephron then folds to make the final PCT, loop of henle DCT and BC
Number of nephrons in a kidney? How?
10^6.
Branching morphogenesis- uroteric bud braches off.
e.g. week 6: 16 branches, week 7- minor calyces formed, week 32: 10^6
Steps of Branching morphogenesis in the kidney?
- Initially proliferation and outgrowth of the uroteric bud ‘tip cells’ under GDNF (to Ret RTK) from the metanephric mesenchyme.
- Arrest in proliferation of the tip cells creating a flatterned cleft
- Continuing growth of lower down cells which create T shape which keeps extending laterally.
- This makes new tip cells of two separate buds, which can either bud again or join to nephrons.
Experimental evidence for kidney braching morphogenesis?
Transgenic animals e.g. GFP transgene to HOXB7 can visuallise the uroteric bud and can see branching.
What is expressed in nephron stem cells?
Six2, which regualtes stem cells in self-renewing state.
Overexpression of Six2? Loss?
Gain: Prevents differentiation in kidney stem cells
Loss: depletes stem cell pool
Why do the Pronephros and Mesonephros fail?
Pronephros- apoptosis and broken down
Mesonephros- some blood vessels form near etc but differentiate into other strucutres.
The lung is derived from how many tissues?
Endoderm: epithelial lining of the trachea, laynx, bronchi
Mesoderm: cartilage, muscle and connective tissue
The lungs from from a bud off what?
Respiritory diverticulum off the ventral foregut
Very briefly how do the lungs form?
Respiritory diverticulum buds off the ventral foregut.
This then forms the trachea and inferiorly undergoes branching morphogenesis to form the lung buds
When in embryogenesis in humans do the lung buds form?
4th week
What induces the growth of the lung buds?
outgrowth from the foregut endoderm induced by adjacent mesodermal mesenchymal cells
What surrounds the lung buds? This becomes?
A sack of mesoderm, inner will become the visceral pleura and the outer the parietal.
Branching times of the lungs in humans?
day 41-44: first two bronchi
week 10: miniature but all formed, got all 5 lobes
6months: 17 branching events
and 6 more post natally until 23 total 2^23 =8million
What causes formation of the respiritory diverticulum?
- Overlaying mesoderm secretes FGF10 which the endothelial epithelial cells respond to bud towards.
- In the tip cells high FGF induces signalling cascade, resulting in MAP Kinases.
- Secondary genes are turned on such as Shh, BMP4, Sprouty and genes encoding signals, making this now a signalling centre.
- BMP4 levels are highest in the leading tip, and these act autonomously to inhibit thier proliferation, causing the flatterning of the bud.
- At the same time Shh diffuses to the mesenchyme and inhibits FGF10 expression in the mesenchyme nearest to the tip.
- Sprouty limits the action of FGF signalling so there is only branching at the tip.
- Where there is still high levels of FGF10 either side, new tips are made either side causing the T shaped branching.
How do the timings work for branching morphogenesis?
There is a lag between the tip cells proliferating under FGF10 (induces expression of cyclins) and it inducing expression of the genes Shh and Sprouty to inhibit it.
Negative feedback loop
How does FGF10 induce proliferation?
induces the expression of cyclins which promote the cell cycle
Wnt sinalling in braching morphogenesis of the lungs?
Canonical Wnt signalling reinforces FGFR2B (FGF10 R) expression in the epithelium, whereas non-canonical WNT5 signalling inhibits FGF10 expression.
BMP4 induce or restrict branching?
BMP4 signalling by the tip epithelium restrict FGF signal transduction and branching, whereas mesenchymal BMP4 enhances local branching
3 periods in the alevolar formation?
Canalicular period- 16-26weeks
Terminal sac period- 24 weeks
Alveolar period- late fetal to childhood
3 periods events in the alveolar formation
FGF acts as angiogenesis factor, attracting endothelilal blood cells to form the capillary network. Surfactant producing cells added. also.
At birth have only …. of alveolar numbers
1/6
If knock what out which gene, braching morphogenesis will be affected?
Rac1
Conditional K/O- baobab duct phenotype with massively enlarged ducts.
What is Rac1?
Ras-related GTPase that regulates many cell movements and differentiation etc.
Rac1 experiments now?
Found raised levels linked to carcinomas, formation of lamellae as a result of which can use to migrate and metastasis-ise. Therapeutic drug target?
Experiment showing mesenchyme is necessary for branching?
If remove the mesenchyme from lung tissue in vitro no branching happens here due to a lack of FGF10.
Branching morphogenesis and cancer?
Key places for cancer where there is branching e.g. lungs, mammary glands, pancreas, prostate etc
What are primordial germ cells?
set of undifferentiated cells set aside in the embryo that give rise to the reproductive gametes enabling the next generation
Location of the Primordial germ cells?
Primordial germ cells are determined in a specific location in extra-embryonic structures posteriorly but later migrate to the gonads.
Requirements of a germ cell?
- totipotent
- capable of meiosis
Example of Primordial germ cells?
P lineage cells in the c elegans-P granules in. posteriorly accumulate.
How do the P granules keep the P cells totipotent?
- Block transcription by binding to the P-cell DNA e.g. epigenetic (no proteins no differentiation)
- Block translation in the cytoplasm
- Promote a stem cell fate- cause cells to undergo meiosis
Where are the PGC’s found in the embryo?
Vertebrates posterior between the epiblast and hypoblast, in the extra-embyonic tissue where they are protected from differentiation signals.
How do the PGCs migrate in the embryo?
Passively travel in the endoderm moving up the gut (as forms by convergent extension) until chemoattractive/repulsive signals cause movement into the gonadal niche laterally along with the gonad precursor cells.
Stem cell niche?
Microenvironment that protects the stem cells in an undifferentiated state.
Drosophila location of the PGS?
in ovaries the cells attatch to the stromal cap, whereas in testes the hub cells.
What if PGS fail to move out of the gut or dont stay undifferentiated?
Teratoma forms- chaotic development of lots of different cell types, can be coming out of bottom or mouth
germ cell migration in mice stem cells are kept undifferentiated how?
In the travelling stem cell niche, with support cells.
Secretes stem cell factor (SCF)
How do the PGC’s migrate out of the gut?
Following a fibronectin trail with chemoattractants (Sdf-1) required.
Embryonic stem cells are taken from where?
Inner cell mass of an embryo(of blastocyst 50-100cells big), then cultured in a lab under conditions to maintain stem cell-ness
Difference between a stem cell and a progenitor?
Progenitors are not totipotent, rather multi as they have a limited number of cell types can become e.g. satellite only muscle.
Also, progenitors have a limited number of times it can self renew, whereas stem cell endless
For every …. stem cells that differentiates how many self renew?
4, 1
so 1/5 self renew
Either make two stem cells when divide or two specialised cells now think- so 1/5 divisions make two stem cells.
use of stem cells throughout life?
Replaces damage/dead cells
or adds cells e.g organ growth
or generate cells needed at specific time in development in life e.g. puberty
Location of stem cells in body?
Bone marrow,gut, skin also brain, liver , muscles
Example of cells that can and cant undergo mitosis?
Can’t: RBC’s, skin, gut lining (nearly all differentiated cells)
Can: stem cells, T-cells, liver cells (rare- stem cells therefore v important for repair, replacement etc, ften from progenitors)
How many new blood cells are needed every day? From where?
100,000 million.
From hematopoietic stem cells in the bone marrow (make blood and immune cells)
cellular homeostasis?
The constant repairing of old or damaged cells,or addition of new cells as needed
Mesenchymal stem cells can form? Found?
Bone, cartilage, fat, muscle
Found in bone marrow, fat, blood
Epithelial stem cells can form? Found?
Skin, gut, other linings
60% of differentiated cells in the body
Found in the bulge region of hair follicles
Neural stem cells can form?
Neurons, retinal, glial
How do stem cells know when to differentiate?
Progenitors can feedback to stem cells to signal about how many progenitors there are, and if more are needed etc (v vascularised so get info)
Change in microenvironment in niche causes the differentiation.
Hematopoietic stem cells developed where? Types?
In bone marrow from v early in development
Myeloid (Monocytes, macrophages, neutrophils)
Lymphoid (T cells, B cells, NK cells)
Where are neural progenitors found?
Subventricular zone lining the lateral ventricles
Subgranular zone of hippocampus
Hypothalamus, lining the 3rd ventricle
Neural S.C
Decisions stem cells have to make? E.g. Hematopoietic stem cell niche learnt from
- stem cells in a cycle between quiencent and active, first decision is whether to enter cell cycle to be active. Adjacent cells in the niche provide factors to determine this.
- When divides, give rise to a differentiated daughter cell or self renew as a HSC.
Hormones and signals in the blood will determine this, so niches have endothelial capillary cells.
(e.g. Stem cell factor)
What is the model of HSCs moving as divide?
Quiescent hematopoietic stem cells (HSCs) are localizated in a zone called the endosteal zone close to osteoblastic lining cells, called the Osteoblastic Niche. When activated (by signals coming from eg endothelial capillary cells/CAR cells), they divide to give rise to active HSCs that move to the central marrow region, in the Vascular Niche.
These can move into the sinusoid blood around body.
Stem cells if mouse pregnant?
Sees lots of oestrogen and makes lots more RBCs frp, progenitors of Hemapoietic stem cells as more are used up.
How are pluripotent stem cells maintained in the cell itself?
Nanos shuts down translation epigenetically, so cant differentiate.
How do progentitor cells move?
By epithelial to mesenchymal transition.
Some stem cell potential uses after disease?
Stoke, baldness, blindness, deafness, myocardial infarction, diabetes, cancers, crohns, arthiritis, SC injury, alzheimers, parkinsons, MS, bone marrow transplantation etc.
Example of stem cell use in disease?
2018, Royal hallamshire hospital for MS
From where is it possible to isolate stem cells in body? Where harder?
Bone marrow relatively easy, gut showing promise
Epithelia much harder
Where are the stem cells in the gut?
Crypt of the gut villi. The crypt is a tube of cells with stem-like cells at the bottom (distal) and as they move up they differentiate into Transient amplifying progenitor cells
Which signals crypt gut promote stemness in the gut crypt?
Crypt base columnar cells see high Wnt (from the neighbouring paneth cells) which induces stem-ness, along with Notch and Noggin.
Which signals cause differentiation of the stem cells in the gut crypt?
BMP, PTEN
Experimental process to separate crypt stem cells?
Genetically label by EGFP (enhanced GFP)
Use Fluorescent activated cell sorting (FACS) to separate the setm cells.
Culture.
A single stem cell can form a new vill crypt and go onto bud into an organoid
Drosophila cell divisions initially? Later?
Initially synchronous cell cycles symmetric only have S and M phases.
Later, after 14 cell cycles: asyncronous, G2 starts and nuclei migrate to the periphery
Early events in drosophila to create synctial blastoderm?
- Syncronous cleavages
- Asynchronous after 14 rounds
- Migration of nuclei to periphery
- Cellularisation as the membrane involutes.
Cell divisions controlled by?
Protein String, a phosphatase that activates cyclin dependent kinases
What inhibits String?
Tribble
Evidence for Tribble?
Mesoderm is one of the first domains to express string (activates the cdks) but one of the last to divide, so although present the cells don’t divide
Meso inducing genes induce Tribble to inhibiit String.
What causes differences between animals e.g. of neck length? (3 methods)
Same strucutres but different growth times, e.g. giraffe’s will grow for longer than humans.
More proliferation is the main cause,
also individual cells may grow larger e.g. cardiac hypertrophy
or Accretion (gradual accumulation of layers of bone)
How can tell if growth of a structure is intrinsically controlled or not?
Graft a limb which is fated to be very large in size, onto another animal which is fated to have smaller limbs, becomes the original large size, showing that it is controlled intrinsically. Not all are.
Examples of organs that are controlled intrinsically vs not?
If add additional thalamus, all organs maintain same size just have a lrger number, intrinsic control.
Whereas, if add another spleen both will only grow to haf the size, giving correct total tissue- show extrinsic control also.
Example of how it isnt just the rate of growth but also duration which causes organ/ structure size? (3)
Head initially v big in babies, but stops growing so comparitively shrinks.
Or puberty- both boys and girls initially grow at the same rate (grils bit earlier) but boys keep growing for longer so end up taller.
In Pygmies- this second growth spurt doesnt happen so remain small.
String is expressed maternally until cycle…?
14
After is zygotic String
Evidence that cells control tissue overal dimention compared to cell numbers?
Experiment with Ploidy (no. of chromosomes)
If haploid- half number of cells, but overall tissue size is the same, same if triploid- smaller cells as more.
What induces the expression of string?
Until 13th cycle: maternally supplied string is induced by the maternal genes -> induces the syncronous divisions
After 14th: Zygotic string induced by zyotic genes e.g. pair rule gap etc -> induces the asyncronous divisions
How are CDK’s activated?
By String phosphatase, then cyclins binding
Why does the mesoderm express Trebble?
To inhibit String, as the mesoderm needs to invaginate , creating the primitive streak, and proliferation will inibit this so until after, it is inhibited.
Why are active cells that are constantly being replaced related to cancer?
Because more chance of error as dividing, through replication etc e.g. epithelia
What is a teratoma?
Cancer cells able to give to give rise to all 3 germ layers. If tranplant these cells into a developing mouse embryo, act normally so are not perminantly altered
What mutations can cause cancer? examples of?
Loss of function of a tumour supressor gene
e.g. Ras, Raf, Myc
gain of function of a proto-oncogene going to an active oncogene.
e.g. Rb- tumour supressor- retinoblastoma
P53- tumour supressor gene-inhibits cell cycle
Patched- Hh- basil cell carcinoma
APC- Wnt- Adenomatous polyposis coli
Two pathways that control cell/organ size?
TOR Pathway- Regulates Cell size (e.g. Tour stops ur cells growing)
Hippo pathway- Regulates Organ size (e.g. a hippos organ is bigger than ours)
How does the Hippo Pathway work?
When it is inactive: The T.F Yki/Yap/Taz is in the nucleus stimulaitng growth and survival
When it is activated these are excluded from nucleus by Phosphorylating them. This inhibits growth.
Hippo pathway nuclear factor?
Yki- Drosophila
Yap/Ta- vertebrate
When would the hippo pathway likely be turned on?
When cells are v crowded, making lots of cell-cell contacts they tend to inibit growth.
If however cells feel stretched they turn it off- so proliferate more.
Wnt/BMP may block
What is Hippo?
It is a kinase, that phosphorylates Yki/Yap/Taz if active, keeping the nuclear factors out of the nucleus.
Hippo -/-?
Loss of control of growth restriction, tissue keeps growing e.g. wing
How is Drosophila adult size determined?
Determined by the size of the larvae.
Larval rate and duration of growth is influenced by insulin
if drosophila larva insulin deficient?
Small cells and fewer.
How does a drosophila larva grow?
It grows through Ecdysis -moults its shell, secretes another shell to grow into etc progressively. These intermoults are called instars and there are 3 instars in drosophila before pupation.
It grows through Ecdysis -moults its shell, secretes another shell to grow into etc progressively. These intermoults are called instars and there are 3 instars in drosophila before pupation.
What causes the molting in the drosophila?
Stretch receptors in the epidermis or cuticle control the molting.
- When the larva has grown too much the receptors will activate and send signals to the brain.
- Protothoracicotrophic hormone released from corpus alatum
- Acts on the Protothoracic gland to release Ecdyson
- The cuticle is released from the epidermis which starts making the new cuticle. Ecdyson causes molting and the old cuticle is broken down by molting enzymes.
Where are the adult structures in the larva?
In imaginal discs
Whereas development of the larva is …, the adult drosophila is more…?
intrinic
Extrinsically controlled e.g. CNS interprets the environment.
Hormones that control metamorphis of the drosophila?
Environmental signals, which the CNS interprets, and secretes PTTH if favourable.
This causes the release of Ecdyson.
If unfavourable Juvenile hormone is released- which prevents metamorphis.
Balance of these two hormones.
Hormones that control metamorphis in the frog tadpole?
Balance between Thyroxin and prolactin, causes metamorphis.
Controlled by environment-> hypothalamus causes the secretion of Corticotrophin releasing hormone->pituitary secretes Thyroid stimulating hormone-> thyroid releases thyroxin.
Positive feedback, if enough is reached then metamorphis is irreversible.
Effect of thyroxin in the tadpole?
Different effects on different tissues, growth in limbs but tail regeneration.
Hormones for Human growth?
Insulin like growth factors 1&2, IGF1 and IGF2 important for both embryo and adult growth
Growth Hormone (IGF’s may monitor) from the pituitary, .
GH stimulated by GH releasing hormone, but inhibited by Somatostatin from hypothalamus.
(GH negatively feeds back on itself)
What hormones affect IGF1 levels?
GH releasing hormone activates GH, whereas Somatostatin inhibits.
GH then activates Insulin like growth factor 1.
GH feeds back on itself to decrease GHRH levels and upregulate somatostatin levels.
Low birth weight in mammals associated with what disease?
Coronal heart disease CHD
Evidence that size of the progeny depends on the maternal environment?
If cross shetland with shire horse, and the mother grows in is a Shetland the birth weight will be small, whereas if a Shire, will be much higher. Both will grow to end up the same size eventuall however.
Evidence in humans of disease link to diet of mother?
Dutch famine WWII winter 1944-45, babies mid-late gestation compensatedbirth size but increased risk in obesity, diabetes, CHD later in life.
Cancer most common in what tissue? Why?
Epithelial tissue-85%
most active tissue continuously being replaced
Misregulation of signalling pathways and cancer?
Wnt- loss of APC- Adenomatous polyposis coli
Hh- basal cell carcinoma and Medulloblastoma
Nodal- melinoma formation
Notch-leukemia
EGF (epidermal GF)- breast cancers and lung
WHat is genome instability?
Frequency of mutations in cell replication etc- lead to cancer.
What actually is cancer on a cellular level?
uncontrolled proliferation without differentiation.
Two types of regeneration?
Morphallaxis: regeneration without growth- dedifferentiation and rearrangement of existing tissues
Epimorphis: New cells formed to replace
How does the hydra regenerate?
Morphallaxis: Cells grow continuously, constantly changing their positional value and repatterning after cells are lost at the budding region or basal disc.
POsitional values of the hydra?
1- head region
6-basal disc
(Budding region between 4 and 5)
Difference in gradients from 1 to 6 in positional values of the hydra?
1- Highest tendancy to become head as it has the highest resistance to the inhibitor, but has highest levels of inhibitor when a head is present.
6- Lowest tendancy, lowest resitance but low conc of inihibtior
Evidence experiment to show head inhibitor?
Take PV 1 tissue and transplant into another hydra- head inhibitor sufficiently strong that it doesnt become head.
However if remove the head, now two heads form, one from this grafted tissue and the existing PV 1 cell.
If transplant a PV 1 to 6 when no head in hydra what will happen?
Low inhibitor levels and high resistance to so another ehad will form near basal tip.
Difference in timings between a PV1 and PV5 to become head after remove head?
PV1- 6hrs
PV5- 30 hours
Longer for the inhibitor gradient to drop to sufficient levels to overcome, and lower resitance to the inibitor than PV1,
What produces the head inibitor signal in the hydra?
The head
What is the head inibitor signal in the hydra?
GSK3 B is inhibition in the head region, which causes an increase in nuclear B catenin causing the head fate.
Example of Epimorph regeneration?
Urodele amphibians
Can regenerate limbs, tail, jaw retina, lens (from iris)
In urodele which surprising tissue can still participate in regeneration?
Muscle, given multinucleate.
Revert back to mononucleate under Thrombin.
Regeneration of urodele limb depends on?
- Epidermal migration after amputation
2. Cells below epithelium dedifferentiate- pluripotent Blastema.
How is regeneration different to differentiation in development?
Much larger scale so morphogens need to work at a much greater distance.
Signals required for dedifferentiation/ regeneration?
Thrombin, Msx1
and Phosphorylation needed of Rb to ihibit the inactivation of the cell cycle- so induces proliferation
What technique can help build a regeneration fate map?
GFP reporter, show muscle will form muscle etc
If cut limb regeneration will happen ….. to the wound? Experiemnt to show?
Distal
If distal limb of a newt is amputated and the stump
Molecular basis of blastemas proximal vs distal?
Differential adhesion properties.
Proximal blastema will engulf distal, as distal cells stick together more (stronger adhesion) whereas proximal cells stick to distal.
Why do Distal cells have higher adhesion to each other than proximal cells?
Prod1 is expressed higher levels in proximal, induced by Retinoic acid which can proximalises a blastema via Prod1 upregulation (GPI Linked protein), or Rard2, Meis homeobox gene upregulation.
Importance of innervation to an amputated limb?
If denervated limb won’t regenerate, however if aneurogenic- never seen nerve cells before, can.
Why difference in regeneration if denervated vs aneurogenic?
nAG- expressed in the nerve sheet after cut normally to damage, and induces regeneration.
In development nAG is expressed in epidermis, and then as nerves develop it stops and is expressed in the nerves. However if aneurogenic, still persists in the epidermis, so can still drive regeneration.
=newt Antior Gradient- binds to Prod1 supporting outgrowth
How does regeneration work in insects e.g. cockroach?
Senses patterning discontinuities in positional vaues, and if there are missing ones it will regenerate those by looking at the patterning before and after to fill in the gaps, irrespective of the overall structure.
Regeneration cockroach experiments?
P1- proximal
P5- Distal
If graft together PV1 to PV5, will regenerate 2,3,4 to fill in the gap.
If graft other way round a PV4 to PV2 will then fill in gaps but keeping a continuum so adding 4,3,2, making it 1,2,3,4,4,3,2,2 etc. as well as the 3,4,5 on the end to finish on the most distal
Mammal regeneration?
Mice or young children, can regenerate finger tips if beyond nail bed.
Can regrow axons but not whole neurons in PNS.
can’t in CNS- Oligos inhibit with Nogo.
vertebrate heart regeneration?
Although cardiomyocytes are present they don’t divide.
instead maladaptive hypertrophy and scar tissue is formed.
Heart regeneration in the zebrafish?
msxB and C expressed, dependent on dedifferentiation of muscle cells, no progenitors or stem cells are used.
Endocardium and epicardium are involved.
Neuregulin may be a signal from the epicardium.
Competiton between scar and regeneration of the vertebrate heart?
Mps1 mutants show scarring, rather than regeneration.
Steps in the regneration of the heart of zebrafish?
- Clot forms where there is damage (prevents circulation coming to complete standstill)
- Epicardial activation (raldh2 mRNA throughout)
- 7 days peak in proliferation of cardimyocytes. Epicardium expands to cover the wound- expressing FGF-like Neurogenin which stimulates cardiomyocyte cell division.
- Vascularisation of regenerating tissue. New muscle expresses FGF, causes reformation of blood vessels.
Mammals heart regeneration?
Neonatal mice can regenerate.
Loss of this ability later, correlates with the loss of Erbb2 a neurogenin co-receptor.
Experiment mammals showing heart regeneration?
Transgenic mice made to have a dominant active form of Erbb2 and were able to induce cardiomyocyte proliferation with limited scar tissue.
Why is there now little use in embryonic stem cells in labs? Difference.
Induced pluripotent stem cells iPS
Previously taken out from the ICM and cultured=ESC’s, now can take an eunucleated egg and insert nucleus of a fibroblast for example.
Who and how were iSC’s found?
1962 Gurdon: Found that if add a fibroblast to an enucleated egg cell- it will now have diploid status and form a normal embryo. (dediffferentiation)
2006 Yamanaka: Estimated the 4 factors that induced pluripotency.
How was the cocktail of 4 TF that induces pluripotency found?
2006 Yamanaka:
Took a fibroblast of mouse, but instead of inserting into an enucleated egg cell injected these 4 TF’s.
This fibroblast was then inserted into a surrogte mother into a blastocyst with female hormones.
Born a normal embryo.
Dedifferentiate into pluripotent stem cells.
Advantages (4) of iPS?
- vastly renewable
- easily acessible
- individual specific so no immune reaction
- can differentiate into many cell types
Disadvantages of iPS?
- More complex e.g. overcoming senescence, epigenesis
- Safety, no FDA approval
- Use of harmful oncogenes when inserting and viral vectors- insertional mutagenesis?
- Low efficiency of insertion.
What can iPS also be used for?
Organoids
Culture them, well oxygenated and nutrients
Two opposing theories of aging and examples of each?
Wear and tear: Elephants die because they can no longer eat as their teeth degrade over time. C elegans decline in muscle function
Genetic programme: Suddenly die after laying their eggs
Disposible soma theory?
Natural selection keeps organism alive until can reproduce and care for young, after there is no natural selection against the animal.
Rate of living theory?
High metabolism, short life span- large animals live longer. (smaller have higher)
What could affect the rate of living theory?
Temperature: Flies 18degrees, 180days, 29degrees 40days- slow metabolism?
3 senescence factors?
Metabolism
Reactive oxygen species
DNA damage
What are reactive oxygen species?
By product of metabolism of oxygen, which can cause damage to cell structures ‘oxidative stress’ can be in response to stress factors too.
e.g. superoxidide radical
Link to aging not completely clear though
High/ low metabolism causes ROS?
High metabolism leads to the formation of ractive oxygen species.
Superoxide evidence against aging? (2)
Against: C elegans- treat with Paraquat or Juglone (compound created by ROS) caused lifespan increase.
If prevent superoxide radicals by adding another antioxidant, shorter life also
Superoxide evidence for aging? (2)
Glucose resitriction ends C elegans life by inducing mitochondrial respiration and increasing oxidative stress
Resistance to oxidative stress induced by ‘longevity genes’ increases life.
Progeria syndromes mutations?
Aging to do with DNA?
e.g
Werner: RecQ DNA helicase mutation, chromosome replication problems
Ehlers Danos- Bgalactosyltransferase mutation
NAD depletion theory?
When DNA becomes damaged upregulate PARP enzyme, which uses NAD to mark damaged cells, but needed for redox reactions e.g. glycolysis
Theories for Aging causes?
NAD depletion
Senescent cell removal
Superoxide radicals
Factors increasing life span? (3)
Dietry restriction
environmental stresses
Signals from gonads
Dietry restriction evidence for lifespan?
If restrict calories- life 40% longer- lower metabolism
What is Hormesis?
A disproportionate response to small stressors, may activate protective mechanisms in which then conveys an advantage
Forward genetics for aging? Found?
Mutations- see which genes affect lifespan- difficult as could just a be a mutation which causes early death, so longlife mutants more sucessful.
Found IGF pathway, TOR, Sirtuins
What is the Dauer stage?
If c elegans crowded or in adverse conditions can enter this stage, can survive 60days under insulin/IGF1 control (insulin growth factor 1)
Insulin for pro-life?
For life: Dauer stage, survives under insulin IGF1 control.
Evidence Insulin causes aging?
IGF1 to IGF R (DAF2) which inhibits DAF16 (FOXO). FOXO regulates genes that respond increase resistance to stressors
Mutations in Insulin/IGF1 pathway double lifespan (flies are in reproductive diapause) linked to resistance to oxidative stress
Female mice with a mutation in IGF1R (IGF1&2 receptor) live 33% longer
Humans and insulin and life?
FOXO1 and 3A, IGF1 varients have been linked with longevity
Evidence of insulin causing aging via GH?
Dietry restriction decreases insulin signalling in mice, and when mice are made to have a GH receptor mutant (which downregulates IGF) dietry restriction makes no impact as already downregulated; shows defintiely IG pathway.
What does FOXO do?
ANITI AGING BY: Inhibits IGF (IGF inhibits FOXO also) Increases anti-oxidant genes Decreases Metabolic genes Increases chaperones Increases antibacterial genes
TOR pathway promotes ageing/life?
When TOR is active (when lots of nutrients and protein synthesis no stress) PRO-LIFE
Stress and TOR pathway?
Stress-> TSC1/2 -I TOR so activates 4E-BP1 (blocks S6 salvage pathway)
