Vertebrate Development

Question 1

Describe the early stages of mouse embryonic development.

Answer 1

Fertilised egg divides multiple times before compacting into a tight sphere
Blastocyst consists of Inner Cell Mass (ICM), Blastocoel (space) and Trophoectoderm (TE) surrounding it
ICM divide within blastocyst and start to pattern before rolling backwards
TE gives rise to placental tissue
ICM differentiation into primitive endoderm and epiblast (most of embryo)
Implantation

Question 2

Describe the early stages of chick embryonnic development.

Answer 2

3 germ layers develop via gastrulation
By end of day 2, neural tube has formed and expanded in head with neurones beginning to differentiate

Question 3

How does the eye develop?

Answer 3

Retina forms from forebrain (CNS) but lens is from surface ectoderm
Eye develops from bulging NS
Neuronal ectoderm induces surface ectoderm to thicken and form lens, neuronal ectoderm forms cup - double cup forms retina

Question 4

How does the ear develop?

Answer 4

Placodes (thickening of ectoderm) fold to form cup
Some cells in cup form neurones in ear but some neurones/accessory cells migrate out

Question 5

Describe human embryological development before the primitive streak.

Answer 5

Divides through fallopian tube
Undergoes compaction and embeds
Outer cells break away and form disc
Primitive streak forms from posterior to anterior
- Streak of cells migrating to middle,down,out
- Lowest layer becomes endoderm, middle is mesoderm and top is ectoderm
- Endoderm rolls up and seals, becoming digestive tube
- Ectoderm rolls and forms outside which becomes skin, brain and spinal cord

Question 6

Describe the formation of neural tissue in vertebrates.

Answer 6

Midline ectoderm thickens and forms neural plate (NP) , starting at the anterior
NP is flanked by neural crest (NC)
NP folds at the centre
Neural folds become Neural Groove
Elevation and Convergance to form NT
Dorsal part breaks away from epidermis and becomes internal
NC cells form at point skin heals over

Question 7

Describe the model for induction of neural fate.

Answer 7

All ectoderm is non-neural until a signal makes it competent for neural development. Fibroblast Growth Factor (FGF) from dorsal mesoderm induces the formation of neural ectoderm in the dorsal midline, leading to the expression of Sox3. Bone Morphogenetic Protein (BMP) inhibits competent ectoderm cells from becoming neural by suppressing Sox2 expression. Antagonists (e.g. Noggin, Chordin and Follistatin) produced by the organiser node in the dorsal mesoderm inhibit this so double inhibition means the competent ectoderm adopts a neural fate.

Question 8

Describe what was found in the Spemann and Mangold experiment (1920s).

Answer 8

Mesoderm from dorsal side (organiser) can induce nearby cells to become nervous system and other dorsal fates.
Only neural induction requires the organiser.
Others result from it and need subsequent signalling
Organiser is only region of embryo with fate determined at an early stage

Question 9

Describe the experimental analysis of neural induction using isolated animal caps.

Answer 9

Isolated animal cap was cultured under 3 conditions.
1. Intact - cells became epidermal
2. Dissociated - Cells became neural tissue
3. +BMP4 and dissociate - cells became epidermal
Suggestion was BMP4 naturally tries to make cells epidermal but dissociating the cells allows them to escape this inhibition and adopt neural fate

Question 10

Describe the BMP Signalling Pathway.

Answer 10

BMP ligand dimer binds to receptor and triggers Smad phosphorylation
pSmad binds to another type of smad to form a transcriptional regulatory complex which enters nucleus and activates/represses target gene by interacting with other transcriptional regulators.

Chordin/Noggin bind to BMP ligands outside of the cell and prevent it binding with the receptor and starting the path
FGF causes further Smad phosphorylation so it can’t enter the nucleus

Question 11

Which experiments are Zebrafish and Chicks good for?

Answer 11

Zebrafish:
- They’re transparent, can easily get hundreds, and anything injected doesn’t dilute
- Treat with signalling molecules - activate or block any pathway without altering 2nd or 3rd pathway
- Early stages have little channels between cells so anything injected go into whole embryo

Chick:
- Good for transplantation of responding or signalling molecules - zebrafish too small

Question 12

Using the neural tube as an example, what is a pseudostratified epithelium of proliferating cells?

Answer 12

Within the vertical cells, the nucleus and most of the cytoplasm shuttle back and forth during different stages of the cell cycle. As such, it looks like it has layers.
They pull the connection away from the outer layer, freeing them up to make plane of cell division then re-establish connection
As they begin to differentiate, they move outwards from the epithelium, so proliferating cells are always lining the lumen

Question 13

Describe the differences in migration in the spinal cord and cortex.

Answer 13

Spinal cord development in “outside-in” gradient of development so oldest cells are on the outside.
Cortex is “inside-out” gradient of development so oldest cells are on the inside. These cells use radial astroglial cells to climb to the outside after they divide

Question 14

What controls neuronal differentiation?

Answer 14

More Posterior - FGF from presomitic mesoderm inhibit Pax6 which is necessary for neural differentiation.
More Anterior - RA from somitic mesoderm (catalysed by Raldh2) promoted differentiation
FGF and RA show mutual inhibition

Question 15

Describe the neurogenic cascade.

Answer 15

Neurogenin is first to be switched on so produces Neurogenin protein.
Protein recognises DNA sequence of promoter of a gene in the NeuroD family.
NeuroD switched on and produces the NeuroD protein which activates neuronal differentiation

Question 16

Describe Inhibition by Notch signalling in Vertebrates and how it affects the neurogenic cascade.

Answer 16

If a cell begins to differentiate in the NT, it will express delta on its surface.
Delta binds to notch receptor on other cell
Enzymes cleave notch, releasing ICD (intracellular domain) which migrates to nucleus and acts as a TF
Hess family of TFs activated and produce proteins which are transcriptional repressors
Blocks expression of Neurogenin, stopping neurogenic cascade so cannot differentiate.

Small proportion of cells can differentiate at any time.
Random process which cell undergoes differentiation but that dictates cells around it

Question 17

Describe the evidence in gain of function experiments showing sufficiency of HES1 and Delta.

Answer 17

Over-express HES1:
- Take virus carrying hess gene and inject into mouse brain where virus will infect dividing cells
- Put in lumen, can only effect innermost layer which are dividing cells (inside NS)
- Control virus (marker gene e.g. Lacz beta galactosidase blue) cells will do pretty much anything in NS
Viruses overexpressing Hess1, cells don’t go anywehere and continue dividing, die and don’t migrate

Over-express Delta:
- Chick retina
- Cells infected with a virus expressing delta and GFP inhibit differentiation

Question 18

Describe the evidence in loss of function experiments showing necessity of HES1 and Delta.

Answer 18

Over-express dominant-negative Delta:
- Lacks ICD
- Putting in disrupted protein blocks signalling in embryo that it would normally do
- Truncated version of delta will interfere with Notch but won’t activate it
- Shows ability to activate notch pathway is absolutely essential to stopping cells from all just immediately differentiating

Knock out HES1 in forebrain
- Other hess in SC but only HES1 in forebrain
- Forebrain no longer has ability for notch delta signalling to be activated and therefore cells cannot be blocked from differentiating
- Forebrain has no growth capability, rest of head breaks apart

Question 19

What is meant by birth dating?

Answer 19

Final mitosis = birth of neuron
Can see if cells born at a specific time have same or different fates:
- Label cells making DNA (add labelled nucleotide that gets incorporated)
- 3H-Thy or BrdU (thymidine analogues)
- Artificial, can get antibodies, stain tissues and look for cells accumulating BrdU

Pulse chase allows labelling over a short period of time but stain gets weaker with each cell division.
In cells that differentiate as soon as you label them, stain will never dilute and will remain strongly labelled until the animal dies

Can transplant cells from E29 to P1
If transplanted before final DNA synth, influenced and react to new environment, migrate to layer 2/3
If finished DNA synth, will still migrate to layer 6
Hypothesis is that migrating cells pause before getting to target layer and communicate with next cells to find out where they are in sequence of events.

Question 20

Describe fate in the dorsoventral axis and how it arises.

Answer 20

DV patterning requires the notochord as it induces floor plate and MNs -FP further induces
V - Shh is released from NC, forms gradient
Nk6.1 requires less Shh than Nkx2.2 so is expressed more dorsally when low conc
No cell will express both genes on either side of the threshold as they inhibit each other

D -BMP family released from RP, forms gradient

Question 21

Describe Yamada’s (1991) experiment that should notochord induced ventralisation of NT, FP and Mns.

Answer 21

Took notochord out of chick embryo in very early stage
Cut next to developing neural tube and transplanted original notochord
Found 2nd FP and MN around where they transplanted notochord - able to override any other signals telling that section they were D
Then removed notochord from chick embryo and saw no ventral features at all

Question 22

Describe the experimental evidence behind the patterning of the dorsoventral axis.

Answer 22

In ovo electroporation of CNS in chick embryo - used to test that TFs repress each other at the border between the two
Hit with square pulses at low voltage several times to make pores in embryo
DNA travels towards positive electrode
One side takes up DNA, other acts as control

Overexpressing Dbx2 inhibits expression of Nkx6.1 but not Pax6 and 7 (already overlap)

Question 23

Describe fate in the anterioposterior (rostrocaudal) axis and how it arises.

Answer 23

Neural tube squishes just behind where brain should be, liquid goes in, brain area swells
Like drosophila, Hox genes encode TFs which determine identity of cells in each segment - wider range of Hox genes, more complex development
Not a lot of variation in spinal cord
Rhombencephalon has segments called rhombomeres - Hox family TFs tend to have sharp boundaries matching segments except between 3/4 and 5/6 (basically the same)
RA synthesised from Vitamin A by RALDH enzymes in mesoderm - RA degraded anteriorly by Cyp26al

Question 24

Describe Neural Crest Formation

Answer 24

Forms from dorsal edge of neural tube around the time of fusion.
Pax7 is early marker but NC is specified prior to Pax7 expression (not committed yet)
Progenitors of neural plate, placodes and neural crest defined early - Epidermis expresses BMPs and Neural Ectoderm plus BMP4/7 also produces crest
Later BMP alone can induce NC formation

Question 25

Describe Neural Crest Migration

Answer 25

Slug causes loss of N-cadherin in dorsal side allowing NC cells to delaminate and escape.
Contact inhibition from surface ectoderm cells which have now fused into one layer.
Some migrate through somites (form DRG) or over outer surface (just under ectoderm to form skin)
Repulsive interactions mediated by Eph-ephrin signalling keep NC cells to anterior portion of each segment

Question 26

Describe the neural crest rotation experiments in chick embryos.

Answer 26

Keynes and Stern
1. Rotate entire neural tube - NC cells still migrate through portion that is now anterior

2. Just rotate somites - NC cells migrate through portion that was anterior

Question 27

What defines neural crest cell fate?

Answer 27

Appears not specified prior to migration (except bone and muscle only made from cranial crest - only express Sox8 and 9)
Only cells arriving in appropriate environment develop further (e.g. Cells which have melanocyte activity will make melanocytes when they get to the skin, others die)

Where placodes are involved, the series of neural and later neurogenic and neuronal genes that control their development is largely the same as those that control CNS development

Question 28

Describe the structure of the growth cone.

Answer 28

Cytoskeleton microtubules with + end pointed towards the tip
Highway to ferry components to tip but Some machinery at end of axon - take too long to move everything from cell body to GC
Meshwork of actin filament directly under PM provides shape and stability

Question 29

Describe what is meant by the molecular clutch.

Answer 29

If the cytoskeleton isn’t tethered by cell surface molecules through the substrate, adding more actin subunits to growing end means molecules in whole structure are pushed back (retrograde throw). If molecular clutch is engaged, cytoskeleton is fixed and adding more actin pushes membrane further out (protrusion)
Under-adhesion = can’t extend
Over-adhesion = can’t push through

Question 30

How do attractive and repulsive signals affect microtubule assembly and disassembly?

Answer 30

Different neurites have different extra/intra cellular machinery so react differently to the same external cues

Rac and Rho are signal proteins in the cytoplasm that response to cell surface signals.
Attractive cues activate Rac and stimulate axon cytoskeleton growth
Repulsive cues activate Rho and inhibit growth by collapsing skeleton.
Rac and Rho inhibit each other.
Switching on/off Rac and Rho is by determining whether/not they’re associated with GTP/GDP

Question 31

How do pioneer axons grow to their target?

Answer 31

Some pattern can be intrinsic e.g. hippocampal neuron dendrite tree - cell acquires identity of being particular neurone and differentiates in particular way

Motor neurones - Initial guidance is non-specific and tend to go where growth is easy (along bv and glial tracts); closer to target, cues get more specific and complex

Question 32

What are some examples of cell surface guidance cues?

Answer 32

Positive: Cell Adhesion Molecules (CAMS):
- Calcium Independent Cadherins e.g. N-cadherin, homophilic (stick to cells expressing same cadherin) or heterophilic (stick to cell with different one on surface)
- Calcium Dependent Integrins

Negative: Semaphorin 1

Question 33

What are some examples of secreted specific guidance cues?

Answer 33

Positive: Netrin 1
Negative: Netrins, Semaphorins

Question 34

Describe how different cells have different responses to signals.

Answer 34

Motor neurones grow right next to FP and direct axon away from it but Commissural interneurons are attracted by FP (FP expresses netrin)

NT-3 repressive neurones find Semaphorin3 attractive but mechanoreceptor neurones don’t (collapse on side of tissue next to signal)

Question 35

Describe how different regions within the same cell can have different responses to signals.

Answer 35

Pyramidal cells follow signal to form shape with distinct axon and branching dendrites.
Gradient of semaphorin3A
Dendrites grow towards high conc (attracted) and axon grows towards low conc (repulsed)

Seems to be due to presence of cGMP
Stain before fully extending
Guanyl cyclase cap seen in cell body
High guanylyl cyclase means high cGMP on one side and makes structures growing from that side attracted to SEMA-3A (dendrites)
Increase cGMP, able to cause switch from repulsion to attraction in other systems

Question 36

Describe how cells can alter their responses to signals.

Answer 36

Netrin from midline attract comm axons towards the midline via receptor DCC.
Netrin KO mouse: comm axons due not cross FP but head for it (Attraction due to shh and repulsion by bmp)

Slit and its receptor robo on axons mediate repulsive properties of midline (in flies lacking robo, axons go round and round).

After crossing midline, axons join 1 out of 3 fasciclin 2 positive fascicles (F).
Expressing robo1 join F closes to midline
Expressing robo1and3 join intermediate F
Expressing robo1,2,3 join lateral F
Moro robo receptors, more sensitive to slit, further grows from slit secreting midline

Question 37

Describe the retinotectal map.

Answer 37

Phenonema by which light forms map on retina and is then connected to back of brain to make the same image

RGC grow along surface of retina to exit point, N-CAM promotes migration and fasciculation to form optic nerve
Netrin1 at nerve exit, made repulsive in combination with extracellular laminin
L1 adhesion and laminin promote growth along tracts to optic tectum, relayed to visual cortex

In retina, gradient of Ephrin A5 signals
Receiving tissue, same gradient of Eph receptor
These dictate target finding and synapsing
Temporal repulsed by high conc, stop early (A)
Nasal not repulsed, travel further (P)

Question 38

Describe the formation of NMJs in vertebrates.

Answer 38

Axonal GC makes contact with target muscle, postsynaptic receptors cluster at contact site.
GC differentiates into presynaptic terminal, each of which contains active zone residing opposite cluster of AChRs on postsyn surface (originally AChRs spaced evenly on muscle before Agrin triggers clustering).
Later expression of genes encoding AChR subunits upregulated near synapse and later downregulated away from synapses.
At same time, axon terminal accumulates synaptic vesicles and a basal lamina forms in synaptic cleft

At the end of embryogenesis, functional NMJs each with few synaptic contacts.
Further synaptic refinement in later dev. stages
Neural activity represses gene expression in non-synaptic areas leading to a reduction in AChR in non-synaptic areas.

Question 39

What regulates AChR clustering?

Answer 39

Agrin acts on muscle specific tyrosine kinase (MuSK) which affects a protein called Rapsyn.
Rapsyn is associated with AChR and is thought to anchor them to the cytoskeleton

Muscles from mice lacking Agrin, MuSK or Rapsyn lack substantial clustering and nerve patterning also disrupted showing need for retrograde signalling.

Question 40

How do nerves use apoptosis to modify connections?

Answer 40

Lack of connection or too many connections for target OR incorrect target connected

Limb bud transplants in chick - remove one, fewer DRG and MNs - add limb bud, more DRG and MN (include more cell death in M/SN in tissue by removing target) - muscle required for survivial and rescuing of neurones

Question 41

What are the mechanisms for modifying connections?

Answer 41

1. Neurotrophic factors tell neurons they have enough target e.g. NGF (nerve growth factor)
   a. Injection rescues dying cells
   b. Antibodies against NGF trigger cell death
   c. In vitro, NGF removal causes cell death
   Most cells programmed to die and require signal telling them not to die
   All neurones need very specific amount and type of neurotrophic factor
2. Electrical Activity
   Increase in muscle, increased MN death, makes neurones dependent on neurotrophic factor
   Decreasing activity has opposite effect
   SO increase/decrease neurotrophic factor

Question 42

Describe what is meant by Wallerian degeneration.

Answer 42

Injury to axon which results in upregulation of neurotrophins (e.g. NGF, BDNF) - schwann cells have receptors and dedifferentiate, proliferate and form permissive environment.
Distal stump degenerates induced by activation of proteases and Ca2+ influx (leaving behind myelin sheath).
Components are recycled by macrophages and Schwann cells which grow into cut and unite stumps
Proximal stump regenerates by sending buds into network of schwann cells and growing along cord of them.

Question 43

Describe regeneration in the dorsal root ganglion.

Answer 43

Pseudo-bipolar - one axon which splits into sense organ and CNS up to brain.
Lesion peripheral branch (leads to increased cAMP) to sensory organs, get full functional recovery but CNS can’t grow into CNS.
Conditioning P lesion allows CNS arm to regenerate even in non-permissive environment.
protein kinase A can inhibit Rho activity and stop Rho inhibiting regeneration

Adding permissive environments (e.g. nerve sheaths) has been helpful but nerves then have issues re-entering normal environment

Question 44

Describe the blocks preventing CNS recovering.

Answer 44

1. Inhibition by myelin-associated inhibitors (when myelin degenerates) e.g. Nogo produced by glia at damage site telling neurons they can’t grow.
2. Astroglial scarring: reactive astrocytes and ECM (CSPG = Chondroitin Sulfate Proteoglycans) chemical and physical barrier

Question 45

How can the glial scar be beneficial to organisms with minor damage in preventing it becoming a widespread issue?

Answer 45

Damaged nerves release ions, excitatory aa and free-radicals causing secondary degeneration of neurones.
Scar separates damaged and healthy tissue.
Astrocytes also provide trophic support for surviving neurones.
KO mice lacking reactive astrocytes suffer greater nerve damage
Some glial cells may support nerve regrowth

Question 46

Describe induction of plasticity as an alternative strategy for regeneration.

Answer 46

After stroke, region around damaged area develops to compensate for the lesioned region. Limited by inhibitory nature of CNS (same mols). E.g. chondroitinase allows visual plasticity to be reactivated in rats

Question 47

What is a neuronal stem cell (NSC)?

Answer 47

Multipotent and self-renewing
Potential to become neuronal progenitor (before neuron) or glial progenitor (before Astrocyte or Oligodendrocyte)

Question 48

Describe making NSCs for therapy.

Answer 48

Spare embryos from IVF
Make ESC with SCNT
Cell fusion, direct reprogamming, cell culture
Issues with ethics (human cloning), efficiency, use of viruses

Question 49

How can NSCs be utilised in treating Parkinson’s disease?

Answer 49

2007 - Fetal midbrain tissue - very variable outcomes, some immune rejection and motor side effects found
Risk of cancer transplanting proliferating cells.
NSCs from midbrain can form dopaminergic cells, not yet in high numbers
Other treatments are improving - deep brain stimulation of subthalamic nuclei; better drugs/growth factors

Question 50

Describe how NSCs can be utilised in treating Strokes.

Answer 50

Loss of blood supply to brain causing death of surrounding tissue
Add various growth factors, high recovery
Issue is damage is where stem cells are
Recent phase 1 clinical trial with secondary progressive MS - SC thought to reduce inflammation that causes disease rather than rebuilding damaged tissue