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Developmental Neuro > Neural Induction and patterning > Flashcards

Neural Induction and patterning Flashcards

1
Q

Discovery of the organiser region

A

Spemann and Mangold Graft blastopore of pigmented newt onto a non-pigmented newt gastrula. Generate embryo with trunks and two nervous systems. This experiment has been repeated and if you graft one organizing region to another developing embryo develop two body axis.

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2
Q

Define neural induction

A

The process of transforming dorsal ectoderm into neuroectoderm is called neural induction.

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3
Q

Describe the work done to discover the role of BMPs in epidermal induction

A

Wilson & Hemmati-Brivanlou

  1. Antibody labelling with the epidermis-specific marker Epi-
  2. In whole gastrula Epi1 is expressed in ectoderm cells fated to become epithelia but not in neural plate.
  3. Exposing dissociated animal cells to exogenous BMP4 induces expression of Epi1.
  4. Therefore, BMP4 promotes epithelial fate in ectoderm cells.
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4
Q

Define organiser region and where is it found in different species.

A

The organiser is critical for formation of the body axes and development of neural tissue (neural indcution).

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5
Q

Name three signals secreted by the organiser region.

A

Chordin Follistain Noggin

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6
Q

What are the two parts of induction

A

1.The inducer (tissue or group of sells secreting inductive signal) 2.The responder (tissue that changes its developmental trajectory in response to the inductive signal via change in genes expression)

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7
Q

What are the four paracrine factor families?

A
  1. FGFs (fibroblast growth factors) 2. Hedgehogs 3. Wnts 4. TGFs (transforming growth factors)
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8
Q

Define gastrulation

A

Gastrulation is a phase in embryonic development of most animals, during which the single-layered blastula is re-organized into a multi-layered structure known as the gastrula.

Results in the production of 3 germ layers + defines the midline and anterior posterior axis of.

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9
Q

Who discovered that the organizer region secrets signals that act as inhibitors of BMP signaling.

A

Zimmerman oCoat beads with noggin. o Expose beads to BMP4 ligand o Wash off BMP4 and run beads on Western blot (separates proteins according to size). o Using antibody labelling to detect for the presence of BMP4. o Band corresponding to BMP4 when noggin coated beads are used. No BMP4 band when control beads used. Conclusion: Noggin binds to BMP4 ligand with high affinity which presumably stops the ligand from binding to the receptor and consequently don’t get any signalling that will get the cells to adopt an epithelial fate

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10
Q

What are the two basic ways in which inducers interact with responders:

A

Juxtacrine signalling: signalling occurs only between juxtaposed (side by side) cells that are in direct contact with one another. No long range signalling, it’s a more contact form of signalling Paracrine signalling: signalling occurs across a field of cells that are within close range of (but not necessarily in direct contact with) the inducer. More long range signalling.

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11
Q

What are the three germ layers

A
  1. Endoderm: gut liver, lungs 2. Mesoderm: skeleton, muscle, kidney, heart, blood 3. Ectoderm: skin, nervous system
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12
Q

What are the two stages the cell must go through before being fully differentiated:

A
  1. Specification: signals secreted and modify gene expression of cell and pushes it to a specified state, e.g bias the cell to neural tissue. This state is labile (it can be reversed) so moving the cell into a different environment and it can be pushed towards another cell type. 2.Determination: Stable state where neural fate is acquired and can no longer be altered.
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13
Q

Who identified Noggin as a neural inducer and how?

A

Richard Harland. Isolate organiser region from Xenopus gastrulae. Generate organiser region RNA from a cDNA library. Inject RNAs into embryo that has been UV radiated so they only have ventral tissue. Determine whether injected RNA can rescue embryo and induce formation of neural tissue. From this Harland identified Noggin as an important neural inducer

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14
Q

Descrieb the role of Retinoic Acid in AP Patterning and where is it induced?

A

A morphogen secreted from the neural ectoderm, that plays a key role in posterior patterninf of the neural tube by establishing boundaries of segements at different concentration thresholds induction of Hox genes.

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15
Q

What are the three paracrine signals important in AP patterning

A
  1. Wnt signals
  2. Fibroblast growth factors (Fgfs)
  3. Retinoic Acid (RA)
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16
Q

What is the default hypothesis?

A
  • Neural fate is the default state for ectoderm cells.
  • Ectoderm cells secrete BMPs which promote epidermal fate.
  • Organiser region secrets proteins that bind BMP ligands, preventing their association with BMP receptors.
  • When this happens, BMP signalling is inhibited and ectoderm cells revert to neural fate
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17
Q

Describe the process of convergent extension

A
  1. Cells initally lieing along axis
  2. Cells polarize: become constricted at ends
  3. Cells converge: migrate towards one another and intercalate
  4. Thsi resortign of cells causes tissue to extend in plane that is at right angles to axis of convergence
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18
Q

Describe the different paracrine signals involved in AP patterning.

A

FGF signalling (retinoic acid) and Wnt signaling posteriorises neural tissue.

Anterior signals are Wnt inhibitors. They block the posterior fate sof the neuroectoderm and induce the anterior nervous system: Cerebus, Dickkopf.

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19
Q

Define neurulation and explain the two types of neurulation

A

Neurulation is when the neural plate develops to form the neural tube

Primary neurulation: the flat neural plate buckles and rolls up to form a neural tube.

  1. Neural plate comprised of columnar-like cells.
  2. Presumptive epidermal cells converge towards neural plate. Apical neural plate cells become wedge shaped, creating folds towards edges of neural plate. Cells at midline of neural plate form medial hinge point (MHP) by anchoring to notochord below. Central region termed “neural groove”.
  3. Surrounding presumptive epidermal ectoderm converges towards midline, elevating neural plate as it does so. Neuroectoderm bends around MHP.
  4. Two sides of neural plate join up leaving a central lumen. Cells at junction between tube and epidermis bud off to become neural crest.

Secondary neurulation: mesenchymal ectodermal cells condense to form a neural rod.

  1. Neuroectoderm mesenchymal cells condense into rod-shaped mass.
  2. Cells undergo mesenchyme to epithelial transition to form a solid rod.
  3. Cavitation establishes a lumen, converting the neural rod into the neural tube.
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20
Q

Explain the formation of the neural tube

A

Neural tube: is the thickening and flattening of the neuroectoderm

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21
Q

List 5 potential causes of neural tube defects

A
  • Therapeutic drugs
  • Environmental pollutants
  • Chronic disease of the mother
  • Genetics: defective planar cell polarity signalling
  • Imbalance in nutrients (e.g. folate)
22
Q

Why do defects in convergent extension cause NTDs?

A

Defects in convergent expansion likely to cause expansion in floor plate (arrowheads).

This expansion is believed to prevent the two halves of the neural tube from being pushed together.

23
Q

Why does loss of polarity affect neural tube formation?

A

If cells cannot polarise, they fail to converge on the midline.

Convergence is essential for constriction of the medial hinge point (red) around which the two halves of the tube come together.

When convergence is disrupted, the hinge point becomes wider and as a result, the two halves of the neural plate fail to meet at the midline.

Gap too wide to facilitate complete neural tube closure.

24
Q

Evidence that Rock-dependent polarisation is critical for neural tube closure

A

In zebrafish embryos, expression of a dominant-negative form of Rock2 causes convergent-extension defects.

Rock2 expression can rescue convergent-extension defects in wnt11 mutants. Therefore Rock2 acts downstream of Wnt signalling.

25
Q

Evidence for the requirement of PCP pathway genes

A

Electroporation of GFP expressing plasmids into midline (enables visualisation of neuroectoderm cells)

In Vangl2 mutant (looptail) mouse: neuroectoderm cells fail to converge and extend. Neural tube does not close.

The neural tube also fails to close in mouse Dishevelled Dvl1/Dvl2 double mutants

26
Q

Define planar cell polarity and describe how defects in PCP can contribute to neural tube defects

A

Planar cell polarity (PCP): the “polarisation of cells within the plane of a cell sheet”

Cells polarise during convergent extension and PCP genes induce cell polarisation during convergent-extension so PCP is believed to be essential for “convergent extension” of neural plate.

27
Q

Describe the mechanism involving PCP genes

A

PCP genes form part of the “non-canonical Wnt signalling” pathway.

  1. Wnt ligand binds Frizzled (Fzd) receptor
  2. Activates Dishevelled (Dvl)
  3. Dvl activates Rock/RhoA which in turn induces cytoskeletal changes.
  4. These result in cell polarisation.
28
Q

Evidence for the effect of follate acid on neural tube defects.

A

Folate acid rescues neural tube defects in splotch (PAX3) mutant embryos (Flemming & Copp, 1998)

  1. PAX3: transcription factor that is essential for regulating gene expression of a myriad of genes during development.
  2. PAX3 mutants have severe neural tube defects.
  3. Mutants have metabolic deficiency in folate supply. Leads to NTD susceptibility (in mice).
  4. Folic acid rescues NTDs in PAX3 mutants
29
Q

Define neural tube defects and explain the different types.

A

Neural tube defects (NTDs) are a group of birth defects in which an opening in the spinal cord or brain remains from early in human development.

Two types:

  1. Open: when the brain and/or spinal cord are exposed at birth
  2. Closed: occur when the spinal defect is covered by skin

If neural tube closure is affected, typically causes “open neural tube” defect where part of CNS is exposed.

If other tissues affected, typically causes “closed neural tube” defect.

30
Q

Describe four types of NTDs.

A
  1. Anencephaly: open brain and lack od skull vault
  2. Spina bifida: closed NTDs, some of the vertebrate isnt completely closed, most common NTD
  3. Crainorachischisis: complete opening of the brain and spinal cord
  4. Myelomeingocele: open spical cord (with a menigial cyst)
31
Q

Give three roles of folate in the body

A

Folate intake is critical for a number of cellular processes including:

  • Mitochondrial protein synthesis
  • Nucleotide synthesis
  • Nucleotide metabolism
  • Amino acid metabolism
32
Q

Describe signals that govern dorsal fates

A

BMPs are morphogens secreted by the roof plate.

They establish a concentration gradient in the developing spinal cord: high dorsally and low ventrally.

Through activation of receptors on competent cells, BMPs regulate gene expression in a concentration dependent manner.

High levels of BMP signalling induce expression of genes associated with most dorsal cell types. Lower concentrations induce expression of genes associated with more ventral cell types.

Gradients set balance of formation of spinal cord.

33
Q

Describe how gradients of paracine factors induce division between the rostral brain and hindbrain/spinal cord.

A

The anteriorposterior divisions of the neural plate stems from the expression of differential transcription factors.

Anterior regions: Wnt inhibitors secreted from the endoderm Posterior regions of the neural plate: high levels of Wnt signaling and lower levels of Wnt inhibitors.
Due to the Wnt signaling gradient, cells in A .and P. regions of neural plate express different transcription factors.

Otx2 at anterior levels (forms forebrain and midbrain domains)

Gbx2 (forms hindbrain domain) at more posterior levels.

Midhindbrain region (MHB): intersection between Otx2 and Gbx2, transcription factors expressed (Engralled)

34
Q

Desrcieb the role of Hox genes in hindbrain segmentation

A

Different Hox proteins are expressed in discrete hindbrain domains, called rhabdomeres.

7 rhombomeres in total (r1-r7). Each rhobomere express a unique set genes that give rise to a unique set of neurons. These genes trigger different cascades of gene expression that give each rhombomere a unique identity.

Genetic studies in the mouse have shown that specifc Hox genes control the identity of neurons in individual rhombomeres.

35
Q

Evidence that Hox genes are required for hindbrain segmentation; Hoxa1

A

Hoxa1

Wt E8.5 mouse

krox20 a marker of r3

kreisler a marker of r5/r6.

Hoxa1 mutant:

krox20 domain is larger than wt (i.e. r3 is expanded).

kreisler restricted to a single domain (r6).

Thus, loss of Hoxa1 causes expansion of r3 and a marked reduction of r4 and r5.

i.e. loss of specific Hox genes alters hindbrain patterning.

36
Q

Evidence that Hox genes are required for hindbrain segmentation; Hoxa2

A

Hoxb1 mutant mice, E10.5

r1, r2 boundary is absent.

Subsequent analysis revealed that r1 is expanded at expense of r2.

37
Q

Desricbe experiement to show what happens if all Hox gene activity is lost?

A

Lose sectioning of future hindbrain rhombomeres

Design morpholino to bind to start codon of mRNA of interest (Pbx2 in this case). Use sequence of nucelotides that is antisense to start codon region.

Inject into 1-4 cell stage zebrafish embryos: morpholinos bind to Pbx2 mRNA and block ribiosomal access, resulting in loss of translation.

Raise embryos and examine hindbrain segmentation.

38
Q

What is identity does hindbrain adopt in the absence of Hox gene expression?

A

WT: Fgfr3 and ephA4a are expressed in r1, r3 and r5

Krox20 is expressed in r3 and r5

When PBX2/4 is abrogated, ephA4a & fgf3 domains expand. However, there is a loss of krox20 domains.

This suggests that in absence of Hox signalling, the entire hindbrain adopts r1 fate.

Therefore r1 is the “ground state” in absence of Hox signalling.

No hox genes expressed in r1 therefore when block hoxgene everything becomes r1

Motor neurons are diminished and those that remain lack segmental organisation

39
Q

Describe the experiment showing how the rhombomeres boundary is maintained via ephrins receptors and ligands.

A

In zebrafish r3 and r5 express Eph receptors while r2, r4 and r6 express ephrin ligands.

Inject ephrin-B2 mRNA into 8 cell stage zebrafish embryos. Leads to stochastic misexpression of Ephrin-B2 ligands.

Co-inject lacZ reporter gene: permits visualisation of cells ectopically (cells that don’t normally) expressing ephrin-B2.

17 hpf stain for lacZ (green) and label r3/r5 with Krox20 riboprobes.

Result: cells overexpressing ephrin ligands remain scattered in domains that normally express ephrins (i.e. r2,r4,r6)

However, cells overexpressing ephrin ligands in domains that normally express only Eph receptors are repelled and move away from their non-expressing neighbours to the rhombomere boundary.

40
Q

Describe how repulsive interactiosn between cells in different rhobdomere segmetns maintaisn boundaries.

A

Eph receptors and Ephrin ligands are expressed in alternating rhombomere boundary domains.

Eph-ephrin interactions induce repulsion between cells in juxtaposed domains.

41
Q

How does Shh affect dorsoventral patterning of neurons.

A

Notochord signals induce the foor plate, which both secrete sonic hedgehog protein (Shh) which induces ventral neurons

Shh concentration gradient in the developing spinal cord: high ventrally and low dorsally.

Through activation of receptors on competent cells, SHH regulates gene expression in a concentration dependent manner.

High levels of SHH signalling induce expression of genes associated with most ventral cell types. Lower concentrations induce expression of genes associated with more dorsal cell types.

42
Q

Descrieb experimental evidence showing that Hedgehog can induce floor plate cells and motor neurons

A

Extract naive neural plate cells from chick embryo.

Culture alongside COS cells transfected with hedgehog cDNA.

COS is a fibroblast-like cell line derived from monkey kidney tissue. Cells from this line are often transfected to produce recombinant proteins for molecular biology, biochemistry, and cell biology experiments.

Transfect with shh dna

Shows that shh can cause cells to adpot ventral fates

43
Q

Evidence that Shh induces ventral neurons

A

Mutant Sonic Hedgehog mice lack ventral progenitors

Nkx2.2 expressed in p3,p2 and pMn domains.

Nkx2.2 expression completely lost in SHH mutants.

Dbx1 is normally p0 domain. Expression expanded ventrally in SHH mutants.

So progenitors fail to express genes associated with ventral fates.

Dbx1 expressed in ventral part of spinal cord

Mutant expression further into ventral region than normal

Cells produce expression more like dorsal phenotypes

The net result is loss of ventral neuron types (such as motoneurons)

44
Q

Describe how Sonic Hedgehog functions

A

No ligand

1) In absence of Hh, patch binds to and inhibits the G-protein coupled receptor “Smoothened”.
2) When Smoothened is inhibited, Slimb and PKA are free to target and cleave the transcription factor Cubitus interruptus (CI).
3) Cleaved CI enters nucleus and represses transcription of target genes.

Shh

  1. When binding Hh, patched undergoes a conformational change. This de-represses (i.e. no longer inhibits) Smoothened.
  2. Activated Smoothened inhibits Slimb and PKA. Thus, CI is not cleaved.
  3. Intact CI enters nucleus and acts as a transcriptional activator of target genes.
45
Q

Evidence that the roof plate can induce dorsal neuron specification in naive neural plate explants

A

Isolate early stage neural plate tissue from chick embryo

Culture with or without quail roof plate explant for 48h.

Stain with antibody markers for dorsal neuron fates (LH2 and Isl1).

Also satin for quail roof plate (with OCPN antibody).

Find that roof plat needed for expression of dorsal neuron types

46
Q

Evicence that roof plate cells express the BMP morphogens

A

ISH against BMP4, BMP5 and BMP7 mRNA in embryonic chick spinal cord.

BMPs induce dorsal cell fates

Culturing naive explants for 48h with BMP4, BMP5 or BMP7 induces specification of D1 neurons (as revealed by anti-LH2 staining).

47
Q

Evidence that roof plate expresses dorsal neuronal types

A

Mice lacking roof plate cells do not express proteins associated with dorsal neuron subtypes

Selectively express diphtheria toxin in roof plate cells of mice.

Taking out roof plate is difficult because you can remove the epidermal cells as well

Take promoter region of diphtheria and fuse with gene encoding for Gdf7 (roof plate marker)

Blocks protein synthesis in these cells and therefore kills them.

Results in loss of roof plate (as detected by loss of roof plate markers).

48
Q

Describe BMP signaling

A

Not fully understood.

TGF receptor activation.

Phosphorylation of SMAD

SMAD regulates gene transcription.

49
Q

Describe how Shh gradient affects induction of transcieption factors.

A

Shh differentially regulates expression of homeodomain transcription factors.

Transcription factors can be segregated into two classes, based on Shh’s effect.

  1. Class I (Pax7, Dbx1, Dbx2, Irx3 and Pax6) expression is repressed by Shh at different threshold concentrations.
  2. Class II (Nkx6.1 and Nkx2.2) expression is induced by Shh at different threshold concentrations.

Class 1 genes for dorsal fates so blocked by shh

Class 2 activated by high con of shh associated with ventral fates

50
Q

Describe the arrangement of spinal neuronal classes in vertebrates

A

Sensory related interneuorns are located dorsally

Motor neurons are ventrally located

And motor-related interneurons lie in between moto and sensory neurons

Developmental Neuro (12 decks)

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