Neurogenesis in the spinal cord

Question 1

what forms the spinal cord

Answer 1

caudal neural plate
tail end

neural tube
transition zone
stem zone

caudal most end
primitive streak - gastrulation still going on
node - organiser region
around node - under influence of factors secreted inhibit BMP signalling

neural tube forms flat ectodermal region flanked on either side by epidermal ectoderm and folds up to form neural tube and is covered by endodermal endoderm

extension of the node =

cells from the node form the axial mesoderm
paraxial mesoderm around the open neural plate = segmental plate
condenses to form the somites

Question 2

what occurs at the stem zone - most caudal neural plate

Answer 2

active proliferation takes place contributing to the mesoderm and ectoderm

Question 3

transition zone

Answer 3

region where the neural plate is determined
ectoderm is defined as neural ectoderm
but neurogenesis is not happening yet

Question 4

the caudal region grows overtime

the anterior region at any time is more mature than the more posterior region because more material is being added posteriorly or caudally

Answer 4

Pax6 is the first marker expressed as neural progenitors exit the transition zone
shortly thereafter, neural determination (neurogenic) and differentiation (NeuroD) genes are expressed and Lateral inhibition (Delta) starts
Pax6 is the first marker expressed
precedes neurogenin 2
NeuroM=NEuroD4

Question 5

what can we tell from this experiment:
microsurgery experiment and looking at the assay
presomatic and lateral mesoderm removed

= premature activation of Pax6
= suppression of Pax6
graft of prismatic mesoderm to the potion of the last formed somite

graft of somite to prismatic mesoderm = premature activation of pax6

Answer 5

extrinsic influence on Pax6 expression

presomtitic mesoderm surpresses pax6 exp

absence of presomitic mesoderm = activation of pax6

somite = positive influence on pax 6

and somite and presomitic mesoderm are both paraxial mesoderm just at different time points of maturation

Question 6

candidate molecules for signalling around the caudal neural tube

Answer 6

Fgf8 expressed in presomitic mesoderm and neural plate

retinoid acid produced in the somites

somatic mesoderm and lateral mesoderm show strong exp of Radldh2 which encodes retinaldihyde dehydrogenase which synthesises retinoid acid from retinaldhyde which is a derivative of vitamin A and oxidation of retinaldehyde = retinoic acid = signal

somites from the last formed somite onwards produce retinoid acid which can influence anything in the neural tube

and in the presomitic mesoderm high expression of FGF8 which reduces as mesoderm matures because transcription of FGF8 stops and little bit of mRNA is degraded

Question 7

candidate molecules for signalling around the caudal neural tube

Answer 7

Fgf8 expressed in presomitic mesoderm and neural plate

retinoid acid produced in the somites

somatic mesoderm and lateral mesoderm show strong exp of Radldh2 which encodes retinaldihyde dehydrogenase which synthesises retinoid acid from retinaldhyde which is a derivative of vitamin A and oxidation of retinaldehyde = retinoic acid = signal

somites from the last formed somite onwards produce retinoid acid which can influence anything in the neural tube

and in the presomitic mesoderm high expression of FGF8 which reduces as mesoderm matures because transcription of FGF8 stops and little bit of mRNA is degraded

there 2 candidates are
FGF8 to suppress pax6 from the presomatic mesoderm
Raldh2 producing retinoid acid which could be the signal promoting pax6 exp

Question 8

pax6 upregulates neurogenin2

what is the effect of high levels of pax6

Answer 8

maintenance of neurogenin2 expression in ventral pole of neural tube is prevented

we would expect delta expression as part of the lateral inhibition system but pax6 represses this
and neuroD4 which marks the start of differentiation of neurons - no neuroD4 expression
- control beta tubulin is a marker for neurons to test for neurogenesis
tubulin protein highly present in the axon makes up the micotubilin network
pax6 electroporation
where no neurogenesis occurred = result interpretation =

Question 9

pax6 induces exp of neurogenic 2 so starts neurogenesis but prolonged exposure. prevents neurgensis

Answer 9

histoenesis in neural tube
proliferating neuro progenitor cells close to neural canal
time progresses
first differentiation of neurons
they move lateral way from central canal - apical side
close to basal side and surrounding mesoderm

if we take a cross section can see where we find exp of proteins pax6 neurogenic in relation to differentiation status and proliferation status

so if left side is apical
right is basal
would expect
proliferating on left
differentiation non proliferating on the right
pax6 expressed more on left mainly in the actively dividing cells in ventricular zone
neurogenin is actvie in the subventricular zone where pax6 levels are reduced

neurod4 is lateral to proliferating zone where pax6 is expressed
neurod4 marks cells that have left the cell cycle and progressing into differentiation

pax6 and neurod4 are exclusive of each other
only some postsomitic cells expressing neurod4 can still express pax6

pax6 expression is not affected by the lateral inhibition signalling system

Question 10

prolonged pax6 is not compatible with neurogeneiss
how is pax6 switched off?
1. hypoethesis
lateral inhibition?
exp:
misexpression of notch intracellular domain or delta
both would activate notch signalling
results
no effect on pax6 = not effected by lateral inhibition system

2.hypoethesis 
neurogenin 2
exp misexpression
expo of pax6 - strong on electroplated side reduced
results 
neurogenin 2 surpasses exp of pax6

Answer 10

what does neurogenin 2 do to neurogenesis
in spinal cord
results of misexp of neurogenin2
effect of neurogenin2 on 2 markers

beta ubulin - marker for neurons 
and pax6
looking at protein levels
control and electroporated side
clear difference = 
massive increase in beta tubulin and decreases in pax6
supports that pax6 inhibits neurogenesis

Question 11

model for spinal cord neurogenesis

Answer 11

1. pax6 expression controlled by fgf8 - supresses it and then retinoid acid inducing it
2. once pax6 is expressed induces expression of neurogenin 2
   firstly low levels for both them
   getting up to high levels of pax6 neurogenin increases more and more but at a slower rate
3. neurogenin 2 now supresses pax6 exp and induces neurod4
4. prolonged pax6 would inhibit neurod4/neuroM expression

2 opposing forces on neurogenesis
pax6 inhibiting neurogenesis and inducing neurogenin which promotes neurogenesis
overcome this
neurogenin 2 down regulates pax6 allowing progression of neurogenesis
= neural precursors progressing to differentiation

Question 12

control of neurogenin2 expression
exp in mouse
to check system in maintained

Answer 12

addition of retinoid acid = increased neurogenin 2 exp

bms492 =
reduction of retinoid acid
= reduction or delayed neuronic 2 exp

fgf8 inhibitor used =
premature neurogenin 2 exp
increased activity of retinoid receptor

affect of shh
cyclopamine
suppress neurogenin 2
genetically
shh knock out
heterozygous siuation = shh still present = control
homozygous mutant whre shh is not expressed = delayed neurogenin2 exp

Question 13

3 signalling systems working on neurogenin 2 expression

Answer 13

fgf8 suppressing neurogenin2 via pax6

retinoid acid and shh being promoters of neurogenesis

gene regulatory network
neurogenin promotes neurod4 and nerogenesis
neurogenin part of lateral inhibition - upredulates delta but itself is down regulated by active notch

neurogenin is regulated by cell cycle regulators
negatively regulated by phosphorylation by cdks
and positively regulated by cdk inhibitors

extrinsic regulation through signals
retinoid acid and fgf8 acting on pax6
shh working on neurogenin
pax6 having the interaction w neurogenin promoting it and then neurogenin down regulating pax6

pax6 downregualtes neuroD4 = prevents neurogenesis

Question 14

role of neurogenin in neurogenesis

Answer 14

interacts w homeodomain proteins to specify motor neurones

forms a hetErodimer with e protein i.e. e12
binds to the e box

specification of particular neuronal fates specifically motor neurones

phosphorylation by cdk serine residues = reduced activity and stability of neurogenin = limits/inhibits neurogenesis early on

neurogenin interacts with LIM homeodomain proteins to specify motor neurones
mediated by NLI = Ldb1 LIM domain binding protein
depends on Ngn2 phosphorylation on specific seriene residues

gsk3 kinase has diff specificity compared to cdk phosphorylates serenes to form lim homeodomain complex which triggers the identity of motor neurones

neurogenesis and fate specification can be linked directly by the same molecule

Question 15

what is the mature spinal cord made up of

Answer 15

incoming axons
sensory neurons send their axons into dorsal part of spinal cord via dorsal roots - afferons = connect to sensory interneurones

efferenn neurons in the spinal cord premotor neurons and exit spinal cord via ventral roots = connect to innerve effectors

afferons and efferons combine laterally to form the spinal nerve

Question 16

dorsal root ganglion

Answer 16

part of the peripheral nervous system where the cell bodies of the sensory neurons are located

Question 17

dorsoventral organisation of the spinal cord

Answer 17

progenitor cells still proliferating at embryonic seas in the nerve canal

next layer out = mantle later contains differentiating and differentiated neurons which send their axons into the marginal layer

Question 18

dorsoventral organisation of the spinal cord

Answer 18

progenitor cells still proliferating at embryonic cells in the central canal

next layer out = mantle later contains differentiating and differentiated neurons which send their axons into the marginal layer

dorsal root ganglion on outside 
alar plate = dorsal
ventral = basal plate
sulcus limiters - indent
floor plate above notochord

Question 19

dorsoventral organisation of the spinal cord

Answer 19

progenitor cells still proliferating at embryonic cells in the central canal

next layer out = mantle later contains differentiating and differentiated neurons which send their axons into the marginal layer

dorsal root ganglion on outside 
alar plate = dorsal - roof plate 
ventral = basal plate
 ventrally floor plate adjacent to notochord
sulcus limitus - indent

Question 20

motor neurons located ventrally

sensory function dorsally they have sensory function and receive the input

controlled by interneurons some ventrally and some further dorsally

dorsal interneurons receive input from sensory neurons

Answer 20

diff homeodomain transcription factors expressed in progenitor cells which are still dividing = diff fates along dorsoventral axis

Question 21

ventralisation of neural tube by signals from the notochord

exp:
1. ectopic grafting of extra notochord
gain of function
results = extra floor plate formed and extra motor neurones on either side of this new floor plate
suppression of pax3

2. early removal of notochord
   loss of function

what is seen
no floor plate 
no motor neurones 
pax3 expressed throughout neural tube =
dorsalised

Answer 21

conclusion
notochord is responsible for ventralisation of neutral tube
induction of floor plate and formation of motor neurons

this induction triggered by the notochord can also be mimicked by shh

Question 22

this induction triggered by the notochord can also be mimicked by shh

Answer 22

shh is first expression in he transition zone in the notochord

shh induces the floor plate in the neural tub which itself expresses shh

initially none
start of shh exp in axial mesoderm in the notochord
afterwards in the floor plate
this means shh induces the floor plate and induces exp of shh in the floor plate inside neural tube

induction

Question 23

what does shh do that is produced by the floor plate

Answer 23

all expressed in progenitor cells in he developing spinal cord
pax7 pax3 Irx3 Nkx6.2 Nkx6.1 Olig2 Shh

expression of all of these genes is correlated with the onset of some exceptional expression in the floor plate

Question 24

notochord or shh can induce the floor plate and ventral characteristics in the neural tube

exp 1.
hypo - concentration dependent response to shh

took neural plate explants
put into cultute
looked at what does the neural plate express on its own and when treated w increased shh

neural plate explants respond to different conc of Shh to express diff homeboy genes
pax7
pax6
Nkx2.2
also time durations

Answer 24

alar plate marker pax7 - expressed in absence of shh
intermediate markers pax6 expressed at low shh conc - high levels of shh are incompatible with their exp
ventral markers Nkx2.2 expressed at high shh conc - require shh

Question 25

specific expression of homeobox genes genes expressed more dorsally and more dorslaly differ in how farventral and how far dorsal they go how are they influenced by shh

Answer 25

``` class 1 repressed dorsally expressed genes class 1 genes like Dbx2,Pax6 or irx3 are generally repressed by high [shh] dorsal ones already respond to low shh conc up to more ventral levels respond to higher levels of shh but eventually get repressed ``` class 2 induced transcription of ventral classs activated/induced by shh Nkx6.1 found lower therfore found further dorsally from shh source since requiring lower levels of shh and Nk2.2 are closer to source requiring higher [shh]

Question 26

class 2 genes induced shh gradient expect to find ~Pax6 exp peters out as shh increases and Nk2.2 exp should be highest close to the shh source and then should peter out sharp boundary between all of the cells that express pax6 and all of the cells expressing nkx2.2 but dont express both cells at the same time hjow do we get from this gradient to sharp distinct boundaries analogue to digital transition ``` there are particular pairs of transcirption factors class 1 and class 2 homebox pairs of genes have a sharp boundary between them i.e. pax6 and kx2.2 ``` pax6 exp domain ventrally and ends sharply and coinciding with the boundary of nkx2.2 expression

Answer 26

gain of function exp cross regualtion of transcription factors mmisexpression of pax6 ectopically express pax6 and look for the effect that it has on nkx2,2 where you misexpress pax6 you lose nkx2.2 ``` the converse exp misexpress nklx2.2 resul;t = control side pax6 nicely expressed where ectpic exp of nkx2.2 = lose pax6 reduction of corresponding gene therefore cocnusion = pax 6 supressed nkx2.2 nkx2.2 supresses pax6 cross regulate each other this interaction is specififc ``` look at nkx2.2 effect on 2 other class 1 genes nkx6.1 and pax7 interaction between nkx2.2 and pax6 is specific cross inhibition is responsible for creating this sharp boundary wherever we have slightly hgher levels of nkx2.2 and pax6 - pax6 wins and supresses nkx2.2 thereby increasing its own expression and vice versa creates sharp boundary

Question 27

model for gene regulation sox genes responsible for regulation of neural genes homeo domain genes ``` ones that respond to shh one class 1 and 1 class 2 create a mutant where we take out the class 2 gene meaning the class 1 gene is no longer repressed by it expression domain will be expanded ``` generated groups of progenitor cells that differ in the expression of homeodomain transcription factors but what does that mean for cell fate? how is that related?

Answer 27

look beyond progenitor cells look at differentiated neurons there are distinct classes of neurons Vo V1 V2 V3 and Mn interneurons and motoneurons ``` each distinct class of neurons is characterised by the expression of particular transcription factor Tfs expressed in differentiating neurons not progenitor cells gene expressiuon in the 5 ventral progenitor domains determines the type of neurone formed ``` cells expresing nkx6.1 and apax6 but not irx3 or nkx2.2 will form motor neurone progenitors neurons correltate w the original progenitor domains

Question 28

which cells will form motor neuiron progenitors

Answer 28

cells expressing nk6.1 and pax6 but not irx3 (which is more dorsal) or nkx2.2 expression of mnr2 in the progenitors determines motor neuron fate = expression of Isl1/2, HB9 in differentiating motor neurones

Question 29

progenitor cells expreing a spcific combination of transcription factors (molceular code ) will differentiate into a specific type of neurone manipulating gene expressonn in progeniotr cells should alter cell fate

Answer 29

gain of function forced ectopic expresson of irx3 by electroporation = transformation of nkx6.1+iIrx3 pMN progenitors to NKx6.1/Irx3+ p2 cells what weve done expanded the expression of irx3 and those cells that co express irx3 and knx6.1 throught the ectiopic extression have been transformed into v2 interneurons rather than motor neursns motor neurones supressed ectopic v2 interneurones formed instead ``` lowss of function create a mutant of nkx6.1 dbx2 counterpart of knx6.1 class 1 gene w crossrepressive interaction q nkx6.1 dbx2 expression markss v1 and v0 interneurons ``` knock out of nkx6.1 = lose nkx6.1 expresion dbx2 expressed more widely becuase not repessed by nkx6.1 so what is the reustltf interms of differnitating neurons ``` looking at interneuron markers v0 - evx1 - no difference v1 interneurons - expransion v2 interneurons - dissapeared mn - massively reduced v3 - maintained = producing v1 interneurons ``` therefore loss of nkx6.1 = transformation of p2 and pmn progenitors into nkx6.1 more dorsal precursors vv2 interneurons and motor neurones supressed ectopic interneurons formed instead

Question 30

patterning the ventral spinal cord

Answer 30

``` formation of a spatial and temporal gradient regional expression of class 1 dorsally9repessed by shh and class 2 (incduced by shh) homebox genes in response to shh gradient ventrally ``` specific class 1 and class 2 genes cross regulate each other nkx2.2 and pax6 nkx6.1 and dbx2 cross regulating each other = sharp expression domains = 3 distinct progenitor domains - within each progenitor domain cells express the same homeobox code between the domains trhere are differences in gene expression the specific code homeobox combination of homedomain TF in a particular group of progenitor cells = determines specifies the fate i.e. motor neurones - pax6 and nkx6.1 but not irx3 and nkx2.2 determines fate of motor neurons happening at the same time as neurogenesis refining of progenitor domain boundaries by cross repressive interaction between pairs of class 1 n 2 genes specification of neuronal fate by homeobox code in the mantle layer due to combined expression of class 1+2 homebox genes in the ventricular zone