Formation and patterning of the nervous system

I. Neural Induction and Neurulation - specification of neural fate and formation of the neural tube.

II. Neural Patterning - patterning of neural progenitors along the dorsoventral and anteroposterior axis

III. Neurogenesis - differentiation of neural progenitors into postmitotic neurons and glia.

IV. Understanding neural patterning in the context of neurogenesis

Basic Organization of the neural tube

Neural stem cell lineage diagram illustrating the generation of different subtypes of neurons and glia

Interneuron's: responsible for the modification, coordination, between sensory and motor neurons.

Motor neurons conduct impulses from the brain and spinal cord to effectors such as muscles and glands

Sensory neurons conduct impulses from receptors to the brain and spinal cord, such as vision, sound, touch, pain etc.

Basic Organization of the neural tube

Progenitors(dividing)

in ventricular zone

Postmitoticneurons in

“mantle” layer

Neuronal differentiation in the caudal neural tube

V0

V1

V2

V3MN

dl1

dl2

dl3

dl5

dl4

dl6

Floor plate

Roof plate

Many different types of neurons are found in the neural tube

Motor neurons conduct impulses from the brain and spinal cord to effectors such as muscles and glands

Interneuron's: responsible for the modification, coordination, between sensory and motor neurons.

Sensory neurons conduct impulses from receptors to the brain and spinal cord, such as vision, sound, touch, pain etc.

Neural stem cell lineage diagram illustrating the generation of different subtypes of neurons and glia

Glial differentiation in the caudal neural tube

• At about E13.5(rat) and E11.5(mouse) Oligodendrocyte precursors arise ventrally in a region that also generates neuronal precursors. Cells migrate dorsally and ventrally before differentiation into oligodendrocyes. Astrocyte differentiation is first detected dorsally though the site of precursor cell differentiation remains unknown.

DORSAL

VENTRAL

vent zone

futureastrocytes

GRPcells

neurons

Basic Organization of the neural tube

Neural stem cell lineage diagram illustrating the generation of different subtypes of neurons and glia

Many different types of neurons are found in the neural tube

Ventral neurons are specified by different combinations of transcription factors

progenitor cells postmitoticneurons

Ventral neurons are specified by different combinations of transcription factors

Pax7

Pax6

Nkx2.2

Pax6-/- Pax6 overexpression

What regulates the expression of these transcription factors?

Start with the ventral spinal cord: notochord and floor plate are organizers

Notochord is necessary and sufficient for floor plate, motoneuron development

floor plate

notochord

dorsal root ganglia

motor neurons

dorsal root ganglia

.

.

Secondary ventralfloor plate

Notochord induces both floor plate and motoneurons. So does floor plate!

Inducer: notochord

Score for expressionof FP marker (green)MN marker (orange)

Responder:Naïve neural tissue

In vitro: Notochord induces both floor plate and motoneurons

So does floor plate

F MNMN

Inducer: floor plate

F MNMN

FF

V0

V1

V2

V3MN

V0

V1

V2

V3MN

Morphogen Signalingrelay

Notochord/Floor plate induce ventral neurons. How?

Testing the morphogen model:

Predictions:•Secreted factor from FP•Should induce neurons in concentration-dependent manner

F

V0

V1

V2

V3MN

Morphogen

Initial test:•FP conditioned medium induces MN without inducing FP

Both the notochord and the floor plate express a possible morphogen, Sonic hedgehog (Shh)

Criteria:1. Secreted2. Right place, right time3. Necessary4. Sufficient

1. Sonic Hedgehog (Shh) is secreted

N-terminus: bioactivity

C-terminus:autocatalytic

Shh precursor:

Autocatalyticcleavage:

Addition of Cholesterol moiety: Is it diffusible?

Artificial solubleform: N-SHH

(45kD)

25kD-no known function 19kD-all Shh signaling

2. Right place, right time: protein?

3. Necessary: spinal cord development in Shh -/- mice

- no floor plate development

- dorsal markersexpand ventrally

Motor neurons also fail to develop in Shh -/- mice

3. Necessity:Floor plate, motor neurons, and ventral interneurons fail to

develop in Shh -/- mice

4. Is Shh sufficientand

is it a morphogen?

4. Sufficiency: Shh can induce floor plate (contact) and motoneurons (diffusible)

4. Sufficiency: can we show clear dose dependent induction?

Attempt to induce cells in concentration-dependent manner

Use artificial soluble N-Shh

Is Sonic hedgehog (Shh) functioning as a Morphogen?

Criteria:1. Secreted

2. Right place, right time

3. Necessary

4. Sufficient

Observations: = Sort of (not very diffusible)

= Sort of (can’t see gradient)

= Yes (but compatible with every other model too) = Yes (pretty good, but not perfect, and done with artificial soluble Shh)

Notochord/Floor plate induce ventral neurons. How?

FF

V0

V1

V2

V3MN

V0

V1

V2

V3MN

Morphogen Signalingrelay

Signalingrelay

Some puzzles - evidence for signaling relay

F

V0

V1

V2

V3MN

F

V3

V1

MN

So: can we devise additional tests, especially to test action at a distance?

Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM.

A motor neuron-dependent step in interneuron differentiation.Cell. 1996 Jan 26;84(2):309-20.

Prediction:

How to distinguish between models?

Mosaic analysis of effect of loss of receptor

Delete receptor for candidate morphogenin a few cells

Prediction:

Phenotype

No phenotype

Tools for manipulating Hedgehog signaling: Patched (Ptc) and Smoothened (Smo) both required

Conventional model of Hedgehog signal reception: Smo (green) has an intrinsicIntracellular signaling activity that is repressed by direct interaction with Ptc(red) within the plasma membrane. This repression is released when HH binds.

To make mosaics: generate chimeric mice from mixing Smo-/- ES cells with wild-type cells

Smo-/- (green) cells fail to express ventral markers (red)(red and green don’t overlap)

Very ventral

A bit moredorsal

Broad ventral

What about dorsal patterning?

Similar logic: epidermal ectoderm induces roof plate, which cooperate to induce dorsal cells.

Inducers: BMPs (perhaps Wnts too?)

dl1dl2

dl3

dl5dl4

dl6

Epidermal ectoderm

Roof plateRP

Dorsal cells

Several BMPs(Wnts too?)

Several BMPs(Wnts too?)

Evidence: in vitro, induce dorsal characteristics

epidermisor

roof plateor

cells expressing BMPs

neural plateR d d ddd d

Evidence: in vivo, How to deal with many BMPs (and Wnts)?

Ablate roof plate genetically

dl1dl2

dl3

dl5dl4

dl6

RP Drive expression of toxinin roof plate in knock-in mice

•Use Diphteria toxin•introduce into GDF-7 locus

Introduce Diphteria Toxin A (DRTA) gene into GDF7 locus

GDF-7

IRES DTA

GDF-7 IRES DTA

Problem:

So: make it conditional

GDF-7 IRES DTAstop

loxP loxP

: silenced

+ Cre recombinase

GDF-7 IRES DTA : active

Dad carries silenced allele+

Mom carries Cre gene activated in early fertilized egg

1/4 of embryos get both, sothey get an activated DTA gene under GDF7 promoter

Expression of silenced allele: same as that of GDF7

In embryos with cre (allele activated): roof plate absent!

No roof plate: lose dl1-dl3, preserve dl4-dl6

Conclusions for dorsal spinal cord:

• Cascade: Epidermis -> Roof plate (like Notochord -> FP)• Lots of BMPs (+maybe Wnts) - different from ventral• No evidence for morphogen effect yet (all other models possible)

What about glial cells?

Summary of spatio-temporal changes in progenitor domains and their relationship to oligodendrocyte production.

Between E3.0 and E7.0, the ventral most expression domain of Pax6 disappears and Nkx2.2Expands dorsally into this region to overlap with Olig2. (data)

Between E3.0 and E7.0, the ventral most expression domain of Pax6 disappears and Nkx2.2 expands dorsally into this region to overlap with Olig2. (data)

Collaboration between Olig2 and Nkx2.2 cell autonomously promotes oligodendrocyte differentiation.

Targeted disruption of Olig2.

Loss of motor neurons in Olig-/- mouse embryos

Spinal Cord Oligodendrocytes fail to develop in the absence of Olig genes

But astrocytes are fine