Formation and patterning of the nervous system
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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

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Formation and patterning of the nervous system

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Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

Basic Organization of the neural tube


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

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.

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


Formation and patterning of the nervous system

Basic Organization of the neural tube

Neuronal differentiation in the caudal neural tube

Postmitotic

neurons in

“mantle” layer

Progenitors

(dividing)

in ventricular zone


Formation and patterning of the nervous system

Many different types of neurons are found in the neural tube

Roof plate

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

dl1

dl2

dl3

dl4

dl5

dl6

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

V0

V1

V2

MN

V3

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

Floor plate


Formation and patterning of the nervous system

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


Glial differentiation in the caudal neural tube

Basic Organization of the neural tube

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.


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

Many different types of neurons are found in the neural tube


Formation and patterning of the nervous system

Ventral neurons are specified by different combinations of transcription factors

postmitotic

neurons

progenitor cells


Ventral neurons are specified by different combinations of transcription factors

Ventral neurons are specified by different combinations of transcription factors

Pax6 overexpression

Pax6-/-

Pax7

Pax6

Nkx2.2

What regulates the expression of these transcription factors?


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

Notochord is necessary and sufficient for floor plate, motoneuron development

floor plate

notochord

dorsal root ganglia

dorsal root ganglia

motor neurons


Formation and patterning of the nervous system

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

Secondary ventral

floor plate

.

.


Formation and patterning of the nervous system

MN

F

MN

MN

F

MN

Inducer: floor plate

In vitro: Notochord induces both floor plate and motoneurons

So does floor plate

Responder:

Naïve neural tissue

Score for expression

of FP marker (green)

MN marker (orange)

Inducer: notochord


Formation and patterning of the nervous system

Notochord/Floor plate induce ventral neurons. How?

Morphogen

Signaling

relay

V0

V0

V1

V1

V2

V2

MN

MN

V3

V3

F

F


Formation and patterning of the nervous system

Testing the morphogen model:

Morphogen

  • Predictions:

  • Secreted factor from FP

  • Should induce neurons in concentration-dependent manner

  • Initial test:

  • FP conditioned medium induces MN without inducing FP

V0

V1

V2

MN

V3

F


Formation and patterning of the nervous system

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

Criteria:

1. Secreted

2. Right place, right time

3. Necessary

4. Sufficient


Formation and patterning of the nervous system

1. Sonic Hedgehog (Shh) is secreted

N-terminus:

bioactivity

C-terminus:

autocatalytic

Shh precursor:

(45kD)

Autocatalytic

cleavage:

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

Addition of

Cholesterol moiety:

Is it diffusible?

Artificial soluble

form: N-SHH


Formation and patterning of the nervous system

2. Right place, right time: protein?


Formation and patterning of the nervous system

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

- no floor plate development

- dorsal markers

expand ventrally


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

3. Necessity:

Floor plate, motor neurons, and ventral interneurons fail to develop in Shh -/- mice

4. Is Shh sufficient

and

is it a morphogen?


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

Attempt to induce cells in concentration-dependent manner

Use artificial soluble N-Shh


Formation and patterning of the nervous system

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)


Formation and patterning of the nervous system

Notochord/Floor plate induce ventral neurons. How?

Morphogen

Signaling

relay

V0

V0

V1

V1

V2

V2

MN

MN

V3

V3

F

F


Formation and patterning of the nervous system

Some puzzles - evidence for signaling relay

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.

V3

Signaling

relay

F

V1

V0

V1

V2

MN

MN

V3

F

So: can we devise additional tests,

especially to test action at a distance?


Formation and patterning of the nervous system

How to distinguish between models?

Prediction:


Formation and patterning of the nervous system

Mosaic analysis of effect of loss of receptor

Phenotype

No phenotype

Delete receptor for candidate morphogen

in a few cells

Prediction:


Formation and patterning of the nervous system

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

Conventional model of Hedgehog signal reception: Smo (green) has an intrinsic

Intracellular signaling activity that is repressed by direct interaction with Ptc

(red) within the plasma membrane. This repression is released when HH binds.


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

Smo-/- (green) cells fail to express ventral markers (red)

(red and green don’t overlap)

Very ventral

A bit more

dorsal

Broad ventral


Formation and patterning of the nervous system

What about dorsal patterning?


Formation and patterning of the nervous system

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

Inducers: BMPs (perhaps Wnts too?)

Several BMPs

(Wnts too?)

Epidermal ectoderm

RP

Several BMPs

(Wnts too?)

Roof plate

dl1

dl2

Dorsal cells

dl3

dl4

dl5

dl6


Formation and patterning of the nervous system

d

d

d

R

d

d

d

Evidence: in vitro, induce dorsal characteristics

neural plate

epidermis

or

roof plate

or

cells expressing BMPs


Formation and patterning of the nervous system

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

Ablate roof plate genetically

RP

  • Drive expression of toxin

  • in roof plate in knock-in mice

  • Use Diphteria toxin

  • introduce into GDF-7 locus

dl1

dl2

dl3

dl4

dl5

dl6


Formation and patterning of the nervous system

GDF-7

IRES

DTA

Introduce Diphteria Toxin A (DRTA) gene into GDF7 locus

IRES

DTA

GDF-7

Problem:


Formation and patterning of the nervous system

So: make it conditional

GDF-7

IRES

DTA

stop

: silenced

loxP

loxP

+ Cre recombinase

: active

GDF-7

IRES

DTA

Dad carries silenced allele

+

Mom carries Cre gene activated in early fertilized egg

1/4 of embryos get both, so

they get an activated DTA gene under GDF7 promoter


Formation and patterning of the nervous system

Expression of silenced allele: same as that of GDF7

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


Formation and patterning of the nervous system

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


Formation and patterning of the nervous system

  • 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)


Formation and patterning of the nervous system

What about glial cells?


Formation and patterning of the nervous system

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 Nkx2.2

Expands dorsally into this region to overlap with Olig2. (data)


Formation and patterning of the nervous system

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

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


Formation and patterning of the nervous system

Targeted disruption of Olig2.


Loss of motor neurons in olig mouse embryos

Loss of motor neurons in Olig-/- mouse embryos


Spinal cord oligodendrocytes fail to develop in the absence of olig genes

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


But astrocytes are fine

But astrocytes are fine


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