Post on 04-Jan-2016
description
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