Transcript Neural Patterning II (Pevny)

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
Sensory neurons
conduct impulses from
receptors to the brain
and spinal cord, such
as vision, sound, touch,
pain etc.
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
Basic Organization of the neural tube
Neuronal differentiation in the caudal neural tube
Progenitors
(dividing)
in ventricular zone
Postmitotic
neurons in
“mantle” layer
Many different types of neurons are found in the neural tube
Roof plate
dl1
dl2
dl3
dl4
dl5
dl6
V0
V1
Sensory neurons
conduct impulses from
receptors to the brain
and spinal cord, such
as vision, sound, touch,
pain etc.
Interneuron's: responsible for
the modification, coordination,
between sensory and motor
neurons.
V2
MN
V3
Floor plate
Motor neurons conduct
impulses from the brain and
spinal cord to effectors such as
muscles and glands
Neural stem cell lineage diagram illustrating the generation of different
subtypes of neurons and glia
Basic Organization of the neural tube
Glial differentiation in the caudal neural tube
DORSAL
•
future
astrocytes
vent zone
GRP
cells
VENTRAL
n
e
u
r
o
n
s
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.
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
postmitotic
neurons
Ventral neurons are specified by different combinations of transcription factors
Pax6-/-
Pax6
overexpression
Pax7
Pax6
Nkx2.2
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
dorsal root ganglia
motor neurons
Notochord induces both floor plate and motoneurons. So does floor plate!
Secondary ventral
floor plate
.
.
In vitro: Notochord induces both floor plate and motoneurons
So does floor plate
Responder:
Naïve neural tissue
MN
F
MN
MN
F
MN
Score for expression
of FP marker (green)
MN marker (orange)
Inducer: notochord
Inducer: floor plate
Notochord/Floor plate induce ventral neurons. How?
Morphogen
Signaling
relay
V0
V0
V1
V1
V2
MN
V3
F
V2
MN
V3
F
Testing the morphogen model:
Morphogen
Predictions:
•Secreted factor from FP
•Should induce neurons in concentration-dependent
manner
V0
V1
V2
MN
V3
F
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. Secreted
2. Right place, right time
3. Necessary
4. Sufficient
1. Sonic Hedgehog (Shh) is secreted
C-terminus:
autocatalytic
Shh precursor:
N-terminus:
bioactivity
(45kD)
Autocatalytic
cleavage:
25kD-no known function
Addition of
Cholesterol moiety:
19kD-all Shh signaling
Is it diffusible?
Artificial soluble
form: N-SHH
2. Right place, right time: protein?
3. Necessary: spinal cord development in Shh -/- mice
- no floor plate
development
- dorsal markers
expand 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 sufficient
and
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
Observations:
= Sort of (not very diffusible)
2. Right place, right time
= Sort of (can’t see gradient)
3. Necessary
= Yes (but compatible with every other
model too)
= Yes (pretty good, but not perfect, and
done with artificial soluble Shh)
4. Sufficient
Notochord/Floor plate induce ventral neurons. How?
Morphogen
Signaling
relay
V0
V0
V1
V1
V2
MN
V3
F
V2
MN
V3
F
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
V3
MN
F
So: can we devise additional tests,
especially to test action at a distance?
How to distinguish between models?
Prediction:
Mosaic analysis of effect of loss of receptor
Delete receptor for candidate morphogen
in 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 intrinsic
Intracellular 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 more
dorsal
Broad ventral
What about dorsal patterning?
Similar logic: epidermal ectoderm induces roof plate, which cooperate
to induce dorsal cells.
Inducers: BMPs (perhaps Wnts too?)
Several BMPs
(Wnts too?)
Several BMPs
(Wnts too?)
Epidermal ectoderm
RP
Roof plate
dl1
dl2
dl3
dl4
dl5
dl6
Dorsal cells
Evidence: in vitro, induce dorsal characteristics
d
d
d
R
d d d
neural plate
epidermis
or
roof plate
or
cells expressing BMPs
Evidence: in vivo, How to deal with many BMPs (and Wnts)?
Ablate roof plate genetically
RP
dl1
dl2
dl3
dl4
dl5
dl6
Drive expression of toxin
in roof plate in knock-in mice
•Use Diphteria toxin
•introduce into GDF-7 locus
Introduce Diphteria Toxin A (DR TA) gene into GDF7 locus
IRES
DTA
IRES
DTA
GDF-7
GDF-7
Problem:
So: make it conditional
GDF-7
IRES
loxP
DTA
stop
: silenced
loxP
+ 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, so
they 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 Nkx2.2
Expands 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