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 Postmitotic neurons in “mantle” layer Neuronal differentiation in the caudal neural tube

V0 V1 V2 V3 MN 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. 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 postmitotic neurons

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

Inducer: notochord Score for expression of FP marker (green) MN marker (orange) Responder: Naïve neural tissue In vitro: Notochord induces both floor plate and motoneurons So does floor plate FMN Inducer: floor plate FMN

F F V0 V1 V2 V3 MN V0 V1 V2 V3 MN MorphogenSignaling relay 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 V3 MN 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. Secreted 2. Right place, right time 3. Necessary 4. Sufficient

1. Sonic Hedgehog (Shh) is secreted N-terminus: bioactivity C-terminus: autocatalytic Shh precursor: Autocatalytic cleavage: Addition of Cholesterol moiety: Is it diffusible? Artificial soluble form: 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 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 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? F F V0 V1 V2 V3 MN V0 V1 V2 V3 MN MorphogenSignaling relay

Signaling relay Some puzzles - evidence for signaling relay F V0 V1 V2 V3 MN 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 Jan 26;84(2):

Prediction: How to distinguish between models?

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?) dl1 dl2 dl3 dl5 dl4 dl6 Epidermal ectoderm Roof plate RP Dorsal cells Several BMPs (Wnts too?) Several BMPs (Wnts too?)

Evidence: in vitro, induce dorsal characteristics epidermis or roof plate or cells expressing BMPs neural plate Rdddddd

Evidence: in vivo, How to deal with many BMPs (and Wnts)? Ablate roof plate genetically dl1 dl2 dl3 dl5 dl4 dl6 RP Drive expression of toxin in 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 IRESDTA GDF-7IRESDTA Problem:

So: make it conditional GDF-7IRESDTA stop loxP : silenced + Cre recombinase GDF-7IRESDTA : 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