Formation and patterning of the nervous system

1. 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

2. Basic Organization of the neural tube

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

4. 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

5. Basic Organization of the neural tube Neuronal differentiation in the caudal neural tube Postmitotic neurons in “mantle” layer Progenitors (dividing) in ventricular zone

6. 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

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

9. 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.

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

11. Many different types of neurons are found in the neural tube

12. Ventral neurons are specified by different combinations of transcription factors postmitotic neurons progenitor cells

13. 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?

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

15. Notochord is necessary and sufficient for floor plate, motoneuron development floor plate notochord dorsal root ganglia dorsal root ganglia motor neurons

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

17. 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

18. Notochord/Floor plate induce ventral neurons. How? Morphogen Signaling relay V0 V0 V1 V1 V2 V2 MN MN V3 V3 F F

19. 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

20. 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

21. 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

22. 2. Right place, right time: protein?

23. 3. Necessary: spinal cord development in Shh -/- mice - no floor plate development - dorsal markers expand ventrally

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

25. 3. Necessity: Floor plate, motor neurons, and ventral interneurons fail to develop in Shh -/- mice 4. Is Shh sufficient and is it a morphogen?

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

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

28. Attempt to induce cells in concentration-dependent manner Use artificial soluble N-Shh

29. 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)

30. Notochord/Floor plate induce ventral neurons. How? Morphogen Signaling relay V0 V0 V1 V1 V2 V2 MN MN V3 V3 F F

31. 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?

32. How to distinguish between models? Prediction:

33. Mosaic analysis of effect of loss of receptor Phenotype No phenotype Delete receptor for candidate morphogen in a few cells Prediction:

34. 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.

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

36. Smo-/- (green) cells fail to express ventral markers (red) (red and green don’t overlap) Very ventral A bit more dorsal Broad ventral

37. What about dorsal patterning?

38. 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

39. d d d R d d d Evidence: in vitro, induce dorsal characteristics neural plate epidermis or roof plate or cells expressing BMPs

40. 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

41. GDF-7 IRES DTA Introduce Diphteria Toxin A (DRTA) gene into GDF7 locus IRES DTA GDF-7 Problem:

42. 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

43. Expression of silenced allele: same as that of GDF7 In embryos with cre (allele activated): roof plate absent!

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

45. 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)

46. What about glial cells?

47. 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)

48. 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)

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

50. Targeted disruption of Olig2.