Neuroinduction

1. Neuroinduction Diffusible morphogen

2. Neural plate (Apposition of Different Germbands) Ant Post Endoderm and Mesoderm Involute with Gastrulation: Induction of the Neural Plate from Neuroectoderm, by the Underlying, Closely Apposed Mesoderm.

3. Hilde Mangold and Hans Spemann • Key experiments performed in 1924 at the • University of Freiburg, Germany. • Hilde Mangold was a 26 year old graduate • student. She died tragically in an accidental • heater explosion. • Hans Spemann was awarded the Nobel Prize • in 1935.

4. A) Mangold-Spemann Organizer B) The “organizer” is sufficient to induce a second nervous system. Hilde Mangold and Hans Spemann Experiments (1924).

5. Explant Experiments with Animal Caps from AmphibianBlastula. (Nieuwkoop, 1969; Grunz and Tacke, 1989; Godsave and Slack, 1991)

6. Isolating Inducing Factors that Promote Neuronal Differentiation. ? + Candidate Neuroinducing Factors

7. Models for Neural Induction Model 1: +”Epidermal factor” Epidermis Presumptive Neuroectoderm Neurons +”Neuronal factor” Epidermis Model 2: (“default”) Presumptive Neuroectoderm Neurons +”Neuronal factor” +”Epidermal factor” Epidermis Model 3: Presumptive Neuroectoderm Neurons (“default”)

8. TGF-b Proteins Signal Through Heterodimeric Receptors and Smad Transcription Factors. (activin)

9. A Dominant-Negative Receptor Subunit Blocks Activation of the Signaling Pathway. Dominant-Negative (i.e. poison) Type II Receptor Subunit (Hemmati-Brivanlou and Melton, 1992)

10. (-TGF-b Signaling) Epidermal Cells TGF-b Signaling is required to promote epidermal fate and inhibit neuronal fate. Neuronal Cells Blocking TGF-b Signaling by Expression of a Dominant-Negative Receptor Causes Isolated Neuroectoderm to Become Neuronal. TGF-b Signaling Blocked by Expression of Dominant-Negative Receptor Subunit (+TGF-b Signaling)

11. BMP-4 / TGF-b Signaling Results in “NeuralEpidermal Induction”. TGF-b: Transforming Growth Factor - b BMP-4: BoneMorphogenic Protein - 4

12. Models for Neural Induction Model 1: +”Epidermal factor” Epidermis Presumptive Neuroectoderm Neurons +”Neuronal factor” Epidermis Model 2: (“default”) Presumptive Neuroectoderm Neurons +”Neuronal factor” +”Epidermal factor” Epidermis Model 3: Presumptive Neuroectoderm Neurons (“default”) (+BMP-4) (-BMP-4)

13. BMP-4 (Secreted by Neuroectodermal Cells) Inhibits Neuronal Fate and Promotes Epidermal Fate. Dissociation dilutes BMP-4 activity.

14. Recombinant BMP-4 promotes epidermal fate and inhibits neuronal fate. Dispersed caps Intact caps Keratin (epidermal marker) NCAM (neuronal marker) (Wilson and Hemmati-Brivanlou, 1995) BMP-4 mRNA is expressed in presumptive ectoderm. (Fainsod, et al., 1995)

15. Are there native anatgonists of BMP-4? Secreted from underlying mesoderm? Yes… chordin / noggin / follistatin.

16. Chordin expresses in mesoderm (Sasai, et al., 1995) +Noggin injection Noggin cRNA injections rescue ventralized embryos. 1pg 10pg 100pg (Smith and Harland, 1992)

17. Differential Substractive Screen Yields Chordin, a BMP-4 antagonist. (1994) Spemann Organizer mRNAs to cDNAs cDNA Library P32 -Labelled cDNA Probes +Li+ Spemann Organizer (+organizer probe) (-organizer probe) (Sasai and DeRobertis, 1994) * *

18. Functional Expression Cloning Yields noggin, a BMP-4 anatagonist. (1992) Fractionated cDNA Library * Spemann Organizer (Inject Pools of cRNAs) neuralization noggin (Smith and Harland, 1992)

19. Chordin / noggin / follistatin anatagonize BMP-4 activity by directly binding and inactivating BMP-4. BMP-4 noggin coupled to beads Mix Wash unbound BMP-4 (keep beads) Separate on gel + BMP-4 (Zimmerman, Jesus- Escoba and Harland, 1996) ?

20. Crystal Structure of Noggin-BMP Complex Confirms Biochemical and Functional Studies noggin Receptor (Type-II) Receptor (Type-I) BMP-7 (Groppe, et al., 2002)

21. Molecular Mechanism of Neuralization. + Epidermal - Neuronal Neuroectoderm induced to become neuronal by suppression of BMP-4, which inhibits the default neuronal fate.

22. TGF-b Proteins Signal Through Heterodimeric Receptors and Smad Transcription Factors. TGF-b antagonists

23. Vertebrates Drosophila BMP-4 Type I Type II Type III noggin chordin follistatin Smad1 Smad2 Smad3 Smad4 Smad5 decapentaplegic (dpp) punt thick veins (tkv), saxophone (sax) Short-gastrulation (sog) Mothers against decapentaplegic (MAD) Medea The Mechanism for Neural Induction is Evolutionarily Conserved between Vertebrates and Invertebrates. Ligand Receptor Antagonist Transcription Factor

24. BMP-4 is a member of the large evolutionarily conserved TGF-b gene family, which mediates many tissue inductive events. (kidney and urinary system) (dorsal neural tube) (brain) (KO with eye defects) (heart, KO is lethal) (KO is lethal) (bone) (Left/Right Symmetry)

25. Neurogenesis: Inductive Mechanisms. 1. Neuroectodermal cells choose either a neuronal or epidermal cell fate. 2. Interactions between mesoderm and neuroectoderm induce neuroectoderm to adopt the neural fate. 3. Induction acts through signaling by a secreted protein, Bone Morphogenic Protein-4 (BMP-4), made by neuroectodermal cells. 4. BMP-4 inhibits neuralization and promotes the epidermal fate in neighboring cells. 5. Mesodermal cells secrete proteins (Chordin, Noggin, Follistatin) which directly bind and antagonizes BMP-4 activity. 6. Neuroectodermal cells become neurons by suppression of BMP-4 activity by secreted proteins from underlying mesodermal cells. 7. The “default” state of neuroectodermal cells is neuronal. 8. This inductive mechanism is conserved between vertebrates and invertebrates. 9. BMP-4 is a member of the Transforming Growth Factor (TGF-b) family of signaling molecules. Similar signaling events maybe locally re-employed later in the developing nervous system.