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Diffusible morphogen

Neuroinduction. Diffusible morphogen. Ligand. Receptor. Kinase Cascade. A. C. B. Effector Proteins ( transcription factors , ion channels, cytoskeletal proteins, enzymes, etc…). Scaffolding Proteins bind multiple signaling molecules to organize specific signaling pathways.

damian-gaines
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Diffusible morphogen

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  1. Neuroinduction Diffusible morphogen

  2. Ligand Receptor Kinase Cascade A C B Effector Proteins (transcription factors, ion channels, cytoskeletal proteins, enzymes, etc…) Scaffolding Proteinsbind multiple signaling molecules toorganize specific signaling pathways Gene Activation/Repression Intracellular Signaling through a Kinase Cascade; Signal Amplification and Multiple Control Points

  3. Ant Post Endoderm and Mesoderm involute with gastrulation: Induction of the Neural Plate from Neuroectoderm, by the underlying, closely apposed Mesoderm.

  4. Hilde Mangold and Hans Spemann • Key experiments performed in 1921-1923 at the • University of Freiburg, Germany. • Hilde Mangold was a 24 year old graduate • student when she performed these experiments. • She died tragically in an accidental alcohol heater • explosion. • Hans Spemann was awarded the Nobel Prize in 1935.

  5. Hilde Mangold and Hans Spemann Experiments (1924)

  6. ! Explant Experiments with Animal Caps from AmphibianBlastula: Puzzling Results…

  7. + Candidate Neuroinducing Factors ? (Intact) Isolating Inducing Factors that Promote Neuronal Differentiation; “Sigma Catalog” Experiments Result in Further Confusion… (Many positives, including apparently non-biological factors!)

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

  9. Vg1 (Melton, 1987; Weeks and Melton, 1987) (K. Mowry Lab, Brown Univ.) Multi-step pathway (kinases, scaffolding proteins) TGF-b Proteins Signal Through Heterodimeric Receptors and Smad Transcription Factors

  10. A Dominant-Negative Receptor Subunit Blocks Activation of the Signaling Pathway (Hemmati-Brivanlou and Melton, 1992)

  11. (+Dominant-Negative Type II Receptor cRNA) TFG-b Signaling Blocked by expression of Dom-Neg Type II Receptor Subunit Animal Cap (Intact) Animal Cap (Intact) + TGF-b Signaling + TGF-b Signaling (Intact) Blocking TGF-b Signaling by a Dominant-Negative Receptor Causes Isolated Neuroectoderm to Become Neuronal

  12. BMP-4 (TGF-b) Signaling Results in “NeuralEpidermal Induction” TGF-b: Transforming GrowthFactor - b BMP-4: BoneMorphogenic Protein - 4

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

  14. + BMP-4 [BMP-4] BMP-4 (Secreted by Neuroectodermal Cells) Inhibits Neuronal Fate and Promotes Epidermal Fate. Tissue Dissociation dilutes BMP-4 activity (Endogenous BMP-4 Diluted) (Wilson and Hemmati-Brivanlou, 1995)

  15. Recombinant BMP-4Promotes Epidermal Fate and InhibitsNeuronal Fate Dispersed caps Intact caps Keratin (epidermal marker) NCAM (neuronal marker) (Wilson and Hemmati-Brivanlou, 1995)

  16. Blastopore Stg 14.0 Stg 11.5 Stg 11.0 A D D D D V V P V V Neural Crest Stg 23.0 Stg 24.0 (Fainsod, et al., 1995) A A D A P P V P BMP-4 mRNA is Expressed in Presumptive Ectoderm

  17. Are there native anatgonists of BMP-4? Secreted from underlying mesoderm? Yes… chordin / noggin / follistatin. And they are enriched in the Spemann-Mangold Organizer!

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

  19. Differential Substractive Screen yields Chordin, a BMP-4 antagonist (1994) Generate cDNA library from oocytes Probe cDNA library with differential probes (Sasai and DeRobertis. 1994)

  20. (Reiterate with positive fraction) Functional Expression Cloning yields noggin, a BMP-4 anatagonist (1992) (Smith and Harland, 1992)

  21. Mechanism: Chordin / noggin / follistatin anatagonize BMP-4 activity by directly binding and inactivating BMP-4 (Migrates into gel) (Stays in loading well)

  22. noggin Receptor (Type-II) Receptor (Type-I) BMP-7 Crystal structure of Noggin-BMP complex confirms biochemical/functional studies and identifies binding sites Binding Sites (Groppe, et al., 2002; Choe Lab)

  23. Molecular Mechanism of Neuralization

  24. TGF-b proteins signal through heterodimeric receptors and Smad transcription factors Multi-step pathway (kinases, scaffolding proteins)

  25. Vertebrates Drosophila Ligand Receptor Antagonist Transcription Factor 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

  26. BMP-4 is only one member of the large evolutionarily conserved TGF-b gene family, which mediates many different tissue inductive events.

  27. 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 antagonists from underlying mesodermal cells.

  28. Neurogenesis: Inductive Mechanisms (cont.) 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 selectively and focally re-employed later in the nervous system to mediate fine tuning at late developmental stages and adult plasticity.

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