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Diffusible morphogen Neuroinduction. Intracellular Signaling through a Kinase Cascade; Signal Amplification and Multiple Control Points Ligand Receptor.

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Presentation on theme: "Diffusible morphogen Neuroinduction. Intracellular Signaling through a Kinase Cascade; Signal Amplification and Multiple Control Points Ligand Receptor."— Presentation transcript:

1 Diffusible morphogen Neuroinduction

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

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

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 Amphibian Blastula: Puzzling Results… !

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

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

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

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

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

12 TGF-  Transforming Growth Factor -  BMP-4  Bone Morphogenic Protein - 4 BMP-4 (TGF-  Signaling Results in “Neural Epidermal Induction”

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

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

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

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

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

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

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

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

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

23 Molecular Mechanism of Neuralization

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

25 The mechanism for neural induction is evolutionarily conserved between vertebrates and invertebrates Ligand Receptor Antagonist Transcription Factor BMP-4 Type I Type II Type III noggin chordin follistatin Smad1 Smad2 Smad3 Smad4 Smad5 Vertebrates decapentaplegic (dpp) punt thick veins (tkv), saxophone (sax) Short-gastrulation (sog) Mothers against decapentaplegic (MAD) Medea Drosophila

26 BMP-4 is only one member of the large evolutionarily conserved TGF-  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-  ) 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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