Presentation on theme: "Diffusible morphogen Neuroinduction. Intracellular Signaling through a Kinase Cascade; Signal Amplification and Multiple Control Points Ligand Receptor."— Presentation transcript:
Diffusible morphogen Neuroinduction
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
Endoderm and Mesoderm involute with gastrulation: Induction of the Neural Plate from Neuroectoderm, by the underlying, closely apposed Mesoderm. Ant Post
Hilde Mangold and Hans Spemann Key experiments performed in 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.
Hilde Mangold and Hans Spemann Experiments (1924)
Explant Experiments with Animal Caps from Amphibian Blastula: Puzzling Results… !
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!)
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:
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.)
A Dominant-Negative Receptor Subunit Blocks Activation of the Signaling Pathway (Hemmati-Brivanlou and Melton, 1992)
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)
TGF- Transforming Growth Factor - BMP-4 Bone Morphogenic Protein - 4 BMP-4 (TGF- Signaling Results in “Neural Epidermal Induction”
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”
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)
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
Are there native anatgonists of BMP-4? Secreted from underlying mesoderm? Yes… chordin / noggin / follistatin. And they are enriched in the Spemann-Mangold Organizer!
Chordin expresses in mesoderm (Sasai, et al., 1995) Noggin cRNA injections rescue ventralized embryos +Noggin injection 1pg 10pg 100pg (Smith and Harland, 1992)
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
Functional Expression Cloning yields noggin, a BMP-4 anatagonist (1992) (Smith and Harland, 1992) (Reiterate with positive fraction)
Mechanism: Chordin / noggin / follistatin anatagonize BMP-4 activity by directly binding and inactivating BMP-4 (Stays in loading well) (Migrates into gel)
(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
Molecular Mechanism of Neuralization
TGF- proteins signal through heterodimeric receptors and Smad transcription factors Multi-step pathway (kinases, scaffolding proteins)
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
BMP-4 is only one member of the large evolutionarily conserved TGF- gene family, which mediates many different tissue inductive events.
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.
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.