Neuroinduction Diffusible morphogen

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 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.

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 +”Epidermal factor” Epidermis Presumptive Neuroectoderm Neurons +”Neuronal factor” Presumptive Neuroectoderm Epidermis Neurons +”Neuronal factor” (“default”) Model 2: Presumptive Neuroectoderm Epidermis Neurons +”Epidermal factor” (“default”) Model 3:

TGF-b 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-b Signaling by a Dominant-Negative Receptor Causes Isolated Neuroectoderm to Become Neuronal (+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 (Intact)

BMP-4 (TGF-b) Signaling Results in “Neural Epidermal Induction” TGF-b: Transforming Growth Factor - b BMP-4: Bone Morphogenic Protein - 4

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

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

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)

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

Noggin cRNA injections rescue ventralized embryos 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) Generate cDNA library from oocytes Probe cDNA library with differential probes (Sasai and DeRobertis. 1994)

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

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

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

Molecular Mechanism of Neuralization

TGF-b 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-b 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-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.