Lecture IV' Mechanisms of Neural Development

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Lecture IV. Mechanisms of Neural Development

• Bio 3411
• Wednesday
• September 2, 2009

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• T. Woolsey
• 3802 North Building
• 362-3601
• woolseyt_at_medicine.wustl.edu

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Readings

• NEUROSCIENCE 4th ed, pp 545-575 (sorta)
• References
• Fainsod, A., Steinbeisser, H., De Robertis, E.
  M. (1994). EMBO J, 13(21), 5015-5025.
• Hemmati-Brivanlou, A., Melton, D. (1997). Annu
  Rev Neurosci, 20, 43-60.
• Melton, D. A. (1987). Nature, 328(6125), 80-82.
• Sasai, Y., De Robertis, E. M. (1997). Dev Biol,
  182(1), 5-20.
• Smith, W. C., Harland, R. M. (1992). Cell,
  70(5), 829-840.
• Weeks, D. L., Melton, D. A. (1987). Proc Natl
  Acad Sci U S A, 84(9), 2798-2802.
• Wilson, P. A., Hemmati-Brivanlou, A. (1995).
  Nature, 376(6538), 331-333.
• Xanthos, J. B., Kofron, M., Wylie, C., Heasman,
  J. (2001). Development, 128(2), 167-180.
• Zimmerman, L. B., De Jesus-Escobar, J. M.,
  Harland, R. M. (1996). Cell, 86(4), 599-606.
• ______________________
• (pdfs on course website http//artsci.wustl.edu
  /sdanker/index.html)

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Embryogenesis 1. Maternal cytoplasmic
determinants. 2. Fertilization creates
dorsal-ventral axis. 3. Cell division. 4.
Blastula created. 6. Ectoderm, mesoderm,
endoderm created. by molecular signals along
the Animal/Vegetal axis. 5. Gastulation. 6.
Spemann organizer creates anterior-posterior
axis. 7. Notocord induces the Neural Plate. 8.
Neurulation forms the Neural Tube. 9. Neural
crest cells form the PNS. 10. Segmentation
Cephalization (anterior enlargement)
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• Cell Signaling
• Discovery of the Organizer
• How Could this Work?
• 4) The Answer
• 5) Blockers
• 6) Current View
• 7) Summary

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• Cell Signaling

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Neuroinduction
Diffusible morphogen
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Intracellular Signaling through a Kinase
Cascade Signal Amplification (Suppression) and
Multiple Control Points
Ligand
Receptor
Kinase Cascade
Effector Proteins (transcription factors, ion
channels, cytoskeletal proteins, enzymes, etc)
Scaffolding Proteins bind multiple signaling
molecules to organize specific signaling pathways
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Endoderm and Mesoderm involute with
gastrulation Induction of the Neural Plate from
Neuroectoderm, by the underlying, closely apposed
Mesoderm.
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• Discovery of the Organizer

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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.

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Mangold Spemann Experiments (1924)
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• How Could this Work?

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Explant Experiments with Animal Caps from
Amphibian Blastula Puzzling Results
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Isolating Inducing Factors that Promote Neuronal
Differentiation Sigma Catalog Experiments
Result in Further Confusion
(Many positives, including apparently
non-biological factors!)
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Models for Neural Induction
Epidermal factor
Neuronal factor
(default)
Neuronal factor
Epidermal factor
(default)
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TGF-b Proteins Signal Through Heterodimeric Recep
tors and Smad Transcription Factors
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• The Answer

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A Dominant-Negative Receptor Subunit Blocks
Activation of the Signaling Pathway
(Hemmati-Brivanlou and Melton, 1992)
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Blocking TGF-b Signaling by a Dominant-Negative
Receptor Causes Isolated Neuroectoderm to Become
Neuronal
TGF-b Signaling
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BMP-4 (TGF-b) Signaling Results in Neural
Epidermal Induction
TGF-b Transforming Growth Factor - b
BMP-4 Bone Morphogenic Protein - 4
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Models for Neural Induction
BMP-4
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BMP-4 (Secreted by Neuroectodermal Cells)
Inhibits Neuronal Fate and Promotes Epidermal
Fate. Tissue Dissociation dilutes BMP-4 activity
(Wilson and Hemmati-Brivanlou, 1995)
(Endogenous BMP-4 Diluted)
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Recombinant BMP-4 Promotes Epidermal Fate and
Inhibits Neuronal Fate
Intact caps
Dispersed caps
Keratin (epidermal marker)
NCAM (neuronal marker)
(Wilson and Hemmati-Brivanlou, 1995)
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BMP-4 mRNA is Expressed in Presumptive Ectoderm
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• Blockers

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Are there native anatgonists of BMP-4? Secreted
from underlying mesoderm? Yes chordin / noggin
/ follistatin. And they are enriched in the
Spemann-Mangold Organizer!
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(No Transcript)
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Differential Substractive Screen yields Chordin,
a BMP-4 antagonist (1994)
Generate cDNA library from oocytes
Probe cDNA library with differential probes
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Functional Expression Cloning yields noggin, a
BMP-4 anatagonist (1992)
(Smith and Harland, 1992)
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Chordin/Noggin/Follistatin directly bind to and
inactivate BMP-4
(Stays in loading well)
(Migrates into gel)
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Structure of Noggin-BMP complex
noggin
Binding Sites
(Groppe, et al., 2002)
Receptor (Type-II)
Receptor (Type-I)
BMP-7
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Molecular Mechanism of Neuralization
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• Current View

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TGF-b proteins signal through heterodimeric recep
tors and Smad transcription factors
Multi-step pathway (kinases, scaffolding
proteins)
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Neural induction mechanisms are conserved
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BMP-4 is only one member of the large
evolutionarily conserved TGF-b gene family, which
mediates many different tissue inductive events.
Relationships between members of the TGF-b super
family. (After Hogan, 1996)
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• Summary

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Neurogenesis Inductive Mechanisms
1. Neuroectodermal cells choose either a neuronal
or epidermal fate. 2. Interactions between
mesoderm and neuroectoderm induce neuroectoderm
to adopt the neural fate. 3. Induction is
signaled by Bone Morphogenic Protein-4 (BMP-4),
a protein made and secreted 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.
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Neurogenesis Inductive Mechanisms
6. Neuroectodermal cells become neurons by
suppression of BMP-4 activity by secreted
antagonists from underlying mesodermal cells. 7.
The default state of neuroectodermal cells is
neuronal. 8. This mechanism is conserved between
vertebrates and invertebrates. 9. BMP-4 is a
member of the Transforming Growth Factor (TGF-b)
family of signaling molecules. 10. Similar
signaling events in the nervous system mediate
changes in later development stages and in adult
plasticity.
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END