Download Lecture 18 – Molecular Mechanisms of Neural

Bio 3411, Fall 2006
Aguan Wei
979 McDonnell Sciences Building, Medical School Campus, WUMS.
email: [email protected]
Lecture 18 – Molecular Mechanisms of Neural Induction.
1. During development, embryonic cells signal to each other with secreted diffusible
molecules that instruct neighboring cells to change their pattern of gene expression.
This signaling event is termed induction. Because the signaling molecule instructs
the developmental fate of the recipient cell, it is called a morphogen.
2. Induction is observed in classic experiments manipulating amphibian blastula:
a. (1924) Hilde Mangold and Hans Spemann. Transplanting the dorsal lip of
the blastopore (presumptive mesoderm) from one embryo (in late blastula
stage) to the ventral pole of a second embryo (same late blastula stage) results
in an embryo with a second neuroaxis! The transplanted tissue is capable of
“organizing” a second neuroaxis, so this region of the embryo is termed the
“Spemann-Mangold Organizer”.
b. (1969) Pieter Nieuwkoop, (1989) Grunz and Tacke, (1991) Godsave and
Slack. Isolated animal caps (neuroectodermal cells) from blastulas can be
cultured in vitro. Unlike the embryo, cultured intact animal caps develop into
epidermal cells, instead of neurons. However, dissociated animal cap cells
develop into neurons. Close contact between developing neuroectodermal
cells results in expression of epidermal cell fate, whereas loss of close contact
results in neuronal cell fate.
c. These two classic experiments form the basis for the molecular analysis of
neural induction.
3. Experimental manipulations show that inductive signaling occurs between
neighboring cells of the developing neuroectoderm, and between developing
neuroectoderm and mesoderm to generate the neural plate. Normal morphogenic
movements during gastrulation permit induction to occur, by placing signaling
and recipient tissues together in close apposition.
4. Developing neuroectodermal cells secrete a protein which signals neighboring cells to
inhibit the neural fate and promote the epidermal fate. This signaling protein is Bone
Morphogenic Protein-4 (BMP-4), a member of the Transforming Growth Factorβ (TGF-β) family of proteins.
Experimental evidence; (1990-1994) Ali Hemmati-Brivanlou and Doug Melton:
a) Expression of exogenous dominant-negative TGF-β receptors, blocks the
function of native TGF-β receptors, leading to neuronal differentiation by
intact animal caps.
b) BMP-4 is expressed in neuroectodermal cells.
c) Addition of BMP-4 to cultures of dissociated animal caps, causes
differentiation of epidermal cells, due to repression of the neural fate.
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5. Developing mesodermal cells secrete multiple protein factors that antagonize the
action of BMP-4. Three main antagonists of BMP-4 are chordin, noggin and
follistatin.
All antagonist factors are:
a) Expressed in the Spemann-Mangold organizer and in developing mesoderm at
the right time to act in neuroinduction.
b) Capable of inducing a second neural axis in embryos, similar to a transplanted
Spemann-Mangold organizer.
c) Capable of inducing the neuronal fate in cultures of intact animal caps.
6. Identification of chordin and noggin relied upon clever molecular cloning strategies.
A) (1992) William Smith and Richard Harland. A functional expression cloning
strategy yields noggin.
a) Ventralized blastula (no neural plate develop) were produced en masse, by
treating blastulas with ultraviolet irradiation. Ventralized blastula used to
assay for candidate factors promoting neural plate formation.
b) mRNAs expressed by blastula were converted to cDNAs, then cloned into a
bacterial plasmid vector to generate a representative cDNA library.
c) Pools of cDNAs clones were transcribed into cRNA, in vitro, then injected
into ventralized blastula.
d) Each positive pool of cDNAs (capable of inducing a neural plate in
ventralized blastula) was fractionated and re-assayed.
e) Reiterative rounds of fractionation and screening for positive pools of cDNAs,
with each successive positive pool containing fewer numbers of clones,
resulted ultimately in the isolation of a single clone capable of full neuralizing
activity. 10 pools of ~10,000 clones screened, yielding one clone for noggin.
B) (1994) Yoshiki Sasai and Eddy De Robertis. A differential screening strategy
yields chordin.
a) An embryonic cDNA library was plated in duplicate, then screened with
probes made from mRNAs enriched for “dorsalized” (expanded Spemann
organizer, with LiCl treatment) or “ventralized” (no Spemann organizer, with
UV treatment) embryos. Clones isolated which were positive for
“dorsalized” probe and negative for “ventralized” probe. ~25,000 clones
screened, yielding 6 clones.
b) Positive clones further assayed for ability to induce neural axis in ventralized
blastula and for appropriate pattern of tissue expression by in situ
hybridization.
c) Chordin identified from 3 of the 6 candidate clones. Sequence of chordin
reveals evolutionary conservation with Drosophila short gastrulation (sog),
which functions in Drosophila neuronal induction.
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7. Chordin/Noggin/Follistatin antagonizes BMP-4 activity by directly binding and
inactivating BMP-4. Evidence provided by in vitro protein association assays.
8. Molecular model of neural induction:
a) The “default” fate of neuroectodermal cells is neuronal.
b) BMP-4 secreted by neuroectodermal cells inhibits neuronal fate and promotes
epidermal fate. This inductive signal between neighboring cells acts to
promote neuroectodermal cells towards the epidermal fate, and maintains the
epidermal fate in intact cultured animal caps.
c) Antagonists of BMP-4 (Chordin, Noggin and Follistatin) are secreted by
mesoderm, which underlies developing neuroectoderm in the gastrula.
d) Chordin/Noggin/Follistatin diffuse to overlying neuroectoderm, and inactivate
BMP-4 activity by directly binding BMP-4.
e) Inactivation of BMP-4 activity in the developing neuroectoderm, releases
repression of neuronal fate, allowing expression of “default” neuronal fate and
formation of the neural plate. Loss of BMP-4 activity is mimicked in
dispersed neuroectodermal cultures by dilution of secreted BMP-4 in vitro,
resulting in expression of the neuronal fate.
9. This model of neuronal induction by a BMP-4 signaling pathway is evolutionarily
conserved in Drosophila. Studies of flies carrying mutations in decapentapelegic
(dpp) [homolog of BMP-4] and short gastrulation (sog) [homolog of chordin],
suggest that these molecules serve the same function in invertebrate neural induction.
10. The TGF-β signaling pathway, exemplified by BMP-4, is reiterated throughout the
body of the developing embryo by other members of the TGF-β gene family to
generate many other vertebrate tissues and organs.
References:
1. Hemmati-Brivanlou, A. and Melton, D. (1997) Vertebrate embryonic cells will
become nerve cells unless told otherwise. Cell 88: 13-17.
2. Harland, R and Gerhart, J. (1997) Formation and function of Spemann’s organizer.
Annual Review of Cell and Developmental Biology 13: 611-67.
3. Gilbert, S. F. Developmental Biology, 3rd Edition. (1997) Chapter 15, Specification of
cell fate by progressive cell-cell interactions. pages 591-633.
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Neurogenesis: Inductive Mechanisms in a Nutshell.
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 proteins from underlying
mesodermal cells.
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-β)
family of signaling molecules. Similar signaling events maybe
locally re-employed later in the developing nervous system.
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