1. Neuroinduction
Diffusible morphogen

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 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
+”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:

9. 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.)

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

11. 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)

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

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

14. 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)

15. 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)

16. 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

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. 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)

19. 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)

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

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

22. 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)

23. Molecular Mechanism of Neuralization

24. TGF-b 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-b 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-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.