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Hedgehog signaling pathway
• Indian hedgehog: expressed in gut and
chondrocytes
• Desert hedgehog: expressed in sertoli cells of
the testes
• Sonic hedgehog: Involved in many
developmental processes. Best characterized
Gilbert, Figure 6.24
Shh and Cholesterol
• Amino-terminal portion is secreted and
functional (also palmitoylated)
• Cholesterol is required for the cleavage of
Shh (N-terminal is active peptide)
• Patched protein requires cholesterol in order
to function (sterol sensing domain)
• Mutations or chemicals that interfere with
cholesterol biosynthesis cause abnormalities
akin to SHH knockout animals
Patched protein (Ptc)
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Is the Shh “receptor”
12 Transmembrane domains
Negative regulator of Shh signaling
2 Vertebrate homologs Ptc-1 and Ptc-2
Ptc-1 is upregulated in response to Shh
Ptc has a sterol sensing domain (SSD)
Smoothened protein (Smo)
• 7 transmembrane domains (like G-protein
coupled receptors)
• Positive regulator of the Shh pathway
• Does not bind to Shh but is signaling
component of the receptor complex
• Smo is constitutively active in the absence
of Ptc
Cubitus interruptus (Ci)/Gli
transcription factor family
• Zn++ finger transcription factors
• Recognize a 9bp consensus sequence in the
promoters of several Hh target genes
• In the absense of Hh the full length protein
(Ci-155) is in the cytoplasm and gets
processed into a 75 kD N-terminal repressor
form (Ci-75)
Modificaiton of Ci in Drosophila
From Munroe, et. al. (1999) Exp. Cell Res. 253:25-33
Ci-75 repressor generation is:
• Triggered in part by protein kinase A (Pka)
phosphorylation of the C-terminus
• Possible participation of Slimb, a ubiquitin
targeting protein and the proteosome
• Cleavage of Ci-155 is blocked by the
presence of Hedgehog (Smo signaling)
Hedgehog activation of
“positively regulating” Ci-155
• Facilitated by a serine/threonine kinase
called fused
• Antagonized by “Supressor of fused” Su(fu)
• Mutant embryos for Fu or Ci have defects
resembling Hh deficiency
• Pka or Ptc mutants display ectopic
expression of Hh target genes
• CBP/p300 is a co-activator for Ci-155
Role of microtuble association
• Cos-2 tethers Ci and Fu to the microtubles
• Cos-2 is a negative regulator of the pathway
• Hh signaling leads to Cos-2 dissociation and
phosphorylation of both Fu and Cos-2
• Unanswered questions:
– How does Smo signal to the microtuble
associated complex?
– What exactly is in the complex?
Hedgehog signaling pathway
From Munroe, et. al. (1999) Exp. Cell Res. 253:25-33
Vertebrate versions of Ci (the Gli
proteins)
• Behave differently in this pathway than Ci
does in Drosophila
• Gli-1, Gli-2 and Gli-3 appear to be
transcriptionally regulated in response to
Shh
• See Munroe et. al. (1999) for several
assorted speculations
Evidence for Sonic Hedgehog
involvement in development
• Mouse knockout results in abnormal limb
development and cyclopia
• Gli-3/Ci mutations lead to Grieg's
cephalopolysyndactyly and other inherited
diseases
• Activating mutations can cause cancers
(basal cell carcinomas-epidermal)
Wnt/wingless pathway
• Ligands, Wnt-secreted glycoproteins
– At least 16 members of the Wnt ligand family
• Receptors are relatives of Frizzled (Frz)
– 11 Frizzled homologs in vertebrates
• Soluble Frz related proteins (Frps) exist
– Antagonists of wnt signaling
– Proposed to play a role in neural development
Wnt signal transduction
Fig 6.23 of Gilbert
Negative regulators of wnt signals
• Glycogen synthase kinase (GSK)-3b
– Binds and phosphorylates several proteins in the
wnt pathway to downregulate b-catenin
• Adenomatous polyposis coli (APC)
– Tumor suppressor in which mutations eliminate
binding sites for Axin and b-catenin
• Axin: Binds APC, b-catenin, GSK-3b and
dishevelled (Dvl)
– Facilitates phosphorylation of APC and b-catenin
by GSK-3b
A closer look at b-catenin regulation
From Polakis (2000) Genes Dev. 14:1837-1851
b-catenin mutations in tumors
Table 1 From Polakis (2000) Genes Dev. 14:1837-1851
Clustered mutations in wnt signaling
components affect negative interactions
• b-catenin mutations in the N-terminal
region affect amino acids necessary for its
phosphorylation dependent interaction with
protein degradation machinery
• APC mutations affect axin and b-catenin
binding
• Axin mutations truncate the protein thus
eliminating b-catenin binding sites
The wnt pathway is loaded with protooncogenes and tumor suppressors
Figure 1 From
Polakis (2000)
Genes Dev.
14:1837-1851
Fibroblast Growth Factors (FGF)
• 19-22 FGF family members (FGF-1 called
acidic FGF, FGF-2 called basic FGF)
• Some lack signal sequences for secretion
• Contain heparin/heparan sulfate binding
domains (critical for the function of some FGFs)
• Four FGF receptors. Several FGFs bind to more
than one FGF receptor with high affinity
• FGFRs are transmembrane tyrosine kinases
Evidence for FGF involvement in
development: KO mice
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FGF2: defects in vascular systems
FGF3: inner ear and tail development
FGF4: early post-implantation lethality
FGF5 and FGF7: Abnormal hair phenotype
FGF8: Early embryonic lethality.
Conditional KO showed effect on brain
development
• FGF10: lung and limb development
FGF Receptor KO phenotypes
• FGFR-1: Postimplantation embryonic lethal
with vertebral malformations
• FGFR-2: Postimplantation embryonic lethal
with abnormal limb development
• FGFR-3: Defective chondrocyte generation
• FGFR-4: Perfectly normal
• Websites 6.2 and 6.6 deal with FGF & FGFR
Facts on Retinoic Acid
• RA is a teratogen, causes birth defects
• Interfering with RA biosynthesis causes
developmental abnormalities linked to Hox
gene expression
• Exerts it effect through the retinoic acid
receptors (RAR) which dimerizes with other
steroid receptors to activate transcription
• Important in development of limbs, neural
tube and in anterior-posterior patterning