PUBLICATION

The Extracellular Domain of Smoothened Regulates Ciliary Localization and Is Required for High-Level Hh Signaling

Authors

Aanstad, P., Santos, N., Corbit, K.C., Scherz, P.J., Trinh, L.A., Salvenmoser, W., Huisken, J., Reiter, J.F., and Stainier, D.Y.

ID

ZDB-PUB-090526-31

Date

2009

Source

Current biology : CB 19(12): 1034-1039 (Journal)

Registered Authors

Aanstad, Pia, Reiter, Jeremy, Stainier, Didier, Trinh, Le

Keywords

none

MeSH Terms

Amino Acid Sequence
Animals
Cilia*/physiology
Cilia*/ultrastructure
Hedgehog Proteins/genetics
Hedgehog Proteins/metabolism*
Molecular Sequence Data
Morpholines/metabolism
Protein Structure, Tertiary
Purines/metabolism
Receptors, Cell Surface/genetics
Receptors, Cell Surface/metabolism*
Receptors, G-Protein-Coupled/genetics
Receptors, G-Protein-Coupled/metabolism*
Signal Transduction/physiology*
Zebrafish/anatomy & histology
Zebrafish/embryology
Zebrafish/metabolism
Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism*

(all 20)

PubMed

19464178 Full text @ Curr. Biol.

Abstract

Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known [1]. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine [2] induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.

Genes / Markers

Marker	Marker Type	Name
cluap1	GENE	clusterin associated protein 1
en2a	GENE	engrailed homeobox 2a
myl7	GENE	myosin, light chain 7, regulatory
myod1	GENE	myogenic differentiation 1
prox1a	GENE	prospero homeobox 1a
ptch2	GENE	patched 2
shha	GENE	sonic hedgehog signaling molecule a
smo	GENE	smoothened, frizzled class receptor

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Figures

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Expression

Gene	Fish	Conditions	Stage	Qualifier	Anatomy	Assay	Figure
myl7	WT	standard conditions	Prim-5		heart tube	ISH	Fig. S9
myl7	WT + MO1-cluap1	standard conditions	Prim-5		heart tube	ISH	Fig. S9
myl7	WT + MO2-cluap1	standard conditions	Prim-5		heart tube	ISH	Fig. S9
myod1	WT	control	Bud		adaxial cell	ISH	Fig. 4
myod1	WT + MO2-cluap1	standard conditions	Bud	Not Detected	paraxial mesoderm	ISH	Fig. 4
prox1a	smo^{hi1640Tg/hi1640Tg}	standard conditions	Prim-5	Not Detected	myotome	IHC	Fig. 1
prox1a	smo^{hi1640Tg/hi1640Tg}	standard conditions	Prim-5	Not Detected	myotome	IHC	Fig. 2
prox1a	smo^s294/s294	standard conditions	Prim-5		slow muscle cell nucleus	IHC	Fig. 1
prox1a	smo^s294/s294	standard conditions	Prim-5		slow muscle cell nucleus	IHC	Fig. 2
prox1a	smo^s294/s294	chemical treatment: purmorphamine	Prim-5		slow muscle cell nucleus	IHC	Fig. 2

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment: purmorphamine	Prim-5	muscle pioneer increased amount, abnormal	Fig. 2
WT	chemical treatment: purmorphamine	Prim-5	smoothened signaling pathway process quality, abnormal	Fig. 2
WT + MO1-cluap1	standard conditions	Bud	adaxial cell physical object quality, abnormal	Fig. 4
WT + MO1-cluap1	standard conditions	Bud	smoothened signaling pathway process quality, abnormal	Fig. 4
WT + MO1-cluap1	standard conditions	14-19 somites	Kupffer's vesicle cilium decreased amount, abnormal	Fig. 4
WT + MO1-cluap1	standard conditions	14-19 somites	Kupffer's vesicle cilium decreased length, abnormal	Fig. 4
WT + MO1-cluap1	standard conditions	Prim-5	determination of left/right symmetry process quality, abnormal	Fig. S9
WT + MO1-cluap1	standard conditions	Prim-5	heart tube bilateral symmetry, abnormal	Fig. S9
WT + MO1-cluap1	standard conditions	Prim-5	heart tube mislocalised, abnormal	Fig. S9
WT + MO2-cluap1	standard conditions	Bud	adaxial cell physical object quality, abnormal	Fig. 4

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
hi1640Tg	Tg(nLacz-GTvirus)	Transgenic Insertion	smo
ml1Tg	Tg(-1.8gsc:GFP)	Transgenic Insertion
s294		Point Mutation	smo
vu16Tg	Tg(nkx2.2a:mEGFP)	Transgenic Insertion

1 - 4 of 4

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
cluap1	MO1-cluap1	MRPHLNO
cluap1	MO2-cluap1	MRPHLNO

Fish

Fish
ml1Tg
smo^{hi1640Tg/hi1640Tg}
smo^hi1640Tg/+
smo^s294/s294
vu16Tg
WT
WT + MO1-cluap1
WT + MO2-cluap1

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
GFP	EFG	GFP

Mapping

Aanstad et al., 2009 ZDB-PUB-090526-31 PMID:19464178

The Extracellular Domain of Smoothened Regulates Ciliary Localization and Is Required for High-Level Hh Signaling

Aanstad et al., 2009

ZDB-PUB-090526-31
PMID:19464178