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Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/beta-catenin activity.

Westfall TA, Brimeyer R, Twedt J, Gladon J, Olberding A, Furutani-Seiki M, Slusarski DC - J. Cell Biol. (2003)

Bottom Line: We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail.The dorsalized phenotypes result from increased beta-catenin accumulation and activation of downstream genes.The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/beta-catenin signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
We provide genetic evidence defining a role for noncanonical Wnt function in vertebrate axis formation. In zebrafish, misexpression of Wnt-4, -5, and -11 stimulates calcium (Ca2+) release, defining the Wnt/Ca2+ class. We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail. Embryos genetically depleted of both maternal and zygotic Wnt-5 product exhibit cell movement defects as well as hyperdorsalization and axis-duplication phenotypes. The dorsalized phenotypes result from increased beta-catenin accumulation and activation of downstream genes. The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/beta-catenin signaling.

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Activated CaMKII suppresses the Wnt-5/ppt−/− tail defect. Tail morphology of (A) wild-type, (B) CaMKIItr-rescued ppt−/−, and (C) ppt−/− embryos are from the same clutch. Arrows denote the length from the end of the yolk tube to the tip of the tail. Zebrafish embryos from a standard ppt heterozygous cross were injected with CamKIItr. Injection sets with increased frequency of wild-type–like morphology (>90% compared with the expected 75%) were individually photographed and PCR genotyped.
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fig7: Activated CaMKII suppresses the Wnt-5/ppt−/− tail defect. Tail morphology of (A) wild-type, (B) CaMKIItr-rescued ppt−/−, and (C) ppt−/− embryos are from the same clutch. Arrows denote the length from the end of the yolk tube to the tip of the tail. Zebrafish embryos from a standard ppt heterozygous cross were injected with CamKIItr. Injection sets with increased frequency of wild-type–like morphology (>90% compared with the expected 75%) were individually photographed and PCR genotyped.

Mentions: If the ppt−/− phenotype is the result of reduced Ca2+ release, it may be possible to suppress the mutant defect by artificially activating a Ca2+ cascade. CaM is a predominant Ca2+ binding protein in the cell, which, when bound to Ca2+, stimulates CaM-dependent enzymes including Ca2+/calmodulin-dependent protein kinases. Due to its proposed role in Xenopus ventral patterning (Kuhl et al., 2000a), we tested whether CaMKII was sufficient to rescue the Wnt-5 loss-of-function phenotype. Injection of full-length CaMKII, which should be under the control of endogenous Ca2+ modulation, did not alter the ppt−/− phenotype. Expression of an activated form of CaMKII, truncated to remove a regulatory domain (CamKIItr; Hansen et al., 2003), suppressed the ppt−/− phenotype (Fig. 7 B) compared with uninjected sibling ppt−/− embryos (Fig. 7 C). However, CaMKIItr expression did not completely rescue the phenotype relative to wild-type (Fig. 7 A). As in the Wnt-5 experiments, embryos from a standard ppt heterozygous cross were injected with CamKIItr and rescue was scored in injection sets with suppressed ppt-like tail and trunk phenotypes (ppt phenotypes in clutch controls are typically at 25%, whereas rescue sets had fewer than 10%). Embryos from injection sets with increased frequency of wild-type–like morphology were individually photographed and PCR genotyped. PCR genotype of wt-looking embryos revealed 19% homozygous ppt in CaMKIItr rescue sets, whereas no homozygous ppt genotypes were identified among phenotypic wild-type clutch embryos. Thus, constitutively active CaMKII can result in straightened tails and increased trunk length (within 90% of wt length). Additional rescued embryos (n > 180) were raised, whereas CaMKII-injected ppt+/− heterozygotes survived, no CaMKII-injected ppt−/− reached maturity.


Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/beta-catenin activity.

Westfall TA, Brimeyer R, Twedt J, Gladon J, Olberding A, Furutani-Seiki M, Slusarski DC - J. Cell Biol. (2003)

Activated CaMKII suppresses the Wnt-5/ppt−/− tail defect. Tail morphology of (A) wild-type, (B) CaMKIItr-rescued ppt−/−, and (C) ppt−/− embryos are from the same clutch. Arrows denote the length from the end of the yolk tube to the tip of the tail. Zebrafish embryos from a standard ppt heterozygous cross were injected with CamKIItr. Injection sets with increased frequency of wild-type–like morphology (>90% compared with the expected 75%) were individually photographed and PCR genotyped.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172822&req=5

fig7: Activated CaMKII suppresses the Wnt-5/ppt−/− tail defect. Tail morphology of (A) wild-type, (B) CaMKIItr-rescued ppt−/−, and (C) ppt−/− embryos are from the same clutch. Arrows denote the length from the end of the yolk tube to the tip of the tail. Zebrafish embryos from a standard ppt heterozygous cross were injected with CamKIItr. Injection sets with increased frequency of wild-type–like morphology (>90% compared with the expected 75%) were individually photographed and PCR genotyped.
Mentions: If the ppt−/− phenotype is the result of reduced Ca2+ release, it may be possible to suppress the mutant defect by artificially activating a Ca2+ cascade. CaM is a predominant Ca2+ binding protein in the cell, which, when bound to Ca2+, stimulates CaM-dependent enzymes including Ca2+/calmodulin-dependent protein kinases. Due to its proposed role in Xenopus ventral patterning (Kuhl et al., 2000a), we tested whether CaMKII was sufficient to rescue the Wnt-5 loss-of-function phenotype. Injection of full-length CaMKII, which should be under the control of endogenous Ca2+ modulation, did not alter the ppt−/− phenotype. Expression of an activated form of CaMKII, truncated to remove a regulatory domain (CamKIItr; Hansen et al., 2003), suppressed the ppt−/− phenotype (Fig. 7 B) compared with uninjected sibling ppt−/− embryos (Fig. 7 C). However, CaMKIItr expression did not completely rescue the phenotype relative to wild-type (Fig. 7 A). As in the Wnt-5 experiments, embryos from a standard ppt heterozygous cross were injected with CamKIItr and rescue was scored in injection sets with suppressed ppt-like tail and trunk phenotypes (ppt phenotypes in clutch controls are typically at 25%, whereas rescue sets had fewer than 10%). Embryos from injection sets with increased frequency of wild-type–like morphology were individually photographed and PCR genotyped. PCR genotype of wt-looking embryos revealed 19% homozygous ppt in CaMKIItr rescue sets, whereas no homozygous ppt genotypes were identified among phenotypic wild-type clutch embryos. Thus, constitutively active CaMKII can result in straightened tails and increased trunk length (within 90% of wt length). Additional rescued embryos (n > 180) were raised, whereas CaMKII-injected ppt+/− heterozygotes survived, no CaMKII-injected ppt−/− reached maturity.

Bottom Line: We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail.The dorsalized phenotypes result from increased beta-catenin accumulation and activation of downstream genes.The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/beta-catenin signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
We provide genetic evidence defining a role for noncanonical Wnt function in vertebrate axis formation. In zebrafish, misexpression of Wnt-4, -5, and -11 stimulates calcium (Ca2+) release, defining the Wnt/Ca2+ class. We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail. Embryos genetically depleted of both maternal and zygotic Wnt-5 product exhibit cell movement defects as well as hyperdorsalization and axis-duplication phenotypes. The dorsalized phenotypes result from increased beta-catenin accumulation and activation of downstream genes. The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/beta-catenin signaling.

Show MeSH