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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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Hyperventralization phenotypes of Wnt-5–injected embryos. Embryos mis-expressing Wnt-5 were evaluated for changes in D-V patterning by morphology and whole mount in situ. (A) Wild-type morphology with the arrow designating the anterior-most region. Loss of dorsal-anterior tissue and expansion of ventral-posterior tissue is evident by lack of head tissue arrow in B and an onion-like mass of ventralized tissue in C. Dual-hybridization whole mount in situ at shield stage with the dorsal-specific chordin domain (red) denoted by arrowheads flanked by the ventral-specific eve (blue) expression domain. Reduction of dorsal domains and expansion of ventral domains compared with (D) wild-type is seen in (E) Wnt-5-injected embryos. (A–C) Lateral orientation with anterior to the left and (D–E) animal pole orientation with dorsal to the right.
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fig2: Hyperventralization phenotypes of Wnt-5–injected embryos. Embryos mis-expressing Wnt-5 were evaluated for changes in D-V patterning by morphology and whole mount in situ. (A) Wild-type morphology with the arrow designating the anterior-most region. Loss of dorsal-anterior tissue and expansion of ventral-posterior tissue is evident by lack of head tissue arrow in B and an onion-like mass of ventralized tissue in C. Dual-hybridization whole mount in situ at shield stage with the dorsal-specific chordin domain (red) denoted by arrowheads flanked by the ventral-specific eve (blue) expression domain. Reduction of dorsal domains and expansion of ventral domains compared with (D) wild-type is seen in (E) Wnt-5-injected embryos. (A–C) Lateral orientation with anterior to the left and (D–E) animal pole orientation with dorsal to the right.

Mentions: For gain-of-function analyses, we manipulated Wnt-5 activity by DNA or RNA injections into wild-type embryos and subsequently monitored Ca2+ release frequency. In this manner, we can correlate the immediate physiological output of Ca2+ release with morphological or molecular changes evaluated hours later. Wnt-5 RNA injected embryos typically demonstrated robust early Ca2+ release activity (from 64- to 1,000-cell) resulting in cell movement defects as described in Slusarski et al. (1997b) and is consistent with morphogenetic defects described for zebrafish Wnt 4 (Ungar and Moon, 1995) and Xenopus Wnt-5A (Moon et al., 1993a). At the highest RNA concentrations, we induce hyperdorsalization defects (unpublished data) consistent with phenotypes described for high dose injections (>100 pg/embryo) in zebrafish (Kilian et al., 2003). Because we have demonstrated that depletion of intracellular Ca2+ stores in zebrafish embryos is sufficient to generate hyperdorsalized phenotypes (Westfall et al., 2003), we were concerned about Ca2+ depletion upon overexpression of Wnt-5 RNA. To this end, we used doses that stimulate a sustained, yet less robust, Ca2+ release frequency during development and in doing so with Wnt-5 DNA, we generated a range of phenotypes, including cell movement defects and ventralization as evaluated by morphology (Fig. 2, B and C) and by whole mount in situ (Fig. 2 E). Wnt-5 DNA injection into embryos is sufficient to activate Ca2+ release just before and beyond 1,000-cell stage. Hyperventralization is demonstrated by reduction of dorsal anterior tissue (Fig. 2 B) and expansion of ventral posterior tissue with the most severe phenotype resembling an onionlike morphology of ventralized tissue (Fig. 2 C). Molecular analysis at 60–80% epiboly demonstrates a reduction of the presumptive dorsal region (labeled by chordin; Miller-Bertoglio et al., 1997) and expansion of presumptive ventral tissue (marked by eve1; Fig. 2 E; Joly et al., 1993) when compared with control-injected embryos (Fig. 2 D). The potency of the Wnt5 ligand is evident as relatively minor dose changes (of both RNA and DNA) have a profound impact on the frequency of Ca2+ release with a concomitant range of developmental defects. Although Wnt-5 misexpression implicates a role in D-V patterning, we wanted to confirm with loss-of-function analyses.


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)

Hyperventralization phenotypes of Wnt-5–injected embryos. Embryos mis-expressing Wnt-5 were evaluated for changes in D-V patterning by morphology and whole mount in situ. (A) Wild-type morphology with the arrow designating the anterior-most region. Loss of dorsal-anterior tissue and expansion of ventral-posterior tissue is evident by lack of head tissue arrow in B and an onion-like mass of ventralized tissue in C. Dual-hybridization whole mount in situ at shield stage with the dorsal-specific chordin domain (red) denoted by arrowheads flanked by the ventral-specific eve (blue) expression domain. Reduction of dorsal domains and expansion of ventral domains compared with (D) wild-type is seen in (E) Wnt-5-injected embryos. (A–C) Lateral orientation with anterior to the left and (D–E) animal pole orientation with dorsal to the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172822&req=5

fig2: Hyperventralization phenotypes of Wnt-5–injected embryos. Embryos mis-expressing Wnt-5 were evaluated for changes in D-V patterning by morphology and whole mount in situ. (A) Wild-type morphology with the arrow designating the anterior-most region. Loss of dorsal-anterior tissue and expansion of ventral-posterior tissue is evident by lack of head tissue arrow in B and an onion-like mass of ventralized tissue in C. Dual-hybridization whole mount in situ at shield stage with the dorsal-specific chordin domain (red) denoted by arrowheads flanked by the ventral-specific eve (blue) expression domain. Reduction of dorsal domains and expansion of ventral domains compared with (D) wild-type is seen in (E) Wnt-5-injected embryos. (A–C) Lateral orientation with anterior to the left and (D–E) animal pole orientation with dorsal to the right.
Mentions: For gain-of-function analyses, we manipulated Wnt-5 activity by DNA or RNA injections into wild-type embryos and subsequently monitored Ca2+ release frequency. In this manner, we can correlate the immediate physiological output of Ca2+ release with morphological or molecular changes evaluated hours later. Wnt-5 RNA injected embryos typically demonstrated robust early Ca2+ release activity (from 64- to 1,000-cell) resulting in cell movement defects as described in Slusarski et al. (1997b) and is consistent with morphogenetic defects described for zebrafish Wnt 4 (Ungar and Moon, 1995) and Xenopus Wnt-5A (Moon et al., 1993a). At the highest RNA concentrations, we induce hyperdorsalization defects (unpublished data) consistent with phenotypes described for high dose injections (>100 pg/embryo) in zebrafish (Kilian et al., 2003). Because we have demonstrated that depletion of intracellular Ca2+ stores in zebrafish embryos is sufficient to generate hyperdorsalized phenotypes (Westfall et al., 2003), we were concerned about Ca2+ depletion upon overexpression of Wnt-5 RNA. To this end, we used doses that stimulate a sustained, yet less robust, Ca2+ release frequency during development and in doing so with Wnt-5 DNA, we generated a range of phenotypes, including cell movement defects and ventralization as evaluated by morphology (Fig. 2, B and C) and by whole mount in situ (Fig. 2 E). Wnt-5 DNA injection into embryos is sufficient to activate Ca2+ release just before and beyond 1,000-cell stage. Hyperventralization is demonstrated by reduction of dorsal anterior tissue (Fig. 2 B) and expansion of ventral posterior tissue with the most severe phenotype resembling an onionlike morphology of ventralized tissue (Fig. 2 C). Molecular analysis at 60–80% epiboly demonstrates a reduction of the presumptive dorsal region (labeled by chordin; Miller-Bertoglio et al., 1997) and expansion of presumptive ventral tissue (marked by eve1; Fig. 2 E; Joly et al., 1993) when compared with control-injected embryos (Fig. 2 D). The potency of the Wnt5 ligand is evident as relatively minor dose changes (of both RNA and DNA) have a profound impact on the frequency of Ca2+ release with a concomitant range of developmental defects. Although Wnt-5 misexpression implicates a role in D-V patterning, we wanted to confirm with loss-of-function analyses.

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