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

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.

Figure 1.

Ca ²⁺ release dynamics in zebrafish embryos expressing Wnts and in Wnt-5/ ppt mutants. Non-canonical Wnt members stimulate Ca²⁺ release in zebrafish and these changes were monitored with Fura-2 in live embryos. The representative embryo shown is a two-dimensional topographic image of the location of all the Ca²⁺ fluxes that occurred during the time course (50 min). Surface plots of Ca²⁺ release activity in embryos mis-expressing (A) Wnt-11 and (B) Wnt-8 RNA. Ca²⁺ release profile of endogenous activity in (C) heterozygous (ppt ^+/−) and (D) mutant (ppt ^{− / −}) embryos from the same clutch. Height and color of peaks indicate the number of Ca²⁺ fluxes observed over the course of the experiment with the embryos oriented in a lateral position. The color bar indicates the pseudocolor representation of the number of transients from low (purple, 1) to high (red, 40).

Figure 2.

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.

Figure 3.

Morphological phenotypes in Wnt-5/ ppt and Ca ²⁺ - inhibited embryos. Lateral view, anterior to the right of 24–36 hpf embryos. (A) In wild-type, the block arrow indicates one eye, the other is out of the focal plane and the dashed lined demarcates the tail extending posteriorly off the yolk. (B) The Wnt-11 mutant (slb ^{− / −}) embryo has a fused eye, the arrow indicates the lens. (C) In Wnt-5 mutant (ppt ^{− / −}), the dashed arrow demarcates the shortened-curled tail defect and in the (D) double mutant (slb ^{− / −}; ppt ^{− / −}), the block arrow indicates the fused eye, whereas the dashed arrow marks the shortened-twisted tail. The notochord and one set of somites are highlighted by a dashed line in (E) IP₃R-inhibited (XeC), and (F) ppt ^{− / −} embryos. The presence of a protruding yolk is an indication of incomplete epiboly cell movements. Fused eyes and shortened twisted trunks can also be observed in (G) Ca²⁺-inhibited (L-690,330-treated) embryos.

Figure 4.

Phenotypes of Wnt-5/ ppt maternal-depleted embryos. Tail and trunk morphology in (A and E) wild-type, (B) zygotic ppt ^{− / −}, and embryos collected from ppt ^{− / −} females in C, D, F, and G. The embryo in C is an example of what was scored as a severe zygotic ppt-like phenotype with a severely shortened trunk. The phenotypes of embryos in D and F resemble two of the extreme classes of dorsalized mutants, whereas the embryo in G has a partial secondary axis (center, foreground) branching off of the primary axis (left, background). The ectopic axis has a beating heart and otic vesicles, an arrowhead highlighting one otic vesicle. Whole mount in situ analysis of embryos from ppt ^{− / −} females. After photographing, genotypes were determined by PCR. mz-ppt represents maternal and zygotic loss of Wnt-5. Lateral view of 36 hpf embryos probed for the heart with Nkx2.5 in (H) wild-type and (I) mz-ppt, the black arrows show the endogenous heart, whereas the ectopic staining is noted with a white arrow. Animal pole view of embryos at 50% epiboly with dorsal to the right and arrowheads marking the lateral extent of the chordin expression domain in (J) wild-type, (K) mz-ppt, and in (L) mz-ppt, the star demarcates an ectopic domain.

Figure 5.

Increased β-catenin protein and boz expression in Wnt-5/ppt mutant embryos. Immunolocalization of β-catenin protein in sphere stage embryos. A panel from a confocal series collected with a 20× objective from (A) wild-type and (B) maternally depleted ppt embryos. White dots identify domains of nuclear β-catenin around the circumference of the embryos in an animal pole orientation. Additional confocal images were collected from the same embryos with a 63× objective. Representative β-catenin–positive nuclei noted by arrowheads with one B-catenin–positive nucleus in (C) wild-type and at least seven in (D) maternally depleted ppt embryo.

Figure 6.

Increased bo z expression in Wnt-5/ppt mutant embryos. Whole mount in situ hybridization with boz; animal pole view of (A) wild-type and (B-D) mz-ppt embryos; dorsal view of (E) wild-type and (F) (ppt ^{− / −} ;slb ^{− / −} ) embryos. The arrows mark the lateral extent of the boz expression domain and plus marks highlight ectopic domains.

Figure 7.

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.

Figure 8.

Wnt signaling network. Schematic of the stimulation of (A) canonical Wnt and (B) non-canonical Wnt/Ca²⁺ and planar cell polarity signaling pathways. (Note: only a few of the known components are outlined.)