Identification and mechanism of regulation of the zebrafish dorsal determinant

Abstract

In vertebrates, the animal-vegetal axis is determined during oogenesis and at ovulation, the egg is radially symmetric. In anamniotes, following fertilization, a microtubule-dependent movement leads to the displacement of maternal dorsal determinants from the vegetal pole to the future dorsal side of the embryo, providing the initial breaking of radial symmetry [Weaver C, Kimelman D (2004) Development 131:3491-3499]. These dorsal determinants induce β-catenin nuclear translocation in dorsal cells of the blastula. Previous work in amphibians has shown that secreted Wnt11/5a complexes, regulated by the Wnt antagonist Dkk-1, are required for the initiation of embryonic axis formation [Cha et al. (2009) Curr Biol 29:1573-1580]. In the current study, we determined that the vegetal maternal dorsal determinant in fish is not the Wnt11/5a complex but the canonical Wnt, Wnt8a. Translation of this mRNA and secretion of the Wnt8a protein result in a dorsal-to-ventral gradient of Wnt stimulation, extending across the entire embryo. This gradient is counterbalanced by two Wnt inhibitors, Sfrp1a and Frzb. These proteins are essential to restrict the activation of the canonical Wnt pathway to the dorsal marginal blastomeres by defining the domain where the Wnt8a activity gradient is above the threshold value necessary for triggering the canonical β-catenin pathway. In summary, this study establishes that the zebrafish maternal dorsal determinant, Wnt8a, is required to localize the primary dorsal center, and that the extent of this domain is defined by the activity of two maternally provided Wnt antagonists, Sfrp1a and Frzb.

Fig. 1.

The secreted Wnt ligand Wnt8a is a strong candidate for the dorsal determinant in zebrafish. Expression of chordin at the sphere stage is shown in (A) wild type (WT), (B) an embryo injected with 500 pg of Frzb mRNA, and (C) an embryo injected with 100 pg of mRNA coding for a dominant-negative Tcf-3 (DN-Tcf3) (34). Embryos are in animal pole view with dorsal to the Right. (D) Expression of the 23 Wnt genes present in the zebrafish genome analyzed by RT-PCR at 1- to 2-cell stage (0.5 h), 1,000-cell stage (3 h), sphere stage (4 h), 40% epiboly (5 h), 60% epiboly (7 h), and at 24 hpf. Arrowheads indicate Wnt genes for which mRNAs are maternally deposited in the egg. (E–J) Whole mount in situ hybridization reveals that Wnt8a mRNAs accumulate in the Balbiani body (bb) in stage I oocytes (E) and are localized in a vegetal position in stage II (F) and stage III oocytes (G). Transcripts of Wnt8a are also observed in cleavage-stage embryos both in blastomeres and in the yolk cortical cytoplasm in vegetal position at (H) the 2-cell stage and (I) 16-cell stage. (J) Treatment with nocodazole, which depolymerizes microtubules, prevents transport of Wnt8a mRNA. Embryos are in lateral view with dorsal to the Right. Arrowheads in H–J indicate the limits of Wnt8a mRNA localization in the cortical cytoplasm.

Fig. 2.

Wnt8a acts as the initial dorsal determinant in zebrafish. (A–C) Expression of chordin, analyzed by in situ hybridization, at the sphere stage in a wild-type (WT) embryo (A), in an embryo overexpressing Wnt8a (100 pg Wnt8a mRNA) (B), and in an embryo overexpressing a dominant-negative form of Wnt8a (500 pg of mRNA coding for DN-Wnt8a ORF2) (C). Embryos are in animal pole view, dorsal to the Right. (D) Nocodazole treatment generates embryos displaying various ventralization phenotypes ranging from weak (lacking notochord), strong (additionally missing the head), and radialized (lacking any dorsal territories). Injection of 25 pg of Wnt8a mRNA into one marginal blastomere at the 64-cell stage efficiently rescues radialized embryos and strongly reduces the frequency of strongly ventralized embryos; injections of 400 pg of Wnt11 mRNA or of a mix of 400 pg of Wnt11 mRNA and 400 pg of Wnt5a mRNA fails to rescue these phenotypes as shown in the graph. Embryos are presented in lateral view with anterior to the Left. Means and error bars (SD) were obtained from multiple experiments (three to five) with at least 50 embryos for each experimental condition. Comparisons between experiments were undertaken using unpaired Student's t tests. P value of <0.05 was considered statistically significant.

Fig. 3.

Negative regulation of the canonical Wnt pathway by Sfrp1a and Frzb. Immunofluorescence localization of β-catenin is shown at 3 hpf (high stage) (A), at the dorsal margin (DM), and (B) at the animal pole (AP) of a wild-type embryo or (C) at the animal pole of an embryo overexpressing Wnt8a (10 pg Wnt8a mRNA). White and yellow arrowheads in A point to the accumulation of β-catenin in blastomeres and yolk syncytial layer nuclei, respectively. (D) Analysis by RT-PCR of the expression of zebrafish Wnt inhibitors during early development. Sfrp (TLC), expressed only during gastrulation (35), is not presented here. Expression of chordin at the sphere stage in lateral view (Upper) and animal pole view (Lower) in (E and I) wild-type embryos, (F and J) Sfrp1a morphants, (G and K) Frzb morphants, and (H and L) Sfrp1a–Frzb double morphants. (M–O) Immunofluorescence localization of β-catenin in nuclei of the animal pole of embryos at the high stage in (J) Sfrp1a, (K) Frzb, and (L) Sfrp1a–Frzb morphants. Embryos are in dorsal view (A), lateral view (E–H), and in animal pole view (B, C, and I–O). (Scale bar, 100 μM.)

Fig. 4.

Sfrp1a and Frzb regulate the activation of the maternal Wnt/β-catenin signaling pathway. Expression pattern of chordin are shown in wild type (A) or in embryos injected with 4 ng (B), 5.5 ng (C), and 7 ng (D) of Frzb morpholinos. (E) At the two-cell stage Sfrp1a and Frzb prevent activation of the maternal Wnt pathway in the blastomeres, whereas the dorsal determinant, Wnt8a, is transported from the vegetal pole toward the blastomeres in a microtubule-dependent manner. At the high stage (immediately after the midblastula transition), Wnt8a translated from these transported mRNAs stimulates the canonical β-catenin pathway. Sfrp1a and Frzb are required to limit this activation to the cells receiving the highest level of stimulation.