Fig. 6

Wnt6a is another dorsal determinant candidate. (A) Maternal expression of wnt6a. Whole-mount in situ hybridization of wnt6a, wnt7ab, and wnt8 in one-cell-stage embryos. Staining with the sense probe for wnt6a was also performed as a negative control. wnt6a and wnt8a were detected in the vegetal pole (wnt6a antisense n=21/87, sense probe n=0/29; wnt8a n=18/19), whereas maternal wnt7ab was not detected (n=0/23). Lateral views. (B) Overexpression of wnt6a posteriorizes the embryos. One-cell-stage embryos were injected with the capped and polyadenylated RNA of wnt6a (5 pg), wnt8a (100 pg), dkk1b (50 pg), or a combination of wnt6a (5 pg) or wnt8a (100 pg), and dkk1b (50 pg). The phenotypes of the resultant embryos were observed at 30 hpf, and were categorized into five classes: no eyes, curled tail (a), no or small eyes (b), wild type-like (c), big head, short tail (d), and big head, no tail (e). Lateral views with anterior to the left. The proportion of embryos showing each phenotype is shown in the right panel. (C) Overexpression of wnt6a induces an expanded expression of dharma. The capped and polyadenylated RNA of wnt6a (1500 pg) or wnt8a (1500 pg) was injected into one-cell-stage embryos, and dharma expression was examined at the sphere stage. Animal pole views with dorsal to the right (upper panels); lateral views with dorsal to the right (lower panels). The dharma expression was restricted to the dorsal-most region (n=20/20), whereas it was expanded in embryos that received an injection of wnt6a RNA (n=12/42) or wnt8a RNA (n=27/64). (D) Overexpression of Dkk1b, a canonical Wnt pathway inhibitor, suppressed the dharma expression. Embryos were injected with dkk1b RNA (375 or 1500 pg) or ΔN-tcf7l1a RNA (900 pg, formerly called ΔN-tcf3a). Lateral views with dorsal to the right. The dharma expression at the sphere stage was reduced in the embryos that received an injection of dkk1b RNA (n=13/35 for 375 pg; n=8/20 for 1500 pg) or ΔN-tcf7l1a RNA (n=32/37). Scale bars: 200 µm in A; 500 µm in B; 200 µm in C.