DEV01733 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. wnt3a MOs can enhance dorsal-ventral (D/V)and anterior-posterior (A/P) patterning defects associated with mild loss of wnt8 function. Embryos were injected with 2 mg/ml wnt3a MO (B,F), 0.25 mg/ml each of wnt8 ORF1 and ORF2 MOs (C,G), or both (D,H). (A-D) Animal pole views, dorsal to the right. Embryos were fixed at 60% epiboly and stained with flh to visualize the dorsal organizer, the limits of which are demarcated by arrows. wnt3a MO injection has no effect on the size of the organizer (B). At these doses, wnt8 MOs result in only a slight expansion of the flh domain (C), while injection of both morpholinos results in a significant expansion of the dorsal organizer (D). (E-H) lateral views, dorsal to the right. Embryos were fixed at 100% epiboly and stained with opl, to mark the prospective telencephalon, pax2, to mark the future midbrain/hindbrain boundary, and tbx6, to mark the margin. Neither wnt3a MOs (F) nor wnt8 MOs (G) caused any defects in A/P patterning of the neurectoderm, but wnt3a/wnt8 morphants showed a dramatic expansion of opl (H), reflecting the increased anterior character of the neurectoderm. With the higher doses of wnt8 MOs used for the other experiments in this paper, co-injection of wnt3a MOs does not enhance D/V and A/P patterning defects.

• Supplemental Figure 2 -

  Fig. S2. The rate of cell proliferation in the tailbud is unchanged in wnt3a/wnt8 morphants. Embryos were injected with 2 mg/ml wnt3a MO (B), 0.5 mg/ml each of wnt8 ORF1 and ORF2 MOs (C), or wnt3a, wnt8 ORF1 and wnt8 ORF2 MOs together (D), and fixed at the 4-somite stage, with uninjected embryos as the control (A). tbx6 expression was visualized with a Fast Red substrate for the color reaction, which yields a fluorescent (red) product. Anti-phosphohistone H3 antibody marks mitotic nuclei (green). To determine the mitotic index, we calculated the number of mitotic cells within the tbx6 expression domain, divided by the total number of cells within that domain. Three embryos were scored for each group of embryos. We calculated a mitotic index of 7.5 (n=1281) for wild type, 6.7 (n=1089) for wnt3a morphants, 7.5 (n=772) for wnt8 MO embryos and 6.8 (n=772) for wnt3a/wnt8 morphants.

• Supplemental Figure 3 -

  Fig. S3. Examination of additional posterior fates in wnt3a/wnt8 morphants. Embryos were injected with wnt3a and wnt8 morpholinos. More mildly affected embryos that made some tail structures were collected and fixed at 27 hpf, along with uninjected wild-type embryos, and stained with the indicated marker. eve1 (A,B) was used as a general marker of the tailbud, msxb (C,D) was used to stain ventral tail fin, gata1 (E,F) was used to visualize the blood, and fli1 (G,H) was used to stain the developing vasculature.