Phylogenetic relationship and embryonic expression of zFmi1 genes. (A) Phylogenetic relationship of zFmi1a and zFmi1b to other flamingo proteins. Estimated evolutionary distance is proportional to line length and numbers indicate confidence. These indicate that both zFmi1a genes are orthologues of the ancestral Fmi1/Celsr1 gene. Sources of predicted protein sequences: zFmi1a and zFmi1b, this study; chick Fmi1/Celsr1 and Fmi3/Celsr3; murine Celsr1, 2 and 3, GenBank accession numbers NM009886, AF031573, AF427498. (B–S) Temporal and spatial expression of zfmi1a and zfmi1b. (B and O) RT-PCR analysis of zfmi1a and zfmi1b expression, respectively (top panel). MW is indicated. Control is EF1α lower panel). (C–N) Wild-type embryos hybridized for zfmi1a at 30% epiboly (C), 40% epiboly (D), shield (E), 65% epiboly (F), 75% epiboly (G and H), 95% epiboly (I and J), 3 somites (K and L), and 6 somites (M and N). Lateral and dorsal views are as indicated on each plate; dorsal is to right and anterior to top, respectively. (H, L, and N) Dorsal views of flat-mounted embryos; anterior is to the top. (E) Arrow indicates expression of zfmi1a in shield. Arrowhead indicates expression of zfmi1a in lateral germ ring. Inset, lateral view; arrow indicates expression of zfmi1a in shield. (H) Small black arrow indicates enriched zfmi1a expression in axial tissue, larger arrow indicates an isolated lateral cell expressing zfmi1a. Arrowhead indicates expression of zfmi1a in head. (J) Inset, dorsal view of animal pole; arrow indicates loss of zfmi1a expression medially. (L) Arrowhead indicates loss of zfmi1a expression within anterior axial tissue, white arrow indicates expression in eye field and black arrow indicates expression within lateral trunk. (P–S) Expression of zfmi1b at sphere (P), shield (Q), 95% epiboly (R), and 16 somite stages. Abbreviations: E, epiboly; s, number of somites.