Harata A , Hirakawa M , Sakuma T , Yamamoto T , Hashimoto C .

             head morphogenesis

Xla Wt + p2ry4 (Fig. 5 e f)
Xla Wt + p2ry4 (Fig. 5 h i)
Xla Wt + p2ry4 TALEN (Fig. 2 c)
Xla Wt + p2ry4 TALEN (Fig. 2 f)
Xla Wt + p2ry4 TALEN (Fig. 2 i)
Xla Wt + p2ry4 TALEN (Fig. 2 l)
Xla Wt + p2ry4 TALEN (Fig. 3 o)
Xla Wt + p2ry4 TALEN (Fig. 3 c f)
Xla Wt + p2ry4 TALEN (Fig. 3 i)
Xla Wt + p2ry4 TALEN (Fig. 3 l)
Xla Wt + p2ry4 TALEN (Fig. 3 r)
Xla Wt + p2ry4 TALEN (Fig. 4 f)
Xla Wt + p2ry4 TALEN (Fig. 4 l)
Xla Wt + p2ry4 TALEN (Fig. S3 col 2)
Xla Wt + p2ry4 TALEN (Fig. S3 col 3)
Xla Wt + p2ry4 TALEN (Fig. S5 C F I L)
Xla Wt + p2ry4 TALEN (Fig. S6 F I)

	Figure 1. Expression patterns of p2ry4 during early development. WISH of p2ry4 from gastrula to tailbud stage. Stages 10.5 and 11 (a and c, dorsal views; b and d, sagittal hemisections). p2ry4 was expressed in the prospective neuroectoderm and the involuting dorsal and ventral mesoderm at gastrula stage. At stage 10.5, p2ry4 was prominently expressed in the head organizer region (b). Stages 13, 17, 19, and 21 (e, f, h, j, anterior views; k, dorsal view; g, sagittal hemisection; i, transverse section). Stage 28 (l, lateral view; m, transverse section; m’, high‐magnification image of m). The extirpated brain of tadpole (n, lateral side; o, dorsal side). nc, neural crest; pl, placode cell; anp, anterior neural plate; mhb, midbrain‐hindbrain boundary; nt, neural tube; not, notochord; som, somite; cg, cement gland; tel, telencephalon; di, diencephalon; mes, mesencephalon; met, metencephalon. Dotted straight line indicates section plane. Black arrowheads indicate blastopore
	Figure 2. Loss of p2ry4 affected neural crest and placode development. Uninjected (a, d, g, j), TALEN‐L injected (b, e, h, k), and TALEN‐L/R injected (c, f, i, l) embryos were subjected to WISH for snail1 (a–f) and hairy2b (g–l). Snail1 expression at early neurula (a–c) and early tailbud (d–f) stages. Snail1 expression was reduced in TALEN‐L/R injected embryos (c, f, red arrowheads). hairy2b expression at early (g–i) and middle (j–l) neurula stages. hairy2b expression at the anterior neural plate border was reduced in TALEN‐L/R injected embryos. Anterior (a–c, j–l), lateral (d–f), and dorsal (g–i) views. ot, otic vesicle; nc, neural crest; fp, floor plate. Red arrowheads indicate migrating neural crest cells. Brackets indicate prospective floor plate
	Figure 3. Loss of p2ry4 affected early neural development. Uninjected (a, d, g, j, m, p), TALEN‐L injected (b, e, h, k, n, q), and TALEN‐L/R injected (c, f, i, l, o, r) embryos were subjected to WISH for sox2 (a–f), otx2 (g–i), pax6 (j–l), rx1 (m–o), and krox20 (p–r) at early neurula stage. The expression of all the neural marker genes was reduced in TALEN‐L/R injected embryos. Dorsal (a–c, p–r) and anterior (d–o) views
	Figure 4. Disruption of p2ry4 reduced expression of head organizer genes. Uninjected (a, d, g, j, m, p), TALEN‐L injected (b, e, h, k, n, q), and TALEN‐L/R injected (c, f, i, l, o, r) embryos were subjected to WISH for dkk1 (a–f), cerberus (g–l), and chordin (m–r) at gastrula stage. The expression of dkk1 and cerberus was reduced in TALEN‐L/R injected embryos at mid‐gastrula stage (f, l, brackets). The change of chordin expression in TALEN‐L/R injected embryos was not significant (o, r) compared to that in uninjected and TALEN‐L injected embryos (m, n, p, q). Dorsal halves of sagittally fractured embryos are shown. Dashed lines indicate the edge of blastocoel. Brackets indicate the area that were reduced in the expression of dkk1 and cerberus. Arrowheads indicate blastopore
	Figure 5. p2ry4 resulted in ectopic expression of head organizer genes. No injection (a, d, g, j) and injection of 500 pg of p2ry4 mRNA (b, e, h, k) and 1,000 pg of p2ry4 mRNA (c, f, i, l) into the ventral side at 8‐cell stage and collection at blastula stage (a–c). Embryos were hemisectioned and subjected to WISH for dkk1 (d–f), cerberus (g–i), and chordin (j–l). Overexpression of p2ry4 led to the accumulation of pigment (b, c, arrows). Dkk1 or cerberus expression was induced by p2ry4 mRNA (e, f, h, i, arrowheads). Animal views (a–c). Arrowheads indicate injected sides
	fig. S1 phylogenetic conservation of P2Y receptors in vertebrate
	fig. S2 Nucleotide sequences of splicing variants of p2ry4.L
	fig. S3 Phenotypes and detection TALEN-mediated mutation in TALEN-injected embryos
	fig. S4 p2ry4 did not induce ectopic head structure in absence of BMP signaling
	fig. S5 The blastopore closer was delayed by deletion of p2ry4
	fig. S6 p2ry4 might be required for involution during gastrulation

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