XB-ART-55162Dev Growth Differ February 1, 2019; 61 (2): 186-197.
Nucleotide receptor P2RY4 is required for head formation via induction and maintenance of head organizer in Xenopus laevis.
Vertebrates have unique head structures that are mainly composed of the central nervous system, the neural crest, and placode cells. These head structures are brought about initially by the neural induction between the organizer and the prospective neuroectoderm at early gastrula stage. Purinergic receptors are activated by nucleotides released from cells and influence intracellular signaling pathways, such as phospholipase C and adenylate cyclase signaling pathways. As P2Y receptor is vertebrate-specific and involved in head formation, we expect that its emergence may be related to the acquisition of vertebrate head during evolution. Here, we focused on the role of p2ry4 in early development in Xenopus laevis and found that p2ry4 was required for the establishment of the head organizer during neural induction and contributed to head formation. We showed that p2ry4 was expressed in the head organizer region and the prospective neuroectoderm at early gastrula stage, and was enriched in the head components. Disruption of p2ry4 function resulted in the small head phenotype and the reduced expression of marker genes specific for neuroectoderm and neural border at an early neurula stage. Furthermore, we examined the effect of p2ry4 disruption on the establishment of the head organizer and found that a reduction in the expression of head organizer genes, such as dkk1 and cerberus, and p2ry4 could also induce the ectopic expression of these marker genes. These results suggested that p2ry4 plays a key role in head organizer formation. Our study demonstrated a novel role of p2ry4 in early head development.
PubMed ID: 30069871
Article link: Dev Growth Differ
Genes referenced: cer1 chrd.1 ctnnb1 dkk1 egr2 eomes gsc hes4 nog nppa otx2 p2ry4 pax6 rax snai1 sox2 sst.1 tbxt
GO keywords: purinergic nucleotide receptor signaling pathway
Disease Ontology terms: spina bifida
Article Images: [+] show captions
|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|