Blanchard C et al. (2019), Comparative Embryonic Spatio-Temporal Expressio...

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Front Cell Neurosci 2019 Jan 01;13:340. doi: 10.3389/fncel.2019.00340.

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Comparative Embryonic Spatio-Temporal Expression Profile Map of the Xenopus P2X Receptor Family.

Blanchard C , Boué-Grabot E , Massé K .

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P2X receptors are ATP-gated cations channels formed by the homo or hetero-trimeric association from the seven cloned subunits (P2X1-7). P2X receptors are widely distributed in different organs and cell types throughout the body including the nervous system and are involved in a large variety of physiological but also pathological processes in adult mammals. However, their expression and function during embryogenesis remain poorly understood. Here, we report the cloning and the comparative expression map establishment of the entire P2X subunit family in the clawed frog Xenopus. Orthologous sequences for 6 mammalian P2X subunits were identified in both X. laevis and X. tropicalis, but not for P2X3 subunit, suggesting a potential loss of this subunit in the Pipidae family. Three of these genes (p2rx1, p2rx2, and p2rx5) exist as homeologs in the pseudoallotetraploid X. laevis, making a total of 9 subunits in this species. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. RT-PCR revealed that all subunits are expressed during the development although zygotic p2rx6 and p2rx7 transcripts are mainly detected at late organogenesis stages. Whole mount in situ hybridization shows that each subunit displays a specific spatio-temporal expression profile and that these subunits can therefore be grouped into two groups, based on their expression or not in the developing nervous system. Overlapping expression in the central and peripheral nervous system and in the sensory organs suggests potential heteromerization and/or redundant functions of P2X subunits in Xenopus embryos. The developmental expression of the p2rx subunit family during early phases of embryogenesis indicates that these subunits may have distinct roles during vertebrate development, especially embryonic neurogenesis.

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Species referenced: Xenopus laevis
Genes referenced: gast odc1 p2rx1 p2rx2 p2rx4 p2rx5 p2rx6 p2rx7
GO keywords: purinergic receptor signaling pathway

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	FIGURE 1. Phylogenetic conservation of the vertebrate p2x receptor subunits. A phylogenetic tree of the p2rx subunits family based on African clawed frogs (Xenopus laevis, Xl and Xenopus tropicalis, Xt), human (Homo sapiens, Hs), mouse (Mus musculus, Mm), chicken (Gallus gallus, Gg), zebrafish (Danio rerio, Dr), Japanese puffer (Takifugu rubripes, Tr) and American bullfrog (Rana catesbeiana, Rc) was constructed on the plastform phylogeny.fr, using the Maximum Likelihood method. Bootstrap percentages (ranging from 0 to 100) are indicated at each node of the tree while branch lengths are representative of sequence substitution rates. P2rx1, 2, 3, 4, 5, 6, 7 subunit subfamilies are respectively colored in purple, blue, green, dark pink, red, light pink, and orange. The Genbank accession numbers of the different receptors are given in the Supplementary Tables S1A,B except for Rcp2rx8 (Accession Number: AAL24075.1).
	FIGURE 2. Temporal expression profile of p2rx genes during embryogenesis. RT-PCR analysis showing the expression pattern of p2rx subunits in X. laevis unfertilized egg and embryos at different stages (St.). Adult brain cDNA was used as positive control and three negative controls (-RT, -RNA, -cDNA) were also performed. The linearity of the signal was controlled by carrying out PCR reactions on doubling dilutions of cDNA from stage 45 embryo, illustrated by the triangle. Odc (ornithine decarboxylase) was used as a loading control. The different phases of embryogenesis and the Mid-Blastula transition (MBT), the switch between maternal and zygotic expression, are indicated above the different embryonic stages. Seg, segmentation; Gast, Gastrulation.
	FIGURE 3. Comparative spatial expression profile of p2rx genes during embryogenesis. Whole-mount in situ hybridization with specific DIG-labeled antisense RNA probes was performed on embryos from stages (St.) 5 to 37/38. Representative embryos were photographed at the magnification X20 for stages 5, 11, and 14 and X16 for later stages. Stage 5: animal pole view, stage 11: vegetal pole view, stage 14: dorsal view except for p2rx1.S (anterior view), later stages: lateral views, with dorsal is up and anterior is right. The arrowheads indicate the staining in the neurogenic placodes and peripheral nervous system detailed in Figure 5. a.p, animal pole; c.g, cement gland; d.c, duct of Cuvier; e, eye; h, heart; h.b, hindbrain; k, kidney; m, mesoderm; n, notochord; n.s, nervous system; p.e.f, presumptive eye field; p.g, pineal gland; s, somite; t.n, trigeminal nerve; v.b.i, ventral blood island.
	FIGURE 4. Comparative staining obtained with the antisense and sense probes of p2rx genes. Lateral views of three representative embryos at stage 37/38 or stage 41stained with the antisense and sense probes for p2rx1.L, p2rx1.S, p2rx2.L, p2rx2.S, p2rx4.L, p2rx6.L, and p2rx7.L. subunits and p2rx5 homeologs. Some unspecific staining, due to probe trapping, can be observed in the head region (brain ventricles, otic vesicle) and in the notochord. For p2r7.L subunit, a dorsal view of a representative embryo completes the lateral views. For lateral views, dorsal is up and anterior is right. For dorsal views, anterior is up.
	FIGURE 5. Specific expression domains of p2rx genes. Representative embryos from stages (St.) 22/23–41 displaying p2rx subunit expression detected by whole-mount in situ hybridization. To further characterize their expression profile, embryos stained for p2rx genes, except p2r6.L and p2rx7.L, were selected for scalpel sections and cryostat sections. Sections were performed on stage 22/23 for p2rx5 stained embryos, on stage 33/34 for p2rx1.L stained embryos, on stage 37/38 for p2rx1.S, p2rx2.S, and p2rx4.L stained embryos and stage 41 for p2rx2.L stained embryos. The red numbered dotted arrows indicate the plan of the sections. For whole mount pictures, dorsal is up and anterior is right except for the p2rx5 and p2rx7 subunits for which a dorsal view completes the lateral view. For section pictures, dorsal is up. The green arrowhead indicates the fused ganglion of middle lateral line and glossopharyngeal nerves, the orange one shows the trigeminal ganglion and ganglion of anterodorsal lateral line nerve and the red one the ganglia of anteroventral lateral line and facial nerves. c.g, cement gland; e, eye; fb, forebrain; g.b, gall bladder; g.l, ganglion layer; h, heart; hb, hindbrain; l, lens; mb, midbrain; n, notochord; o.n.l, outer nuclear layer; o.v, otic vesicle; p.g, pineal gland; p.gl, pronephric glomus; p.t, pronephric tubules; s, somite; t.n, trigeminal nerve; v.b.i, ventral blood island.

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