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.


| INTRODUC TI ON
Characteristic head structures are thought to define vertebrate species. The vertebrate head structure is basically derived from the brain, the neural crest, and placode cells. Vertebrate head formation originates from the establishment of an organizer (Niehrs, 2004;Spemann & Mangold, 1924, 2001. At the onset of gastrulation, the organizer is formed in the dorsal equatorial region. The organizer ectopically induces dorsal structures, including the central nervous system, when transplanted ventrally (Spemann & Mangold, 1924, 2001. The organizer can be subdivided into two regions: the head and trunk organizers (Sasai & De Robertis, 1997). BMP antagonists (Noggin, Chordin, and Follistatin) emanating from the organizer directly induce neural tissues in the ectoderm layer (Fainsod et al., 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.
P2Y receptors have been investigated from both physiological and pathophysiological perspectives, but little is known about how they function in early development. Some P2Y receptors are known to function in processes related to head morphogenesis.
In X. laevis tadpole, pyrimidinergic receptor P2Y, G-protein coupled, 4 L homeolog, p2ry4.L, is expressed in basal cells of the main olfactory epithelium and involved in the regulation of cell turnover in the olfactory epithelium (Hassenklover, Schwartz, Schild, & Manzini, 2009;Hassenklover et al., 2008). A study provided evidence that the supporting cells of the vomeronasal organ express p2ry4.L and the basal cells express multiple P2Y receptors (Dittrich, Sansone, Hassenklover, & Manzini, 2014).
We show herein the involvement of P2RY4 receptor in head formation in Xenopus development. p2ry4 is expressed in tissues necessary for head formation, such as the head organizer, the anterior neural tissues, the head neural crest, and placode cells, during Xenopus early development. Loss of p2ry4 function impaired head structure development, revealing the functional involvement of p2ry4 in head formation. p2ry4 seems to function in multiple steps of head formation, such as involution movement, head organizer formation, and neural crest and placode cell establishment. In addition, P2Y receptors are highly conserved in vertebrates and their homologs cannot be found in any invertebrates. Taken together, as the head structure is thought to define the vertebrate (Ota, Kuraku, & Kuratani, 2007), it might not be a simple coincidence that the vertebrate-specific receptor is involved in the formation of a vertebrate-specific structure, namely, "the head". This has further motivated us to think of how vertebrate species emerged in the course of evolution.

| Embryo manipulation
Adult Xenopus laevis was purchased from Watanabe Zosyoku (Hyogo, Japan) and embryos were obtained by artificial fertilization.
Digoxigenin-labeled-antisense RNAs were generated by in vitro transcription with a MAXIscript Kit (Ambion) and DIG RNA Labeling Mix (11277073910, Roche).

| Spatial expression patterns of p2ry4
Xenopus laevis is known to be allotetraploid species, and the L and S chromosomes exist in X. laevis genome. However, genes have been lost asymmetrically between the two subgenomes. Some genes are lost from one subgenome and arose singleton, and the size and number of deletion are greater on the S subgenome (Session et al., 2016).
We confirmed that p2ry4 exist only in L chromosome and no other p2ry4 related sequence could be found in S chromosome. Therefore, in this report we call p2ry4.L just p2ry4. First, we performed a phylogenetic analysis of P2Y receptors. P2Y receptors were found in only vertebrate and some ancestral genes were found in lamprey (see Discussion, Supporting information Figure S1). Some P2Y receptors were involved in the head morphogenesis of X. laevis (for example, see Masse et al., 2007;Hassenklover et al., 2008;Geach et al., 2014).
We also reported that p2ry1 and p2ry11 were involved in the head formation (Harata et al., 2016). Furthermore, the phylogenetic analysis showed that these genes were classified into four groups and p2ry1, p2ry2, p2ry4, p2ry6 and p2ry11 were included in the same group.
Then, to clarify whether p2ry4, whose ancestral gene was found in lamprey, is actually related to the Xenopus head formation, we

| p2ry4 disruption in TALEN mRNA injected embryos
As shown above, p2ry4 is expressed in regions important for head morphogenesis. To investigate the role of p2ry4 in early development, first, we used a knockdown approach by using morpholino antisense oligonucleotides (MOs). However, we could not down-  Figure S3A). On the other hand, embryos treated with TALEN-L (Supporting information Figure S3A) or TALEN-R alone (data not shown) developed normally and their survival rates were consistently higher than 90%, indicating that TALEN mRNAs did not exhibit toxicity under our experimental conditions. Phenotypes of TALEN-L/R injected embryos were divided into two groups: moderate and severe.
The moderate group showed the small head phenotype (Supporting information Figure S3A, L/R-1) and the severe group exhibited a defect in blastopore closure that led to the spina bifida phenotype with a small head (Supporting information Figure S3A, L/R-2). Then, we determined whether the target gene was disrupted or not by DNA sequencing analysis (Supporting information Figure S3B) and found that p2ry4 TALEN possessed the ability to disrupt the target gene at an early stage. Therefore, our results suggested that p2ry4 plays a crucial role in early development.

| p2ry4 is important for head organizer formation
p2ry4 was prominently observed in the head organizer region at early gastrula stage (Figure 1b). In order to clarify whether p2ry4 functioned in the head organizer region during neural induction, we examined the effect of p2ry4 depletion on the expression of organizer genes at gastrula stage ( Figure 4). It is known that to determine epidermal fate in the presumptive ectoderm, BMP activities should be inhibited by such antagonists as Chordin, Noggin, and Follistatin, all of which are secreted by the organizer (Fainsod et al., 1997;Sasai et al., 1994;Smith & Harland, 1992). Then, head neural tissue is determined by the expression of such Wnt antagonists as Dkk1 and Cerberus (cer) (Glinka et al., 1997(Glinka et al., , 1998Piccolo et al., 1999). Although the expression of dkk1 or cer in TALEN-L/R injected embryos was hardly affected at early gastrula stage (dkk1, 11.8%, n = 17, Figure 4c; cer, 18.8%, n = 16, Figure 4i) (dkk1, n = 19,  (Figure 5a) and thus, we were able to confirm that injected p2ry4 was located in the area.
We found that the expression of dkk1 and cer was induced ectopically by p2ry4 (dkk1, n = 15, Figure 5e, n = 16, Figure 5f; cer, n = 10,  Figure 5d; cer, Figure 5g), we considered that p2ry4 could induce the expression of these head organizer genes, possibly in a cell-autonomous manner. These data suggested that p2ry4 is required for the expression of Wnt antagonists, such as dkk1 and cer, and may play a key role in the head organizer formation. Therefore, it is expected that the co-injection of p2ry4 with the truncated BMP receptor (TBRII) (Suzuki et al., 1994) would induce a complete secondary axis with head structures (Glinka et al., 1997) but not a secondary head structure (see Discussion and Supporting information Figure S4). Furthermore, the depletion of p2ry4 resulted in the spina bifida phenotype (Supporting information Figure   S3A, TALEN-L/R-2), which was caused by the defect of involution movement during gastrulation (Chung et al., 2004;Tao et al., 2005).
Therefore, we analyzed the effect of p2ry4 depletion on mesoderm involution by observing the expression patterns of brachyury (bra) as a chordamesoderm marker (Supporting information Figure S5) and goosecoid (gsc) as a prechordal mesoderm marker (Supporting F I G U R E 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 information Figure S6) at gastrula stage. We found that p2ry4 disruption inhibited the involution movement although mesoderm induction occurred (see Discussion and Supporting information Figures   S5 and S6).

| p2ry4 knockout using TALENs enables targeted mutagenesis in vivo for functional analysis
Our results revealed that the TALEN-mediated knockout of p2ry4 af- depletion specifically. Interestingly, when all four MOs were injected at once, most of the embryos died during gastrulation. Although the blastopore appeared at the early gastrula stage in the four MO injected embryos and the TALEN injected embryos, normal blastopore closure was gradually suppressed. Therefore, it is possible that the complete loss of p2ry4 may lead to developmental defect prior to gastrulation, as seen in the TALEN injected embryos.

| p2ry4 is required for induction of neural tissues
We showed that p2ry4 depletion resulted in the reduced expression of genes specific for the neuroectoderm (sox2, otx2, pax6, rx1, and krox20), the anterior neural border (snail1 and hairy2b), and the head organizer (dkk1 and cer). In addition, the ectopic expression of p2ry4 induced the expression of the head organizer genes. These findings indicate that p2ry4 primarily induces and/or maintains the expression of genes specific for the head organizer, and induces the formation of head neuroectoderm. Therefore, the incomplete establishment of the head organizer in p2ry4 TALEN-L/R injected embryos may result in the defect of anterior neural tissues. If this is the case, p2ry4 should induce a complete secondary axis when expressed ventrally with BMP antagonists. To check this possibility, a truncated BMP receptor (TBRII) was injected with cer or p2ry4 mRNA ventrally at the 8-cell stage (Supporting information Figure S4). It is known that the inhibition of BMP by TBRII expression induces an ectopic dorsal structure (Suzuki et al., 1994) and the simultaneous inhibition of both BMP and Wnt signaling induces a complete secondary axis with head structures, including eyes and cement glands (Glinka et al., 1997).
TBRII injection with cer induces a complete secondary axis (Piccolo et al., 1999). However, contrary to our expectation, p2ry4 could not induce ectopic head structures when BMP signaling was suppressed (Supporting information Figure S4). These results suggested that p2ry4 may be necessary but insufficient for the head organizer formation although p2ry4 is required for the expression of dkk1 and cer.
It was recently shown that two structurally and functionally related Xenopus GPCRs flop1 (also known as Xgpr4; Chung et al., 2004) and flop2 (also known as xflop; (Tao et al., 2005) induced the expression of Wnt antagonists, such as dkk1 and cer, cell-autonomously and functioned in head formation via an RhoA-dependent or independent pathway (Miyagi, Negishi, Yamamoto, & Ueno, 2015). P2RY4 receptor is responsive to uridine nucleotide (UDP, UTP, and UMP) and ATP (Bogdanov, Dale, King, Whittock, & Burnstock, 1997), but f1op1/2 ligands have not been identified yet. We expect that p2ry4 would also have similar functions to flop1/2 and contribute to head formation. Another Xenopus GPCR, lpar6 (also known as p2y5 ;Choi et al., 2010), is required for telencephalon development (Geach et al., 2014). Thus, p2ry4 co-expressed with these genes may induce a complete secondary axis when TBRII is also expressed, whereas p2ry4 alone cannot. Therefore, the head induction in Xenopus may be regulated by the synergistic effects of several GPCRs. Furthermore, even though the expression of dkk1 and cer was not affected in TALEN-L/R injected embryos at early gastrula stage, ectopic p2ry4 expression induced the expression of these head organizer genes.
F I G U R E 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 We interpreted these contradictory observations as indicating that siamois and twin, the downstream targets of the early Wnt pathway, transiently induced dkk1 and cer expression at early gastrula stage so that p2ry4 depletion might not lead to the downregulation of dkk1 and cer. The reduction of head organizer gene expression at mid gastrula stage in TALEN-L/R injected embryos was also consistent with the possibility that p2ry4 has the ability to maintain the expression of head organizer genes.

| p2ry4 is involved in involution movement during Xenopus gastrulation
We also found that p2ry4 depletion yielded the spina bifida phenotype, which was caused by a defect of tissue movement during gastrulation. p2ry4 was expressed in the dorsal marginal zone, which was composed of the anterior and posterior mesoderm, suggesting that p2ry4 might be also involved in such morphological movements as mesoderm involution and convergent extension. To examine the effect of p2ry4 on involution movement at gastrula stage, we checked the expression patterns of bra (Supporting information Figure S5) and gsc (Supporting information Figure S6) and found that p2ry4 disruption inhibited the involution movement without affecting the mesoderm induction. In normal development, the gsc domain internalized fully and the bra domain localized at the tip of the blastopore lip in mid gastrula embryos (Supporting information Figure S6D, E). However, in p2ry4 depleted embryos, the gsc domain failed to internalize (Supporting information Figure S6F) and the anterior mesoderm faced the outside of embryo (Supporting information Figure S6I), suggesting that p2ry4 was involved in the involution. It was reported that a member of the same cluster, p2ry11, was required for the convergent extension movement of the chordamesoderm, but not for the involution movement (Shindo et al., 2010).
Therefore, p2ry4 may participate in the mesoderm involution and then p2ry11 cooperatively functions in the convergent extension.

| p2ry4 may function in multiple steps of X. laevis head formation process
As mentioned above, we demonstrated that p2ry4 functioned in neural induction through the appropriate formation of the head organizer.
Embryos lacking the p2ry4 function could not form the head structures properly. This means that the proper formation of the neural crest and placode cells is inhibited by the depletion of p2ry4. As neural crest formation is fully dependent on neural induction, it is quite natural to think that failure of head organizer formation can affect neural crest formation. In addition, p2ry4 was prominently expressed at the neural boundary at neurula stage, and it was shown that pax3 and zic1 activated p2ry4 as the target gene in neural crest cells (Bae et al., 2014), suggesting that this receptor might play a role in the establishment of neural crest identity. [Ca 2+ ]i increases in response to ATP stimulation in various cell types were shown to play roles in regulating cell proliferation, differentiation, and migration (Berridge, 1993;Lauder, 1993). In X. laevis, multiple P2Y receptors were expressed in basal cells of the olfactory epithelium in which stem cells and various progenitors were present, and regenerative capacities were maintained. The increases of [Ca 2+ ]i in basal cells were involved in the stimulation of cell proliferation to regenerate damaged tissues (Hassenklover et al., 2008(Hassenklover et al., , 2009Hegg, Greenwood, Huang, Han, & Lucero, 2003). Neural crest cells are multipotent progenitor cells and need to be maintained in proliferative and undifferentiated states (Nagatomo & Hashimoto, 2007). In this regard, p2ry4 condensed into the neural crest at neurula stage might regulate cell proliferation and differentiation to maintain progenitor cells.

| Evolutionarily conserved p2y genes may be involved in acquisition of head in vertebrates
We found that P2Y receptors were highly conserved in vertebrates although their homologs were not present in any invertebrates, even in ascidian or amphioxus (Supporting information Figure S1).
We found five ancestral p2y genes in lamprey and many paralogs in jawed vertebrate species. Phylogenetic analysis showed that p2y genes were classified into four clusters by comparative analysis of amino acid sequences. These four clusters might be generated by two rounds of whole genome duplication.
p2ry1, p2ry4, and p2ry11 are classified in the same cluster (Supporting information Figure S1A). It has been shown that p2ry1, p2ry4, and p2ry11 are involved in head formation (Harata et al., 2016;this report). In addition, p2ry5, which belongs to another cluster (Supporting information Figure S1D), is required for the initial specification and/or maintenance of the telencephalon (Geach et al., 2014). The expression patterns of p2ry12, p2ry13, and p2ry14 are very similar to those of p2ry1, p2ry4, and p2ry11 in various regions important for head formation (data not shown), suggesting that p2ry12, p2ry13, and p2ry14 may be also required for head formation. Thus, the ancestral genes of these p2y could originally have crucial roles in head formation. It is known that vertebrates came to possess "the head" by acquiring the neural crest and placode cells during evolution (Gans & Northcutt, 1983).
In addition, p2y, which is involved in head formation processes, is found only in vertebrates. This coincidence indicates that further studies of the role of P2Y receptors in head formation would give exciting hints to understand how vertebrate species emerged during evolution.
K. Umesono for TBRII/pCS2. We also thank Dr. K. Suzuki for invaluable help and our laboratory members for stimulating discussions and animal maintenance.