Selective Expression of Purinoceptor cP2Y1 Suggestsa Role for Nucleotide Signalling in Developmentof the Chick EmbryoMARTIN P. MEYER,1* JONATHAN D.W. CLARKE,1 KETAN PATEL,3 ANDREA TOWNSEND-NICHOLSON,2AND GEOFFREY BURNSTOCK2

1Department of Anatomy and Developmental Biology, University College London, London, England2Autonomic Neuroscience Institute, Royal Free Hospital School of Medicine, London, England3Department of Zoology, School of Animal and Microbial Sciences, University of Reading, Reading, England

ABSTRACT Responses to extracellular nu-cleotides (e.g., ATP, ADP, etc.) have been demon-strated in a number of embryonic cell typessuggesting they may be important signalling mol-ecules during embryonic development. Here theauthors describe for the first time the expressionof a G-protein–coupled receptor for extracellularATP, chick P2Y1 (cP2Y1), during embryonic devel-opment of the chick. During the first 10 days ofembryonic development, cP2Y1 is expressed in adevelopmentally regulated manner in the limbbuds, mesonephros, brain, somites, and facialprimordia, suggesting that this receptor may havea role in the development of each of these sys-tems. Dev Dyn 1999;214:152–158.r 1999 Wiley-Liss, Inc.

Key words: ATP; chick embryo; P2Y receptors

INTRODUCTION

The role of P2 purinoceptors and their ligands (ATPand other nucleotides) is well established in manyareas of pharmacology and physiology. Our aim is toinvestigate the possibility that ‘‘purinergic’’ signallinghas a role to play during embryonic development.Extracellular ATP has been shown to increase intracel-lular Ca21 (Cai

21) in a number of embryonic cell types(for review see Burnstock, 1996), and increases in Cai

21

can regulate cell proliferation, migration, and differen-tiation (Berridge, 1993; Lauder, 1993). Thus extracellu-lar ATP has the potential to regulate important pro-cesses in embryonic development. In order to investigatethis possibility further, we looked at the expression ofP2 receptors in the chick embryo.

P2 receptors are divided into two families, P2X andP2Y, based on molecular structure, transduction mecha-nisms, and pharmacological properties (Abbracchio andBurnstock, 1994; Burnstock and King, 1996). P2Xreceptors are members of the ligand gated ion channelsuperfamily, and activation of these receptors by extra-cellular ATP elicits a flow of cations (Na1, K1, and Ca21)across the plasma membrane. To date seven P2X recep-tor subtypes have been cloned from mammalian species(Burnstock and King, 1996).

P2Y receptors constitute a family of G-protein–coupled receptors for extracellular nucleotides. Physi-ologic and pharmacologic experiments have demon-strated that P2Y receptors are linked to signallingmechanisms that have been implicated in mitogenesisand differentiation, such as Ca21 mobilisation, polyphos-phoinositide metabolism, and protein phosphorylation(Dubyak and El-Moatassim, 1993). For example, ATPhas been shown to activate mitogen activated protein(MAP) kinases via P2Y1 and P2Y2 receptors in ratastrocytes (Neary and Zhu, 1994). The MAP kinasecascade is a key element of signal transduction path-ways involved in cellular proliferation and differentia-tion. (Avruch et al., 1994; Davis, 1993). Acting throughP2Y receptors, ATP has previously been shown to bemitogenic for 3T3, 3T6, A-431 cells (Huang et al., 1989),and also chick, human, and rat astrocytes (Rathbone etal., 1992; Abbracchio et al., 1994; Ciccarelli et al., 1994).In these cells, ATP also acts synergistically in combina-tion with a variety of polypeptide growth factors tostimulate proliferation (Huang et al., 1989; Neary etal., 1994). These studies in adult systems and on celllines have therefore implicated P2Y receptors in pro-cesses that are vital during embryonic development.

Eight P2Y receptor subtypes have been proposedfrom mammalian, avian, and amphibian species (Burn-stock and King, 1996; Bogdanov et al., 1997). Of these 8proposed P2Y subtypes, only two have been identifiedin chick. These are chick P2Y1 (cP2Y1) and chick P2Y3(cP2Y3). cP2Y1 was isolated from an E10 whole chickbrain cDNA library, and was found to be preferentiallyactivated by ATP and 2MeSATP (a synthetic derivativeof ATP), and to a lesser extent ADP (Webb et al., 1993).cP2Y1 shows 39% amino acid sequence homology tocP2Y3 (Webb et al., 1996), but the two receptors havedistinct pharmacologies as cP2Y3 is activated by ADP,UTP, and UDP (Webb et al., 1996). Here we describe theexpression pattern of cP2Y1 during development of thechick embryo. We show that cP2Y1 is expressed in adevelopmentally regulated manner in the limb buds,

*Correspondence to: Martin Meyer, Department of Anatomy andDevelopmental Biology, University College London, Gower Street,London WC1E 6BT, England. E-mail: ucgampm@ ucl.ac.uk

Received 9 September 1998; Accepted 29 October 1998

DEVELOPMENTAL DYNAMICS 214:152–158 (1999)

r 1999 WILEY-LISS, INC.

mesonephros, brain, somites, and facial primordia ofthe chick embryo, indicating a potentially importantrole for purinergic signalling during embryonic develop-ment.

RESULTSOverview of cP2Y1 Expression

Northern blotting of Poly A1 mRNA isolated fromdifferent stage embryos and embryo tissues showedthat cP2Y1 transcripts were present between stages 7and 12. A single band of 2.7kb was detected thatpersists until hatching, with particularly intense expres-sion seen in the brain and skeletal muscle of stage 41and 45 embryos. Transcripts were also detected in theheart and liver at stage 41, and in the liver and eye atstage 45 (Fig. 1). High levels of discrete expression arefirst detected by whole-mount in situ hybridisation atstage 19. The Northern analysis demonstrates expres-sion before this stage, but our failure to detect it by insitu hybridization suggests that early expression levelsmay be low, although present in many tissues and cells.Here we describe in detail the pattern of cP2Y1 expres-sion in the limbs, mesonephros, somites, branchialarches, and brain.

cP2Y1 Expression in Chick Limb Buds

Expression of cP2Y1 was first detected in mesodermalcells in the posterior third of the wing buds in stage 19embryos. By stage 21, the expression had intensifiedand spread within the limbs to all but the anterior thirdof both wing and leg buds (Fig. 2A). Sectioning revealedan asymmetric pattern of expression along the dorso-ventral axis, with expression increased dorsally (Fig.2B,C). cP2Y1 was absent from the mesodermal cellsimmediately subjacent to the limb ectoderm and fromthe limb bud core.

In a stage 25 limb, cP2Y1 transcripts formed a distalband of expression running anterior to posterior, just

proximal to the progress zone, which is the most distalstrip of mesenchyme underlying the apical ridge (Fig.2D). This pattern was seen on both the dorsal andventral surfaces of the limb. cP2Y1 was not expressed inthe ectoderm or immediately subjacent mesenchyme,nor in the cartilage condensations (Fig. 2F). However,cP2Y1 was expressed in the rest of the limb mesen-chyme, which surrounded the condensing cartilaginouselements. Proximal expression formed a uniform ringaround the limb bud core (Fig. 2E).

By stage 29, expression was strongest around thedistal periphery (Fig 2G). At the proximal limit of astage 29 wing (not shown) the expression pattern wasvery similar to that seen in equivalent proximal sec-tions at stage 25 (Fig. 2E). At the level of both ulna andradius (Fig. 2H) and wrist (Fig. 2I), expression wasstrong in the anterior and posterior mesenchyme butwas less intense dorsally and absent ventrally. In asection through the developing digits, strong expres-sion surrounded the cartilage condensations (Fig. 2J),the strongest expression being at the posterior margin.

In stage 31 limbs, the relationship of cP2Y1 expres-sion to the developing skeleton and tendons was visible,particularly in the digits (Fig. 2K). In a proximalsection (Fig. 2L), there was uniform but faint expres-sion in the dorsal and ventral mesenchyme. A sectionthrough the proximal digit region (Fig. 2M) revealedfaint expression in dorsal mesenchyme, but ventrallyexpression of cP2Y1 was absent. Expression was stron-gest in the mesenchyme surrounding the most posteriordigit. Strong expression was also seen in the otherdigits (Fig. 2N,O) surrounding cartilage and tendoncondensations (arrowed). cP2Y1 expression was absentfrom the core mesenchyme in the interdigital regions.Expression of cP2Y1 in a stage 35 leg (Fig. 2P) borderedthe rods of cartilage that will later form the bones of thedigits, but there were gaps in expression, which corre-spond to the sites of joint formation.

Fig. 1. Northern blot analysis of cP2Y1 expression during chick embryo development. Stage and tissue areindicated at the top. GAPDH was used as a control. Northern blotting of polyA1 mRNA isolated from wholeembryos and embryo tissues showed that transcripts appear between stages 7 and 12, and persist untilhatching. Note the strong expression in stage 41 and 45 brain and skeletal muscle.

153SELECTIVE EXPRESSION OF PURINOCEPTOR cP2Y1

Fig. 2. Expression of cP2Y1 during limb development visualised bywhole-mount in situ hybridisation. In A,D,G,K, and P, specimens areoriented with anterior toward the top. All other specimens are oriented withthe dorsal surface uppermost and anterior to the right. Stage is indicatedin the bottom right corner and the dotted lines indicate the level at whichsections were taken. A: Dorsal view showing expression of cP2Y1 in thewing buds. B: Section through wing demonstrating dorso-ventral differ-ence in cP2Y1 expression. C: Longitudinal section through stage 21 wingshowing expression beneath distal tip. D: Dorsal view of stage 25 wingshowing a more restricted pattern of expression of cP2Y1. E, F: Proximaland distal sections through a stage 25 wing showing expression inmesenchyme surrounding cartilage condensations, c. G: Dorsal view ofstage 29 wing showing strong expression of cP2Y1 around the distal

periphery. H–J: Proximo-distal series of transverse sections through astage 29 wing. Expression is strong in the anterior and posterior marginsin I, and was absent from the ventral surfaces of H and I. K–P: Thesespecimens show expression in the leg. The expression pattern was thesame in the wing, but the leg better demonstrates the pattern ofexpression in the digits. K: Dorsal view of stage 31 leg, showing strongexpression of cP2Y1 surrounding the digits, joints, and tendons, andweaker expression in the interdigital areas. L–O: Proximo-distal seriesthrough a stage 31 leg. The arrows in M and N indicate the tendons.Expression in the core of the interdigital areas is absent in specimen N. P:Expression of cP2Y1 in the digits of a stage 35 leg. Areas of joint formationdid not express cP2Y1. Scale bars 5 100 µm.

Figures 3–6.

In summary, cP2Y1 was expressed in limb bud mesen-chyme, but was absent from ectoderm, developingcartilage condensations, tendons, and areas of jointformation. Expression within mesenchyme was dy-namic; position and strength of expression varied accord-ing to stage and position within the limb. Particularlystrong expression was seen in the mesenchyme sur-rounding the most distal cartilaginous elements andtendons.

cP2Y1 Expression in the Mesonephros

Expression of cP2Y1 in the mesonephros was firstdetected at stage 19 level with somites 14–17. Therewas a cephalocaudal gradient of expression and, bystage 20, transcripts were detected at the level ofsomites 16–22, while expression anterior to somite 16was now absent (Fig. 3A). Expression was character-ised by rows of discrete oval shaped areas of intensestaining in cells that lie at the proximal end of thes-shaped mesonephric tubules (Fig. 3B). This area ofmesenchyme is the mesonephric corpuscle and willbecome the glomerulus. By stage 25, expression ofcP2Y1 in the mesonephric corpuscle had reached thelevel of the leg buds but was absent at the level of thewing buds (Fig. 3C). The pattern of expression reflectsthe maturity of the developing mesonephros. Rostrally,expression of cP2Y1 was more diffuse and less intense.The close proximity of cells expressing cP2Y1 to thevasculature of the developing glomeruli was clearlyvisible in the more mature rostral mesonephros (Fig.3E). Meanwhile the more recently established areas ofexpression, located more caudally (Fig. 3D), were con-fined to the discrete oval clusters of cells describedabove. The glomeruli at this caudal position had not yetbegun to form. By stage 29, expression of cP2Y1 wasdetected in only the most caudal region of the mesoneph-ros.

cP2Y1 Expression in the Somites

cP2Y1 expression in the somites was first detected atstage 21. Expression was strongest in the six mostanterior somites (Fig. 4A), but by stage 25 all but themost posterior somites at the tip of the tail expressedcP2Y1. Sectioning revealed that expression was con-fined to the myotome, within which expression wasstronger ventrally (Fig. 4B).

cP2Y1 Expression in the Branchial Arches

In stage 21 embryos, cP2Y1 was expressed in acrescent shaped distribution of cells in the mandibularprocess of the first arch, and to a lesser extent thesecond arch. (Fig. 5A). Transcripts were restricted tothe mesenchyme in these structures. In the anteriorhalf of the mandibular process, expression of cP2Y1 wasintense and confined to a triangular shaped group ofcells on the dorsal surface immediately subjacent to theendoderm lining the pharynx (Fig. 5B). In the posteriorhalf of the mandibular process, expression was lessstrong, and here expression extended across to theventral surface.

cP2Y1 Expression in the Central Nervous System

Expression in the central nervous system (CNS) wasfirst detected by in situ hybridisation at approximatelystage 33. The largest area of expression was in thetelencephalon, with smaller patches in dorsal diencepha-lon and posterior midbrain (Fig. 6A,B). A small triangu-lar patch of expression was also seen in the anteriorhindbrain either side of the midline (Fig. 6B), which wetentatively identify as the trochlear motor nucleus.Expression in the telencephalon, dorsal diencephalon,and posterior midbrain strengthened by stage 36 (Fig.6C). Telencephalic expression was fairly uniform withinthe superficial cell layers of the pallium (Fig. 6D) and

Fig. 3. Expression of cP2Y1 in the mesonephros. Stage is indicated in thebottom right corner. Dotted lines indicate the position from which sections weretaken. A: Ventral view of stage 20 embryo showing expression in mesoneph-ros and limb buds. B: High power photo showing localisation of expression tomesonephric corpuscle (mc). m, mesonephric tubule. C: Ventral view of stage25 embryo showing that expression of cP2Y1 in the mesonephros hadprogressed caudally and was down-regulated rostrally. D: cP2Y1 in the mostrecently established areas of expression located caudally. Expression wasstrong and restricted to tight oval clusters of cells. E: The most rostrally locatedareas of cP2Y1 expression were associated with the newly formed glomeruli(g). The bright areas are blood cells within the glomerulus. Expression herewas weaker and less restricted than seen caudally. Bars indicate 200 µm in Aand D, 100 µm in B, and 50 µm in all other specimens.

Fig. 4. cP2Y1 expression pattern in the somites. Stage is indicated inthe bottom right corner. A: Lateral view showing expression in the anteriorsomites. The dark area in the head region is due to an artefact ofphotography and slight trapping of probe in head mesenchyme; very weakstaining in this region was also seen with the negative control senseprobe. B: Transverse section showing localisation of expression to themyotome (m). sc, spinal cord; bar indicates 30 µm in each photo.

Fig. 5. cP2Y1 expression in the branchial arches. Stage is indicated inthe bottom right corner. A: Lateral view of expression in the mandibular

(man) process of the first arch, and the second branchial arch (2nd ba).The line drawing indicates the region shown in the photograph. Anterior isto the left. B: Transverse section through the anterior mandibular process.Dorsal is uppermost. nc, notochord; ph, pharynx. Expression was con-fined to a triangular shaped group of cells on the dorsal surface. Barindicates 100 µm.

Fig. 6. Expression of cP2Y1 in the CNS. In A–C, specimens areoriented with anterior to the left, dorsal surface uppermost. In D and E,dorsal is at the top. Stage is indicated in the bottom right corner and dottedlines indicate levels at which sections were taken. A: Dorsal view offorebrain showing expression in telencephalon and dorsal diencephalon.B: Dorsal view of midbrain and hindbrain. cP2Y1 was expressed inposterior midbrain and in triangular patches of cells either side of themidline in anterior hindbrain. hb, hindbrain. C: Dorsal view of stage 36brain showing increased levels of expression in telencephalon, dorsaldiencephalon, and posterior midbrain. tel, telencephalon; mes, mesen-cephalon; cb, cerebellum. D and E are sections through telencephalon ofstage 33 brain. D: Expression is confined to the superficial layers of thepallium. s/p, striatum-pallium boundary. Expression close to the septalregion (sr) is also shown. E: Dorsal diencephalic expression. vz, ventricu-lar zone. The scale bar in C represents 1 mm (for A–D). The scale bar in Erepresents 300 µm.

156 MEYER ET AL.

extended up to but not into the striatum. cP2Y1 wasalso found in a small patch of the ventricular zone closeto the septal regions and extended from just the lateralaspect of the ventricle at stage 33 to include its medialaspect by stage 36. The dorsal diencephalic expressionlay within developing grey matter and did not includeventricular zone cells (Fig. 6E).

DISCUSSION

The idea that extracellular nucleotides may play arole in the process of embryonic development is not new.In this study, however, we present the first detaileddescription of the selective expression of an extracellu-lar ATP receptor in multiple tissue systems throughoutdevelopment of a vertebrate embryo. The expressionpatterns are dynamic and their appearance in diversesystems suggests ATP may play multiple roles through-out embryogenesis.

The expression pattern of the cP2Y1 receptor de-scribed here belongs to a family of G-protein–coupledreceptors that, in adult systems and cell lines, has beenshown to be involved in regulating proliferation anddifferentiation. For example, extracellular nucleotides,the ligands for these receptors, are potent regulators ofdifferentiation of astrocytes. They have been shown toincrease the expression of the astroglial-specific markerglial fibrillary acidic protein (GFAP), promoting astro-cytic hypertrophy and elongation of astrocytic pro-cesses, events that may participate in the remodellingand functional recovery of brain circuitries followingtrauma and ischemia (Abbraccio et al., 1995; Neary etal., 1994; Neary et al., 1996). P2Y receptors have alsobeen shown to regulate the proliferation of these cells(Rathbone et al., 1992; Abbracchio et al., 1994; Cic-carelli et al., 1994), and of the 3T3, 3T6, and A-341 celllines (Huang et al., 1989). Extracellular nucleotides canalso synergise with fibroblast growth factors (FGFs) inexerting these effects on proliferation of astrocytes(Neary et al., 1994), and with FGF, epidermal growthfactor (EGF), and platelet derived growth factor (PDGF)on proliferation of 3T3, 3T6, and A-341 cells (Huang etal., 1989).

These studies show that these receptors are capableof regulating mechanisms important for embryonicdevelopment. Their ability to modulate the actions ofsignalling molecules already known to be important inthis field, such as FGFs, has prompted us to search formore direct evidence for P2 receptor signalling indevelopment of the embryo. We have shown here thatduring early development of the chick embryo, cP2Y1 isexpressed in a developmentally regulated manner in anumber of tissues where it has the potential to regulateproliferation and differentiation of cells by the mecha-nisms described above.

In the early limb bud (HH stage 21) cP2Y1 is moreheavily expressed dorsally. Activation of cP2Y1, leadingto increased proliferation in expressing cells, may con-tribute to the dorsal curvature that begins to appearsoon after cP2Y1 transcripts are first detected. In

general, at stage 25 and later stages, cP2Y1 expressionis consistently strong distally surrounding the mostrecently differentiating cartilage elements and is main-tained at lower levels surrounding both cartilage andtendon in more proximal regions of the limb mesen-chyme. This pattern of expression suggests this purino-ceptor may be marking areas of relatively undifferenti-ated mesenchyme where it has the potential to regulateproliferation and/or differentiation of these cells. Thelength of time that expression persists, coupled withthe dynamic expression within limb mesenchyme, raisesthe possibility that cP2Y1 fulfills more than one func-tion within the developing limb.

The expression of cP2Y1 within the mesonephros iscephalocaudally regulated and expression is down-regulated as red blood cells become visible in thecapillaries of the newly formed glomeruli. This patternsuggests a role in development rather than in thefunction of the glomerulus. We have not yet studiedstages that include metanephric kidney developmentbut it would be interesting to see if transcripts aredetected at the time of glomerular vascularisation inthe metanephros.

In contrast to the recently isolated Xenopus xl-P2Y8receptor, which is expressed at very early stages ofneural plate development (Bogdanov et al., 1997), thechick P2Y1 receptor does not appear to play a role inearly development of the CNS. It is expressed mostly inregions of post-mitotic neurons (e.g., superficial layersof the telencephalon) at a stage when the telencephalonis beginning its morphogenetic expansion to envelopthe dorsal diencephalon. Proliferative cells in the CNSare located in the ventricular zones, but with theexception of the region close to the developing septalnuclei (Fig. 6L), the ventricular cells do not expresscP2Y1.

Activation of a P2Y receptor has previously beenshown to be involved in differentiation of rat astroglialcells (Neary et al., 1994; Abbraccio et al., 1995; Neary etal., 1996). cP2Y1 therefore has the potential to play arole in the differentiation of chick embryo CNS cells inwhich it is expressed. It will be interesting to comparethis receptor’s expression in the embryo with its pat-tern in the more mature bird brain in order to establishits developmental and/or functional roles.

In summary we have introduced the concept ofextracellular ATP as an important signalling moleculeduring early embryonic development. We have shownthat the ATP receptor cP2Y1 is expressed in a number ofembryonic chick tissues. The precise functions of thisreceptor in these tissues remain to be explored.

EXPERIMENTAL PROCEDURESEmbryos

Eggs were incubated at 37°C and staged according tothe Hamburger and Hamilton (HH) series (Hamburgerand Hamilton, 1951). Embryos were removed from theegg, rinsed, and dissected free of extra embryonicmembranes in sterile PBS. Embryos were then either

157SELECTIVE EXPRESSION OF PURINOCEPTOR cP2Y1

fixed in 4% paraformaldehyde for use in whole-mountin situ hybridisation or processed for RNA extraction.

Northern Blots

Total RNA was isolated from staged embryos andembryo tissues using guanidium isothyocyanate (CPLaboratories). Poly A1 mRNA was prepared from thetotal RNA using a Poly A tract mRNA isolation system(Promega). The Poly A1 mRNA was then electropho-resed through a MOPS formaldehyde 1% agarose gel,and transferred to Hybond N1 membrane (Amersham)using standard techniques (Sambrook et al., 1989). Thehybridisation probe was a 1.4 kb cP2Y1 cDNA fragmentlabelled with [a-32P] dCTP to a specific activity ofapproximately 13109 dpm/µg. Probe synthesis wascarried out using a Ready to Go DNA labelling kit(dCTP), and unincorporated nucleotides were removedusing a Sephadex G-50 NICK column (both from Phar-macia). Hybridisation was performed overnight at 42°Cin 50% formamide, 53SSC, 53Denhardts, 0.5% SDS,100 µg/ml salmon sperm DNA, and 50 mM phosphate.The hybridisation filter was subjected to a final wash at65°C in 0.23SSC, 0.1% SDS for 20 min before beingexposed to X-ray film for 24 hr at 270°C with anintensifying screen. The same blot was probed with a250bp fragment of chicken GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA as a control (Pana-bieres et al., 1984).

Generation of Riboprobes and Whole-MountIn Situ Hybridisation

A 1.4kb cP2Y1 cDNA fragment was subcloned into theHindIII site of pRC CMV (Invitrogen). Sense andantisense digoxygenin (Boehringer Manheim) labelledprobes for cP2Y1 were made by transcription with T7(sense) and Sp6 (antisense) RNA polymerase accordingto the manufacturer’s instructions (Promega). The anti-sense probe was a 1.2kb transcript made from thecoding region 161–1407 of the published cP2Y1 nucleo-tide sequence (Genbank accession number x73268).Whole-mount in situ hybridisation was performed ac-cording to the protocol described by Nieto et al. (1996).Although sequence homology between cP2Y1 and theother known chick P2Y receptor (cP2Y3) is relativelylow, the post-hybridisation washes were performed athigh stringency (50% formamide, 53SSC, 1% SDS, for 1hr at 75°C, followed by 50% formamide, 23SSC, for 30min at 70°C) to eliminate the possibility of crossreactivity. To ensure complete penetration of the probeat stage 29–36, whole-mount in situ hybridisation wasperformed on brains dissected free of surrounding headtissue. A sense riboprobe was used throughout as anegative control. Stained embryos were embedded ingelatin-albumin for cutting 50 µm vibratome sections.

ACKNOWLEDGMENTS

We thank Les Dale for advice throughout this workand for helpful comments on the manuscript.

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