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Keywords:

  • ATP;
  • rat embryo;
  • P2Y receptors;
  • somites;
  • skeletal muscle;
  • heart;
  • nervous system;
  • RT-PCR;
  • immunohistochemistry

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Extracellular ATP mediates diverse biological effects by activating two families of receptors, the P2X and P2Y receptors. There is growing evidence to show that activation of G protein-coupled P2Y receptors can produce trophic effects in many cell types. Yet the expression and function of the P2Y receptors in development has rarely been studied and has never been investigated in mammalian development. This study used the reverse transcription-polymerase chain reaction and immunohistochemistry to demonstrate the abundant and dynamic expression of P2Y receptors in rat development. These receptors were expressed in a wide range of embryonic structures, notably somites, skeletal muscle, the central and peripheral nervous system, the heart, lung, and liver. All the P2Y receptors studied were expressed as early as embryonic day 11, when most embryonic organs were far from being functional and still in the process of being formed. P2Y receptor proteins were strongly expressed in temporary, developmental structures that do not have a correlate in the adult animal, including the somites (P2Y1, P2Y2, and P2Y4) and the floor plate of the neural tube (P2Y1). P2Y receptors were also dynamically expressed, with receptor mRNA and protein being both up- and down-regulated at different developmental stages. The down-regulation of the P2Y1, 2, and 4 receptor proteins in skeletal muscle and heart, and the disappearance of the P2Y4 receptor from the brainstem and ventral white matter of the spinal cord postnatally, demonstrated that many P2Y receptors were likely to be involved in functions specific to embryonic life. Thus, these findings strongly suggest that P2Y receptors play an important role in the development of many tissues, and pioneer further studies into the role of purinergic signalling in development. Developmental Dynamics 228:254–266, 2003. © 2003 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Extracellular nucleotides evoke responses by activation of two families of purinoceptors, namely the P2X and P2Y receptors (Abbracchio and Burnstock, 1994). Whereas P2X receptors are ligand-gated ion channels, P2Y receptors are G protein-coupled receptors. Eight P2Y receptor subtypes have been cloned to date in mammals (P2Y1,2,4,6,11,12,13, 14) (Communi et al., 2001; Abbracchio et al., 2003). These receptors couple predominantly to phospholipase C activation, leading to the formation of inositol phosphate and mobilization of intracellular Ca2+ (Ralevic and Burnstock, 1998; Vassort, 2001). In addition to increased intracellular Ca2+, a variety of signal transduction pathways, involving protein kinase C, phospholipase A2, and the mitogen-activated protein kinases, mediate the effects of P2Y receptors. Thus, activation of the P2Y receptors can cause long-term, trophic effects on cell activity (Neary et al., 1996; Abbracchio and Burnstock, 1998, Burnstock, 2002a).

P2Y receptors have been implicated in the regulation of cell proliferation and differentiation. Activation of P2Y receptors on a variety of cell types, including astrocytes and vascular smooth muscle cells, results in cell proliferation (Burnstock, 2002b; Neary et al., 1998, 1999; Neary, 2000). In contrast, activation of the P2Y11 receptor has been strongly implicated in neutrophil differentiation (Jiang et al., 1997; Conigrave et al., 1998; Communi et al., 2000). Although these findings strongly suggest a role for purinergic signaling in development, there have been few studies focusing on the expression and function of P2Y receptors in embryonic and postnatal development.

The Xenopus P2Y receptor, the XIP2Y (also called P2Y8), was shown in the neural plate during neurulation but not detected after neural tube closure (Bogdanov et al., 1997), suggesting a role for this receptor in the process of neurulation. More recently, expression of P2Y1 receptor (cP2Y1) mRNA and protein has been demonstrated during chick embryonic development (Meyer et al., 1999a; Choi et al., 2001). Meyer et al. (1999a) reported strong expression of cP2Y1 receptor mRNA in undifferentiated limb mesenchyme cells, but expression was lost as the cells differentiated. The same group also demonstrated that ATP acting by means of cP2Y1, significantly inhibited cartilage formation in micromass cultures (Meyer et al., 2001).

These studies are limited to the P2Y1 and P2Y8 receptors. Other P2Y receptor subtypes have rarely been studied in development, and none have ever been studied in mammalian in vivo development. In this study, we used the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the expression of four P2Y receptors, P2Y1, P2Y2, P2Y4, and P2Y6, in rat embryonic and postnatal development. Expression of all the P2Y receptors cloned in rat to date was studied by RT-PCR. In addition, by using specific antibodies that have only recently become commercially available, the localization of P2Y1, P2Y2, and P2Y4 receptor proteins was investigated. Thus, it has been possible to demonstrate for the first time, the abundant and dynamic expression of P2Y receptors in mammalian development.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

RT-PCR for P2Y Receptors

By using RT-PCR, the developmental expression profiles for P2Y1, P2Y2, P2Y4, and P2Y6 were studied in rat embryos (Fig. 1; Tables 1, 2). All the P2Y receptor subtypes examined were detected as early as embryonic day 11 (E11), and mRNA transcripts were present throughout development (E12–E18). From E11 onward, clear bands were observed for P2Y1 and P2Y4 mRNA transcripts (Fig. 1). P2Y2 receptor expression was very weak at E11 but became progressively stronger with development. Although P2Y6 receptor expression was present as early as E11, expression was only barely detectable and strong expression was observed only at E18.

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Figure 1. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to investigate the expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptor transcripts during rat development from embryonic day 11 (E11) to E18. Total RNA from whole embryos (E11, E12, E14, and E18) or isolated embryonic organs (brain, heart, liver, lung, and muscle) was extracted and subsequently used for RT-PCR experiments.

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Table 1. Summary of the Expression of P2Y Receptor mRNA at Different Developmental Agesa
 E11E12E14E18
  • a

    The + indicates clear positive bands; − indicates absence of expression; and ± indicates barely detectable bands. E, embryonic day.

P2Y1++++
P2Y2±+++
P2Y4++++
P2Y6±±±+
Table 2. Summary of Expression of P2Y Receptor mRNA in Tissue at Two Different Agesa
 BrainHeartLiverLungMuscle
E14E18E14E18E14E18E14E18E14E18
  • a

    The + indicates clear positive bands; − indicates absence of expression; and ± indicates barely detectable bands. E, embryonic day.

P2Y1+++++++++
P2Y2++++++++
P2Y4+++++±+++
P2Y6++++±+

To determine more precisely the location of P2Y receptor expression, brain, heart, liver, lung, and muscle were taken from both E14 and E18 embryos for RT-PCR analysis (Fig. 1; Table 2). All embryonic tissues examined expressed at least one P2Y receptor subtype. However, different embryonic organs demonstrated different P2Y receptor profiles. For example, at E14 only P2Y4 receptor mRNA was detected in the brain, whereas P2Y1, P2Y2, and P2Y4 were expressed in lung, and all the P2Y receptors tested (P2Y1, 2, 4, 6) were expressed in heart. Furthermore, the expression of P2Y receptor mRNA was up- and down-regulated during the course of embryonic development. In the brain, no P2Y1 was detected at embryonic day (E) 14, but at E18, RT-PCR demonstrated strong expression. Similarly in muscle, P2Y6 expression only began at E18. In contrast, whereas P2Y4 was expressed in the lung at E14, no P2Y4 transcripts were detected at E18.

Immunohistochemistry Demonstrated Expression of P2Y1, P2Y2, and P2Y4 Receptor Protein in Somites

P2Y receptor immunoreactivity was first detected in embryos at E11. At this stage P2Y1 and P2Y4 receptor proteins were detected in the somites. Whereas the P2Y4 receptor was detected in the dermomyotome, P2Y1 receptor expression appeared to be restricted to the myotome (Fig. 2A,C,D). The P2Y2 receptor was also expressed in the somites at E12 (Fig. 3A), a day later than the expression of P2Y1 and P2Y4. By using MF20 as a marker for the myogenic cells of the myotome (Fig. 3B), the expression of all the P2Y receptors was demonstrated specifically in this area of the somite. As in the case of P2Y1 and P2Y4, double-labeling for P2Y2 and MF20 showed colocalization of these proteins in the myotome as opposed to the sclerotome (Fig. 3C).

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Figure 2. Immunoreactivity for P2Y receptors in somites and developing skeletal muscle. A: Transverse section with hematoxylin and eosin staining showing the morphology of embryonic day (E) 11 somites (sm). B: Transverse section showing hematoxylin and eosin staining of an E14 embryo at the mid-liver level. The rectangular insert shows an area of developing skeletal muscle. C: Transverse section taken at the level of the embryonic heart at high magnification showing limited P2Y1 receptor expression in E11 somites (sm). To aid orientation the ventral (v) and dorsal (d) aspects are marked. P2Y1 receptor expression was detected in the myotome. D: Transverse section taken at the caudal region at high magnification showing widespread P2Y4 receptor expression in E11 somites (sm). P2Y4 receptor expression was detected in the dermomyotome. E: Higher magnification (of the rectangular insert shown in B) demonstrating P2Y1 receptor expression in the developing skeletal muscle (skm). F: Higher magnification (of the rectangular insert shown in B) demonstrating P2Y4 receptor expression in the developing skeletal muscle (skm). G,H: Transverse sections of E18 embryo showing the intercostal muscle between ribs (rb). The P2Y1 receptor was not detected in the skeletal muscle fibers (G), whereas strong P2Y4 receptor immunoreactivity was detected in the intercostal muscle (H). Scale bar in H = 50 μm in C,D, 200 μm in A,E–H, 750 μm in B.

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Figure 3. Immunofluorescence of P2Y receptors during skeletal muscle development and in the embryonic spinal cord. A: Transverse section showing P2Y2 receptor immunoreactivity (green) in E12 myotomes (my). B: The same section also showed immunoreactivity for MF20 (red), a marker of the differentiated myogenic cells. C: Double labeling showing that the P2Y2 receptor and MF20 were coexpressed in the myotome (yellow). D–F: Double-labeling experiments for P2Y receptors (green) and acetylcholine receptors (red) on the tibialis anterior muscle of 3-week-old animal. D: P2Y1 receptor immunoreactivity was localized at the smooth muscle layer of the blood vessel (bv) between the skeletal muscle fibers. E: P2Y2 receptor immunoreactivity was detected in scattered cells (arrowheads) located between skeletal muscle fibers. F: P2Y4 receptor protein was detected on the membranes of the peripheral but not the central muscle fibers. None of the P2Y receptors were detected at the neuromuscular junctions (nmj), as demonstrated by acetylcholine receptor expression (red). G: Transverse section showing P2Y4 receptor immunoreactivity (green) in the spinal cord of an E14 embryo. Arrows showing the spinal nerves emerging from dorsal (dh) and ventral horns (vh). H: PGP9.5 immunoreactivity (red) on the same section (G). I: Double labeling showing the colocalization (yellow) of the P2Y4 receptor and the PGP9.5 in the ventral spinal horn but not the dorsal spinal cord. J: Transverse section showing expression of the P2Y4 receptor (green) in embryonic skeletal muscle (skm). K: PGP9.5 stains nerves (red) innervating the muscles in the same section. L: Double labeling experiment showing that P2Y4 receptor protein was expressed in the muscles only but not in the peripheral nerves innervating the muscles. Scale bar in L= 105 μm in A–C, 50 μm in D–F, 200 μm in G–L.

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Immunohistochemistry Demonstrated Expression of P2Y1, P2Y2, and P2Y4 Receptor Protein in Skeletal Muscle

Consistent with the expression of P2Y1, P2Y2, and P2Y4 in the myotome, the primary skeletal muscle fibers were also immunopositive for these P2Y receptors (Fig. 2B,E,F). However, in the case of the P2Y1 and P2Y2 receptors, expression was not maintained. By E18, no P2Y1 or P2Y2 immunoreactivity was detected in skeletal muscle fibers (Fig. 2G). In contrast, at this stage, strong P2Y4 receptor expression was found in all skeletal muscle masses (Fig. 2H).

Examination of postnatal skeletal muscles (3-week- and 2-month-old rats) demonstrated the down-regulation of P2Y4 receptor expression. Immunoreactivity for P2Y4 was detected only in the peripheral muscle fibers. P2Y1 and P2Y2 receptor expression was detected in cells in between muscle fibers. The smooth muscle layer of blood vessels showed strong P2Y1 receptor staining (Fig. 3D), as identified by double labeling with smooth muscle actin (data not shown). The P2Y2 receptor was expressed in scattered cells adjacent to the skeletal muscle fibers (Fig. 3E). Although the identity of the cells was not confirmed, this expression pattern would suggest that these cells were skeletal muscle satellite cells. None of the P2Y receptors showed any immunoreactivity at the neuromuscular junction (identified by staining with Texas Red–labeled α-bungarotoxin) in either the prenatal or postnatal skeletal muscle examined (Fig. 3D–F).

Immunohistochemistry Demonstrated Expression of Only P2Y1 and P2Y4 Receptor Protein in the Brain

Consistent with RT-PCR results, which demonstrated the expression of P2Y4 receptor mRNA alone at E14, only P2Y4 receptor protein was detected in embryonic brain at this age (Fig. 4A–F). Receptor expression was demonstrated in the olfactory system, diencephalon, amygdala, and brainstem. In the olfactory system, P2Y4 receptor staining was detected in the olfactory nerve and lateral olfactory tract (Fig. 4A,B). In the diencephalon, expression was restricted to the anterior hypothalamus, dorsal geniculate nucleus, and lateral hypothalamic area (Fig. 4C,D). In the amygdala, only the cortical amygdaloid nucleus showed P2Y4 receptor expression (Fig. 4D). P2Y4 receptor expression in the brainstem was widespread. The midbrain, pons, and medulla all showed P2Y4 receptor immunoreactivity (Fig. 4E,F). The cerebral cortex, the basal ganglia, the hippocampus, and the cerebellum did not show any P2Y4 receptor staining at E14.

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Figure 4. P2Y receptor immunoreactivity in embryonic and neonatal rat brain. Coronal sections of an embryonic day 14 brain are shown in an anteroposterior direction (A–F). A,B: P2Y4 immunoreactivity was detected in the olfactory tubercle (A, ot) and the lateral olfactory tract (B, lot). C,D: In the diencephalon, P2Y4 receptor protein was expressed in the anterior hypothalamus (C, ah), the dorsolateral geniculate nucleus (D, dgn), and the lateral hypothalamic area (D, lh). The cortical amygdaloid nucleus also stained for P2Y4 (D, ca). E,F: In the brainstem, the P2Y4 receptor expression was detected in the midbrain (E, mb), the pons (E, pn), the isthmus (F, is) and the medulla (F, md). G,H: Coronal sections of a neonatal rat brain showing P2Y4 receptor immunoreactivity in the septum (G) and the neuroepithelium (ne) adjacent to the ventricles (H). I: Coronal section showing P2Y1 receptor immunoreactivity in the cerebral peduncle. Scale bars = 500 μm in F (applies to A–F), 100 μm in G; 200 μm in I (applies to H,I).

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Expression of P2Y4 receptor protein was not maintained in all parts of the brain postnatally. In the neonatal brain (postnatal day 1, P1), P2Y4 receptor expression disappeared from the midbrain, isthmus, and medulla. However, receptor protein was still detected in the olfactory system, the amygdala, the diencephalon, and the pons. In addition, areas such as the septum and the neuroepithelium (adjacent to the ventricles), which did not previously show any staining for P2Y4, became immunopositive for this receptor (Fig. 4G,H). Consistent with the up-regulation of P2Y1 receptor mRNA in late embryonic development, P2Y1 receptor protein was also detected in the postnatal day (P) 1 brain. Receptor expression was restricted to the cerebral peduncle (Fig. 4I). As would be predicted from RT-PCR, no P2Y2 receptor protein was expressed at any stage examined.

Immunohistochemistry Demonstrated Expression of P2Y Receptor Protein in the Spinal Cord and Peripheral Nervous System

P2Y1, P2Y2, and P2Y4 receptor proteins were all expressed in the neural tube and peripheral nervous system from E12. At this stage, P2Y1 receptor protein was detected in the floor plate of the spinal neural tube (Fig. 5A). Subsequently, expression was detected in the ventral commissure of both E14 and E18 spinal cord (Fig. 5B). P2Y1 receptor protein was also detected weakly in the gray matter but not the white matter at E18 (Fig. 5B). The P2Y2 receptor was first detected in the spinal motor nerves in E12 embryos (Fig. 5C). At E14, P2Y2 receptor protein was expressed heavily in the white matter of the intermediate and ventral horns, and the dorsal column of the spinal cord (Fig. 5D). The gray matter showed clear but relatively weaker P2Y2 immunoreactivity. This pattern of expression was maintained in late embryonic development (Fig. 5E). At E18, the presence of P2Y2 receptor expression in the dorsal root ganglia was also clearly apparent (Fig. 5E). P2Y4 immunoreactivity was first weakly detected in the ventral horn of the spinal neural tube at E12. Staining for P2Y4 in the ventral horns increased in strength at E14. Both the spinal motor neurons and the white matter showed immunoreactivity for this receptor (Fig. 3G). However, double labeling with the neural marker PGP9.5 showed that P2Y4 was not expressed in any of the peripheral nerves (Fig. 3J–L). In the E18 spinal cord, P2Y4 expression disappeared from the ventral white matter and was only weakly expressed in the gray matter and dorsal root ganglia (Fig. 5F).

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Figure 5. Immunohistochemical expression of P2Y receptors in the embryonic nervous system. A: Transverse section of embryonic day (E) 12 embryos (at the level just caudal to the hindbrain neural tube) showing P2Y1 receptor immunoreactivity specifically located in the floor plate region (fp, arrow). B: Transverse section showing P2Y1 receptor expression in the ventral commissure (arrowhead) of the E18 spinal cord. Note the very weak P2Y1 immunoreactivity in the gray matter (gm). C: Transverse section of E12 embryos demonstrating P2Y2 receptor expression in spinal nerves (arrows). D: In E14 spinal cord, P2Y2 receptor protein was heavily expressed in the white matter of the ventral horns (vh) and in the peripheral nerves. The gray matter and dorsal root ganglia (drg) also stained weakly for the P2Y2 receptor. E: At E18, widespread P2Y2 receptor expression was detected in the spinal cord, with very strong immunoreactivity in the white matter of the intermediate and ventral horns (arrows). Immunostaining for P2Y2 was also detected in the dorsal root ganglia. F: The P2Y4 receptor was expressed in the gray matter of the spinal cord at E18. Weak staining was also observed in the white matter and dorsal root ganglia (drg) at this stage. Scale bar in F = 50 μm in A, 200 μm in C, 450 μm in B,E,F; 750 μm in D.

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Immunohistochemistry Demonstrated Expression of P2Y Receptor Proteins in the Embryonic Cardiovascular System, Liver, Lung, and Lens

P2Y1, P2Y2, and P2Y4 receptor proteins were strongly expressed in the cardiovascular system, liver, lungs, and lens. The P2Y1, P2Y2, and P2Y4 receptors were first weakly expressed in the heart at E11 and expression became stronger at E12 (Fig. 6A,B). Immunostaining was localized to both the primitive atria and ventricles at this stage. By E14, the P2Y receptors (P2Y1, P2Y2, and P2Y4) were detected only in the atria and the inner trabecular layer of the ventricles (Fig. 6C). The outer myocardial layer and the interventricular septum showed either weak or no expression. At E18, P2Y1, P2Y2, and P2Y4 receptor expression was restricted to the atria and the inner trabecular layer of the ventricles. No P2Y receptor proteins were detected in the myocardium and interventricular septum (Fig. 6D). In the case of all three P2Y receptors detected, P2Y1, P2Y2, and P2Y4, expression appeared to be restricted to the myocardium, with no immunostaining present in the endocardium. This finding was confirmed by double-labeling experiments, performed for P2Y1,2,4 and muscle myosin/von Willebrand factor (a marker for endothelial cells), which showed that expression of the P2Y receptors was localized to the myocardial and not the endocardial cells (data not shown).

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Figure 6. Immunoreactivity for the P2Y receptors in the embryonic cardiovascular system. A: Transverse section showing the expression of the P2Y2 receptor in the developing heart at embryonic day (E) 12. B: Transverse section showing the expression of the P2Y4 receptor in the developing heart at E12. C,D: Transverse sections showing P2Y4 receptor immunoreactivity in the hearts of E14 (C) and E18 (D) embryos demonstrate that this receptor was specifically expressed in the trabeculated layer of the ventricles and atria of the developing hearts but was absent from the interventricular septum (ivs). E,F: Transverse sections showing P2Y1 receptor immunoreactivity in the smooth muscle layers of dorsal aorta at E12 (E) and E18 (F). Scale bar in F= 200 μm in A,B, 450 μm in C, 750μm in D, 50 μm in E, 200 μm in F.

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In the case of P2Y1, receptor protein was also detected in the blood vessels from E12 onward (Fig. 6E,F). Colocalization experiments, performed for P2Y1 and smooth muscle actin/von Willebrand factor (a marker for endothelial cells), confirmed that expression of the P2Y1 receptor was localized to the smooth muscle cells of the dorsal aorta and not the endothelial cells (data not shown).

P2Y receptor expression in the liver began at E14 (Fig. 7A). At this stage, only the P2Y1 receptor was expressed. By E18, P2Y2 receptor expression could also be detected. Whereas the P2Y1 receptor showed distinct staining in scattered cells (Fig. 7B), very weak P2Y2 receptor expression was detected in the general embryonic liver parenchyma (data not shown). The P2Y4 receptor was not detected in the liver in any of the stages examined.

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Figure 7. Immunoreactivity of P2Y receptors on embryonic liver, lung, and lens. A,B: Transverse sections showing P2Y1 receptor expression in embryonic day (E) 14 (A) and E18 (B) liver. The P2Y1 receptor was expressed weakly in E14 (A) and clearly in E18 (B) liver. Scattered cells showed clear P2Y1 receptor immunoreactivity in E18 liver. C–E: Transverse sections showing P2Y1 (C), P2Y2 (D), and P2Y4 (E) receptor-stained E18 lung tissues. Note that the P2Y1 receptor was expressed in the smooth muscle layer of the bronchi (arrows, C) and the P2Y2 receptor expressed in the epithelial cells (arrows, D). The P2Y4 receptor was not expressed in the lung (E). F,G: Transverse sections of the lens at E14 (F) and postnatal day (P) 1 (G) show that the P2Y1 receptor was expressed in the elongating lens fiber cells (lf) but not the epithelial cells (ep) inside the anterior capsule of the developing lens. H: The P2Y4 receptor was also expressed in the elongating lens fiber cells at P1 (lf). Scale bar in H = 200 μm in A–E, 50 μm in F, 100 μm in G, 50 μm in H.

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In the lung, the smooth muscle layer beneath the bronchial epithelium showed P2Y1 receptor immunoreactivity weakly in E14 and clearly in E18 embryos (Fig. 7C). The P2Y2 receptor was not expressed in the lung until E18. Some, but not all, of the epithelial cells of the bronchus within the fetal lung showed P2Y2 immunoreactivity (Fig. 7D). P2Y4 receptor expression was absent from the lung (Fig. 7E).

The P2Y1 receptor was strongly expressed in the primary lens fibers at E14 (Fig. 7F). Although these fibers differentiate from the epithelial cells in the anterior part of the lens (between the lens capsule and the elongating primary lens fibers), the epithelial cells showed no P2Y1 receptor immunostaining. Immunoreactivity for the P2Y1 receptor was maintained in the lens fibers postnatally (Fig. 7G). At this stage, the P2Y4 receptor was also expressed in the lens fibers but was absent from lens epithelial cells (Fig. 7H). No P2Y2 receptor expression was detected in the lens at any stage examined.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Despite the fact that P2Y receptors have been shown to regulate cell proliferation and differentiation (key processes in tissue formation), few studies have investigated the expression and function of P2Y receptors in embryonic development. Prior to this study, the expression of the P2Y2, P2Y4, and P2Y6 receptors had never been investigated in development and none of the P2Y receptors had ever been studied in mammalian development. By using RT-PCR and immunohistochemistry, it was possible to demonstrate the abundant and dynamic expression of the P2Y1, P2Y2, P2Y4, and P2Y6 receptors in many tissues and organs, including skeletal muscle, heart, brain, spinal cord, liver, lung, and lens, in rat embryonic and postnatal development. The pattern and timing of receptor expression strongly suggested a role for these receptors in development.

All the P2Y receptors studied were expressed as early as E11, when most embryonic organs far from being functional were still in the process of being formed. Furthermore, P2Y receptor proteins were strongly expressed in transient, developmental structures, including the somites (P2Y1, P2Y2, and P2Y4) and the floor plate of the neural tube (P2Y1). While both these structures play an essential role in embryonic development, the floor plate in patterning the ventral neural tube and the somites in the formation of mesodermal tissues, neither are retained. P2Y receptors were also dynamically expressed, with receptor mRNA and protein being both up- and down-regulated. The down-regulation of the P2Y1, 2, and 4 receptor proteins in skeletal muscle and heart, and the disappearance of the P2Y4 receptor from the brainstem and ventral white matter of the spinal cord postnatally demonstrated that many P2Y receptors were developmentally regulated and were involved in functions specific to embryonic life. Thus, these findings strongly suggest that, whereas there are many well-recognized functions for P2Y receptors in mature, adult tissues, P2Y receptors may also play a role in tissue formation and development.

Skeletal Muscle

The transient expression of the P2Y1, P2Y2, and P2Y4 receptors in skeletal muscle strongly suggests a role for these receptors in the formation and differentiation of skeletal muscle. The onset of P2Y1 and P2Y4 receptor expression (E11) was similar to that of MyoD and Myf5, the two myogenic transcription factors responsible for defining the muscle precursor cells (myoblasts; Buckingham, 2001). The expression of both receptors was ultimately confined to the myotome, as opposed the dermatome that will form the dermis of the skin. It is still difficult to determine whether the expression of P2Y1 and P2Y4 receptors was required for myogenic specification or the expression follows the specification, unless the P2Y receptor expression has been positioned in the signaling pathway of myogenesis. The P2Y2 receptor was expressed a day later (E12) in the myotome. P2Y receptor expression in muscle was not maintained. Staining for the P2Y1 and P2Y2 receptors disappeared by E18 and P2Y4 receptor expression was down-regulated postnatally. These findings suggested that, although the P2Y receptors were unlikely to be involved in myogenic specification, these receptors could regulate subsequent processes in muscle formation, including the proliferation of myoblasts, migration or fusion to form primary and secondary myotubes (Buckingham, 2001). In fact, a recent study by Ryten et al. (2002) demonstrates that myoblast proliferation in vitro can be potentiated by application of UTP, an agonist at both P2Y2 and P2Y4 receptors. P2Y receptors have also been implicated in the migration of several cell types, including vascular smooth muscle cells and human dendritic cells (Idzko et al., 2002; Pillois et al., 2002).

The pattern of P2Y receptor expression in skeletal muscle also suggested a role specifically for the P2Y4 receptor in myotube maturation and differentiation. P2Y4 receptor expression was maintained during postnatal development of muscle fibers, which included muscle fiber hypertrophy and maturation of the neuromuscular junction. Because these processes are largely dependent on changes in intracellular Ca2+ concentration and P2Y receptor activation will result in the mobilization of intracellular calcium, this receptor has the potential to be involved in any of these processes (Olson and Williams, 2000a, b; Sanes and Lichtman, 2001). However, because neither the P2Y4 receptor nor for that matter the P2Y1 and P2Y2 receptors were found to be expressed specifically at the neuromuscular junction, it is more likely that P2Y4 receptor activation plays a role in muscle fiber hypertrophy or fiber-type determination. Although these findings do not entirely agree with a recent study by Choi et al. (2001), they are consistent with the work of Meyer et al. (1999a). Choi et al. (2001) have demonstrated the up-regulation of the P2Y1 receptor during the course of embryonic muscle development in chick and expression of P2Y1 receptor protein at the adult neuromuscular junctions in chick and rat, whilst our findings and those of Meyer et al. (1999) suggest that this receptor is down-regulated in development and is not expressed at the neuromuscular junction.

Heart

The dynamic expression of the P2Y receptors in embryonic heart also suggested a role for these receptors in the development of cardiac muscle. Immunoreactivity for the P2Y1, P2Y2, and P2Y4 receptors was detected in both the primitive atria and ventricles of the embryonic heart from E11, just before trabeculations first become evident along the inner myocardial layers (E11.5) (Sedmera et al., 2000). At E14, when trabeculations develop and become compressed within the ventricular wall, P2Y receptor expression was restricted to the trabeculated layers of the atria and ventricles, no immunoreactivity being found in the compact layer of the ventricular myocardium. Consistent with previous reports, P2Y receptors were down-regulated with further development (Webb et al., 1996).

Because formation of the trabeculated layer of the heart and its fusion with the compact layer is vital to heart development, the expression of the P2Y receptors in the trabeculated layer, specifically, is likely to be of functional significance. In fact, trabeculations are so vital to cardiac morphogenesis that the absence of these structures in neuregulin-null mice results in embryonic death at E11 (the tubular heart stage) (Gassmann et al., 1995; Lee et al., 1995). Thus, the pattern and timing of P2Y receptor expression in the heart might suggest a role for these receptors in the differentiation of the trabeculated layer and the formation of the ventricular myocardium.

Nervous System

Expression of the P2Y receptor proteins, and particularly the P2Y4 receptor, was prominent in the embryonic nervous system. Among all the P2Y receptors examined, the P2Y4 receptor was the first to be expressed in the embryonic brain at E14 on the basis of both RT-PCR and immunohistochemistry. Instead of a general or weak expression throughout the whole brain, the receptor expression was intense and localized. At E14, P2Y4 immunoreactivity was detected in the telencephalon (olfactory system, pallidum, and amygdala), diencephalon (lateral hypothalamic area and dorsal geniculate nucleus), and brainstem (midbrain, pons, and medulla). After birth, additional regions of the brain, such as the septum and the neuroepithelium, adjacent to the ventricles showed P2Y4 receptor expression, and P2Y1 receptor staining was detected in the cerebral peduncle. However, of greatest developmental significance was the disappearance of P2Y4 immunoreactivity from the brainstem after birth. Thus, the P2Y4 receptor appeared to be the dominant P2Y receptor present early in the brain. The early expression of P2Y4 receptor in specific brain regions and its subsequent down-regulation in some areas, suggested that this receptor has a role to play in prenatal brain development, particularly within the brainstem.

The spinal cord showed immunoreactivity for the P2Y1, P2Y2, and P2Y4 receptors. Expression of all these receptors was related either directly or indirectly to motor neuron development. The P2Y2 and P2Y4 receptors were expressed in the ventral horns of the embryonic spinal cord, in the case of the P2Y4 receptor only transiently (E14–E18). Both P2Y2 and P2Y4 receptor expression was mainly localized to the white matter of the ventral horns, although the spinal motor neurons also showed weak expression. P2Y1 expression was localized to the floor plate of the spinal neural tube and subsequently the ventral commissure of the spinal cord, the former an important structure in the differentiation of the ventral neural tube. The floor plate expresses a powerful signaling molecule, Sonic hedgehog (Shh), which specifies the identities of motor neurons and interneurons in a concentration-dependent manner (Dodd et al., 1998). Thus, the expression of the P2Y1 receptor in floor plate, and the P2Y2 and P2Y4 receptors in the ventral horns, suggests that purinergic signaling could regulate motor neuron development at multiple sites.

It seems likely that not all P2Y receptor expression had a role in embryonic development, but rather some receptor expression was related to adult function. The lack of dynamic or transient P2Y receptor expression in the embryonic and fetal liver suggested that P2Y receptors were not involved in liver formation. As in adult hepatocytes (Dixon et al., 2000), experiments conducted on embryonic liver demonstrated the expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptor mRNA transcripts and immunoreactivity for the P2Y1 and, to a lesser extent, the P2Y2 receptor. Expression of the P2Y1 and P2Y4 receptors in the lens fibers is also unlikely to be involved in lens development. The P2Y1 receptor is strongly expressed in both the adult (Merriman-Smith et al., 1998) and embryonic lens, but the P2Y4 receptor was only expressed postnatally. Nonetheless, this study does demonstrate for the first time the expression of the P2Y4 receptor in lens fibers, and expression of this receptor could account for the reports of responses of adult lens cells to ATP (Churchill and Louis, 1997; Collison and Duncan, 2001). Similarly, expression of the P2Y1 and P2Y2 receptors in the fetal lung is unlikely to be related to lung development. In the fetal (E18) lung, the P2Y2 receptor was expressed in the epithelial cells of the bronchi. Adult lung epithelial cells also express this receptor and can respond to the P2Y2 receptor agonist UTP, with an increase in transepithelial chloride secretion and mucus secretion (Rice and Singleton, 1987; Rice et al., 1995; Burnstock, 2002b). The P2Y1 receptor, which is known to be expressed on many types of smooth muscle cells, was also expressed in the smooth muscle layer of the bronchi. Thus, although P2Y receptors are undoubtedly essential for proper lung function, it is very unlikely that embryonic P2Y receptor expression is related to organogenesis of the lung.

The question of the source(s) of ATP involved in activating these P2 receptors should next be raised. Many cell types are known to release ATP in response to mechanical disturbance (Burnstock, 1999; Bodin and Burnstock, 2001a, b), and since there is much cellular movement during embryogenesis, several cell types might be releasing ATP. In addition, the presence of apoptotic cells in several developing tissues might represent another source of ATP.

In summary, we have demonstrated for the first time the early and dynamic expression of the P2Y1, P2Y2, P2Y4, and P2Y6 receptors during rat embryonic development. Although not all P2Y receptor expression is likely to be related to embryonic development, these findings suggest the involvement of purinergic signaling in skeletal muscle, heart, and central nervous system development. Thus, purinergic signaling is likely to be an important signaling system in embryonic development and in particular organogenesis.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

RT-PCR

Total RNA was extracted from fresh embryonic tissues at E11, E12, E14, and E18 by using SV Total RNA Isolation system (Promega, Madison, WI). Reverse transcription and cDNA amplification for all the P2Y receptors was carried out with a thermal cycler (Hybaid, UK) in a two-step protocol using Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech, Buckinghamshire, UK). Every sample was further treated with Amplification Grade DNase I (Sigma, UK) to remove any residual DNA present that could generate false-positive results. Briefly, 1 μg of total RNA was reverse transcribed using the pd(T)12–18 as the first-strand primer at 42°C for 30 min and the enzyme was denatured at 95°C for 5 min. The sequence specific primers (Life Technologies, Bethesda, MD) for P2Y receptors (Bailey et al., 2000, 2001) were then added to the reaction mixtures, and the PCR cycling parameters were 95°C for 30 sec, 58°C for 1 min (58°C for P2Y1, 60°C for P2Y2 and P2Y6, 64°C for P2Y4), 72°C for 1.5 min for 35 cycles (40 cycles for P2Y4), followed by a further stage of 10-min extension at 72°C. The resulting PCR products were resolved in a 2% agarose gel containing ethidium bromide and observed under ultraviolet illumination.

RT-PCR results for all tissues were confirmed by repetition with at least three separate RNA samples, prepared from embryonic tissues obtained from separate rat litters. Furthermore, a minimum of three RT-PCR experiments were performed for each P2Y receptor on each individual embryonic tissue. Control experiments were conducted by denaturing the reverse transcriptase (95°C for 15 min) before the RT-PCR reaction. These experiments demonstrated that, on denaturation of the reverse transcriptase, no P2 receptor mRNA could be detected. Thus, we were able to verify that the results obtained were due to the presence of P2 receptor mRNA in a sample and not as a result of genomic DNA contamination.

Tissue Preparation

The embryonic expression of P2Y receptor proteins was studied in Sprague-Dawley rat embryos of E10.5–18.5 by using fluorescence immunohistochemical techniques. The day of identification of the presence of a vaginal plug was designated as E0. Pregnant Sprague-Dawley rats were killed by asphyxiation with a rising concentration of CO2 (between 0% and 100%), and death was confirmed by cervical dislocation according to Home Office (UK) regulations covering Schedule 1 procedures. Embryos collected were either freshly cryoembedded or fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C. Fixed embryos were cryoprotected in 20% sucrose until they sank. The embryos were embedded in Tissue-Tek and kept at −80°C until cryosectioning. Tibialis anterior muscles from both 3-week- and 2-month-old animals were collected and freshly embedded in Tissue-Tek. Frozen sections (12 μm) were cut in a cryostat and mounted.

Immunohistochemistry

Air-dried sections were post-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 2 min at room temperature. Nonspecific binding sites were blocked by incubating sections in 10% normal horse serum (NHS) in phosphate buffered saline (PBS) for 1 hr. The sections were then incubated with primary antibodies, diluted in 10% NHS in PBS, overnight at room temperature. The primary antibodies used were rabbit anti-P2Y1,2,4 (diluted 1:200 for P2Y1, and P2Y2; 1:100 for P2Y4; Alomone, Israel), rabbit anti-PGP9.5 (UltraClone, Ltd., UK), mouse anti–α-smooth muscle actin (1:400, Sigma), and mouse anti-MF20 (1:100, Developmental Studies Hybridoma Bank). For labeling of acetylcholine receptors, sections were incubated with Texas Red–conjugated α-bungarotoxin (1:800, Sigma). The sections were washed in 0.1 M PBS and incubated for 1 hr at room temperature in either fluorescein isothiocyanate–conjugated or Cy3-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). In immunohistochemical experiments using 3,3′-diaminobenzidine to perform a color reaction, the procedures were performed according to the protocol previously described in Ryten et al. (2001).

For control experiments, the sections were incubated with the primary antibodies preabsorbed with the control peptide antigens or with NHS only. To distinguish between specific and nonspecific immunoreactivity, preabsorption experiments were performed for P2Y receptors on all tissues and at every developmental stage examined. Only immunostaining, which could be displaced by preabsorption with the relevant peptide, was reported as positive immunoreactivity. Immunohistochemical results were representative of findings from at least five embryos, taken from at least three litters. All images of immunohistochemical staining were taken with the Leica DC 200 digital camera (Leica, Switzerland) attached to a Zeiss Axioplan microscope (Zeiss, Germany).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. Chrystalla Orphanides for editorial assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES