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.
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.
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.
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.