Association of cell and substrate adhesion molecules with connexin43 during intramembranous bone formation.

Prior studies in our laboratory have demonstrated an association of specific gap junction proteins with intramembranous bone formation in the avian mandible. The purpose of the present study was to extend these observations by determining if there was a relationship between the expression of one of the gap junction proteins examined previously (connexin43) and the expression of specific cell adhesion (CAM) and/or substrate adhesion (SAM) molecules [i.e. NCAM, A-CAM (N-cadherin) and tenascin (tenascin-C)] that have previously been shown to be associated with bone formation. Immunohistochemical localization of connexin43, tenascin, NCAM and N-cadherin was performed on serial sections of mandibles of chick embryos from 6 to 12 days of incubation. Analysis of adjacent serial sections revealed that the NCAM and tenascin immunostaining that appeared initially on the lateral aspect of Meckel's cartilage preceded the overt expression of trabecular bone. At subsequent stages, NCAM and tenascin staining gradually overlapped the region of connexin43 expression. In contrast, the expression of N-cadherin was found to colocalize with that of connexin43 from the first appearance of connexin43 expression. Most significantly, although the domains of NCAM and tenascin expression were initially separate from that of connexin43, bone formation originated only in the region where these domains intersected. These findings suggest that, of the CAMs and SAMs examined, N-cadherin appears to be associated with the establishment of cell contacts responsible for the presence and/or maintenance of connexin43-mediated gap junctional communication, while tenascin and NCAM appear to be associated, in a more specific manner, with processes that accompany the overt expression of the osteogenic phenotype.

Expression patterns of connexin43 protein during facial development in the chick embryo: associates with outgrowth, attachment, and closure of the midfacial primordia.

BACKGROUND: In a prior report, evidence was presented for the presence of gap junction proteins [connexin32 and connexin43 (Cx43)] in embryonic facial primordia. The purpose of the present study was, first, to examine in detail the patterns of distribution of Cx43 protein in embryonic chick facial primordia and, second, to consider the possible roles played by this protein during midfacial development. METHODS: Chick embryo heads were serially sectioned and processed for immunofluorescent localization of Cx43. The developmental stages examined encompassed the period of formation, enlargement, and union of the facial primordia. Western blot analysis of the facial primordia was also performed. RESULTS: Analysis of serial sections revealed the presence of signal in both epithelium and mesenchyme at sites of attachment in each of the midfacial primordia (i.e., the medial nasal, lateral nasal, and maxillary processes). Furthermore, although signal was concentrated in mesenchyme in the distal tips of the primordia at sites of attachment, immunoreactivity was absent, sparse, or less intense outside the areas of attachment. In some cases (i.e., the maxillary process), immunoreactive signal in mesenchyme did not appear in the distal tip until the primordia approximated each other or contact of the primordia was initiated. Most significantly, signal was also found between the facial primordia in nonprimordial epithelium and mesenchyme at sites where the primordia were joined. CONCLUSIONS: These data suggest that the expression of Cx43 protein is spatially and temporally regulated in the facial primordia and that the patterns of expression that were observed are significant to the cascade of events that ultimately lead to the attachment and union of the primordia that form the midface.

Immunolocalization of connexin 43 in the tooth germ of the neonatal rat.

Rabbit polyclonal antibodies to amino acids 346-360 of connexin 43, the 'heart' gap junction protein, were employed to immunolocalize connexin 43 gap junctions in the neonatal rat molar tooth germ. Connexin 43 appears early in the differentiation of both ectodermally derived and ectomesenchymally derived cells of the developing tooth. Connexin 43 immunoreactivity is present in the epithelial components of the enamel organ, including the area of the proximal and distal junctional complexes of the ameloblast layer, and the stratum intermedium, stellate reticulum and outer enamel epithelium. Secretory odontoblasts and developing alveolar bone also display a pattern of connexin 43 immunostaining. Both the epithelial and ectomesenchymally-derived components of the developing tooth acquire connexin 43 channels in a manner that correlates with cell differentiation. In addition, three regions can be defined by connexin 43 immunostaining: the epithelia of the enamel organ that are derived from the oral epithelium, the odontoblast layer derived from the ectomesenchyme, and the alveolar bone. The results suggest that connexin 43 may provide the mechanism for functional compartmentalization of the tissues associated with tooth formation. Compartmentalization suggested by connexin 43 expression could play important roles in the development and functions of these tissues.

Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible.

The spatial and temporal expression of three closely related members of the connexin family of gap junction proteins (connexin42, Cx42; connexin43, Cx43; and connexin45, Cx45) was evaluated during bone formation in the mandibular process of the chick embryo. Mandibles of chick embryos from Hamburger and Hamilton stage 25 (approximately 5 days) through 19 days of development were dissected, serially sectioned and processed for immunocytochemical localization, employing site-specific anti-connexin antibodies. Our data revealed that (1) Cx43 was present throughout mandibular bone formation; (2) although it appeared to be associated with all bone cell types, Cx43 was concentrated in mesenchymal cells during the earliest stages in the osteogenic lineage; (3) most importantly, the localization of Cx43 at sites of bone formation appeared to precede the overt expression of the osteogenic phenotype; (4) by contrast, Cx45 was more restricted, spatially and temporally, in its distribution; (5) Cx42 expression was not detected in osteogenic tissue during mandibular bone formation. From all of the data obtained, Cx45 appeared to be associated with stages of bone formation characterized by the elaboration of matrix and the progressive expression of the differentiated osteogenic phenotype. Cx43 appeared to be associated with condensation of mesenchyme and the earliest stages of osteogenesis. Because of these associations, we propose that connexin expression may be necessary for the initiation of bone formation and the full expression of the osteogenic phenotype.

Connexin expression in the developing avian cardiovascular system.

Recent observations have suggested that the patterns of expression of the gap junction protein connexin43 in the developing cardiovascular system of the avian embryo diverge significantly from the patterns previously seen in mammalian species. Therefore, a detailed analysis of connexin43 expression in the chicken embryo was performed by use of immunofluorescent localization with two different connexin43-specific antipeptide antibodies as well as Western and Northern blot analysis. Connexin43 protein was not detected in the avian myocardium, the venous system, or the smaller vessels of the arterial system. Rather, it was limited exclusively to the vessels of the arterial outflow tract in a concentric pattern that became evident by embryonic day 8. Double staining with anti-alpha-smooth muscle actin and connexin43 demonstrated colocalization in the media of outflow tract vessel walls. The developmental expression of connexin43 was found to mirror the spatial patterning of secondary actin; connexin43, however, preceded the expression of secondary actin by a period of 1-2 days. In contrast, antibodies to a related gap junction protein (connexin42) revealed an absence of immunostaining in the avian outflow tract. Double staining with anti-connexin42 and anti-A-cell adhesion molecule (specific for avian intercalated discs) demonstrated colocalization between cardiac myocytes, indicating that connexin42 is a constituent of avian myocardial gap junctions. In light of these findings, developmental expression of differing myocardial connexins may reconcile previous studies showing different physiological properties of avian and mammalian cardiac gap junctions.