Antisense oligonucleotide blockade of connexin expression during embryonic bone formation: evidence of functional compensation within a multigene family.

Prior studies in our laboratory demonstrated the presence of gap junction proteins (connexins) throughout intramembranous bone formation [Minkoff et al. (1994) Anat Embryol 190:231-241]. In addition, two members of the connexin family of gap junction proteins, connexin 43 (Cx43; Gj alpha 1) and connexin 45 (Cx45; Gj alpha 6), were found by Civitelli et al. [1993; J Clin Invest 91:1888-1896] to be associated, specifically, with osteogenesis. Recently, however, a null mutation in the gene encoding Gj alpha 1 in mice has been produced by Reaume et al. [1995; Science 267:1831-1834]. Gj alpha 1 null homozygotes survived to term but died at birth of heart abnormalities. Examination of the null homozygous embryos, surprisingly, did not reveal overt histological or anatomical abnormalities in any organ system other than the heart. In view of this, the present investigation was initiated in order to evaluate bone formation under conditions in which the expression of Gj alpha 1 and Gj alpha 6, the connexins specifically associated with osteogenesis, had been perturbed, individually as well as in combination. An in vitro system employing organ cultures of dissociated embryonic chick mandibular mesenchyme was employed. Mesenchyme was cultured in the presence and absence of sense and antisense oligodeoxynucleotides (ODN), ranging in length from 15 to 24 mer and containing sequences that included the initiation codon of Gj alpha 1 and of Gj alpha 6. In cultures of mesenchyme, grown for 6 to 13 days in the presence of the combined antisense ODNs to Gj alpha 1 and Gj alpha 6, bone formation was markedly reduced or absent. By contrast, in cultures grown in medium containing the combination of corresponding sense ODNs to both Gj alpha 1 and Gj alpha 6, bone formation was evident. In addition, when cultures were grown in the presence of antisense or sense ODNs to either Gj alpha 1 or Gj alpha 6, individually, bone formation was seen. Immunohistochemical analysis of connexin expression revealed intense immunoreactive signal to Gj alpha 1 and Gj alpha 6 in bone of the control explants, in which no ODNs were present; in those cultures in which either Gj alpha 1 and Gj alpha 6 antisense ODNs were present, however, the expression of the respective connexin protein was either significantly reduced or absent. Further, in those explants in which Gj alpha 1 expression was blocked, immunoreactive signal to Gj alpha 6 appeared to have been amplified in regions of developing bone. These results suggest that, in avian osteogenic tissue, when Gj alpha 1 protein expression has been impeded another related connexin protein (Gj alpha 6) may subserve the functions of the missing connexin. The findings of this study, therefore, support the hypothesis that, within the connexin gene family, functional compensation can occur.

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.

Sonic hedgehog participates in craniofacial morphogenesis and is down-regulated by teratogenic doses of retinoic acid.

The face is one of the most intricately patterned structures in human and yet little is known of the mechanisms by which the tissues are instructed to grow, fuse, and differentiate. We undertook a study to determine if the craniofacial primordia used the same molecular cues that mediate growth and patterning in other embryonic tissues such as the neural tube and the limb. Here we provide evidence for the presence of organizer-like tissues in the craniofacial primordia. These candidate organizers express the polarizing signal sonic hedghog (shh) and its putative receptor, patched, as well as fibroblast growth factor 8 and bone morphogeneic protein 2. Shh-expressing epithelial grafts functioned as organizing tissues in a limb bud assay system, where they evoked duplications of the digit pattern. High doses of retinoic acid, which are known to truncate the growth of the frontonasal and maxillary processes and thus produce bilateral clefting of the lip and palate, inhibited the expression of shh and patched but not fgf8, in the craniofacial primordia, and abolished polarizing activity of these tissues. From these studies we conclude that the embryonic face contains signaling centers in the epithelium that participate in craniofacial growth and patterning. In addition, we discuss a novel mechanism whereby retinoids can exert a teratogenic effect on craniofacial morphogenesis independent of its effects on Hox gene expression or neural crest cell migration.

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.

Modulation of gap junction-mediated intercellular communication in embryonic chick mesenchyme during tissue remodeling in vitro.

Gap junction-mediated intercellular communication was analyzed in a model system in which tissue necrosis and remodeling could be modulated. This in vitro system, previously used for analysis of epithelial-mesenchymal tissue interaction, was modified to permit analysis of the presence and extent of intercellular communication by monitoring intercellular transfer of the microinjected fluorescent dye, Lucifer Yellow. Light and transmission electronmicroscopy were employed to correlate the presence and degree of gap junctional communication (coupling) with tissue morphology. Digital image analysis was used to determine cell density and mitotic indices within the outgrowths of explants. Our results indicated that cell communication in outgrowths adjacent to necrotic foci within an explant was minimal or absent. Cell-coupling in outgrowths adjacent to a compartment of viable mesenchyme was significantly higher - equivalent to unseparated control cultures. A time-course study demonstrated correlation of increased levels of cell-coupling in outgrowths with the level of tissue remodeling within an explant. Our conclusions from these studies are that embryonic mesenchymal cell populations may be selectively uncoupled as a result of alterations in the microenvironment produced by a proximate impaired cell population. It is proposed that endogenous factors in the microenvironment ("wound signals"), emanating from impaired cell populations, regulate gap junction-mediated intercellular communication in adjacent viable tissue. Normal, unimpaired populations of cells surrounding an area of injury are thereby isolated from the effects of a potentially toxic environment. This could serve as a protective function in development and may represent, in a more general sense, part of the repertoire of events associated with tissue repair and remodeling.

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.

Cell proliferation during formation of the embryonic facial primordia.

Cell proliferation of mesenchyme in the developing primary palate of the chick embryo was analyzed by tritiated thymidine autoradiography. Pulse labeling, repeated labeling, and label dilution techniques were employed to determine generation times, transit times, growth fractions, and other parameters of the cell cycle. In vivo and in vitro studies were performed to evaluate the role of tissue interactions during outgrowth of the facial primordia. These studies indicated that initially, during early stages of primary palate formation, virtually all mesenchymal cells are in the division cycle with relatively short generation times. As development proceeds, mesenchymal cell populations in the facial primordia, such as the maxillary process, retain cycle characteristics comparable to those of the progenitor cell populations. In regions adjacent to the facial primordia, such as the roof of the stomodeum, cell cycle times become more heterogeneous and result in removal of cells from rapidly cycling cell populations into subpopulations that are cycling more slowly and that, in some instances, become quiescent. Regional analysis of cell proliferation in the maxillary process indicated that growth rates of mesenchyme differ based on proximity to the overlying epithelium. Correlative in vitro studies of epithelial-mesenchymal separation and recombination experiments in organ culture revealed that the viability of mesenchyme was dependent on the presence of epithelium and that this effect was strongly stage-dependent. These and other results lead us to the conclusion that epithelial-mesenchymal interaction is significant to the maintenance of growth rates in the facial primordia and that the effects observed are mediated, at least in part, by developmental signals at the epithelial-mesenchymal interface.

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain.

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 x 10(3) Mr, connexin 32) and heart (43 x 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 x 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the 'heart' 43 x 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

Influence of epithelial-mesenchymal interaction on the viability of facial mesenchyme. II: Synthesis of basement-membrane components during tissue recombination.

The presence of basement-membrane components during tissue separation procedures was determined employing monoclonal antibodies to laminin and type IV collagen. In addition, the reconstitution of basement-membrane components and the formation of the basement-membrane were examined in isolated epithelium and mesenchyme and in tissue recombination. Epithelium and mesenchyme of maxillary processes of chick embryos were separated by a variety of protocols, including those employed in a prior study (Saber et al: Anat. Rec. 225:56-66, 1989). Results indicated that the protocol previously employed did not remove basement-membrane components after enzymatic tissue separation. A revised protocol in which the basement-membrane components (i.e., laminin and type IV collagen) were removed from isolated tissues prior to recombination revealed that a developmental compartment and a gradient of cell viability, comparable in size and dimensions to that observed in the study of Saber et al. (ibid.) was present in the mesenchyme of recombined explants. Type IV collagen and laminin, therefore, do not appear to be required initially during tissue recombination in order for subsequent growth-sustaining effects to be expressed. Additional studies revealed, however, that synthesis of basement-membrane components occurred not only in isolated tissues but was altered markedly by tissue recombination. Culture of isolated tissues demonstrated induction of laminin synthesis in separated epithelium by 24 hours and induction of collagen synthesis in isolated mesenchyme by 24 hours. Recombination of epithelium and mesenchyme, however, resulted in rapid induction of laminin synthesis within 1 hour. Recombination of epithelium and mesenchyme after 24 hours resulted in the presence of laminin not only in epithelium but in mesenchyme as well. Both tissues were required for basement-membrane formation which appeared to be fully reconstituted by 24 hours in culture. These observations indicate that recombination in culture alters the pattern of synthetic activity of these basement-membrane components. These can be characterized as "early" (temporal) and "late" spatial) responses by the recombined tissues.

Distribution of type IV collagen, laminin, and fibronectin during maxillary process formation in the chick embryo.

The presence and distribution of laminin, type IV collagen, and fibronectin were analyzed in the facial primordia and developing primary palates of chick embryos from stages of development corresponding to maxillary process formation and primary palate closure. Frozen sections through the maxillary process and roof of the stomodeum were prepared for indirect immunofluorescence employing a biotin-avidin system using monoclonal antibodies against laminin, type IV collagen, and fibronectin. Light microscopic examination of sections stained with antibodies against type IV collagen revealed a much stronger fluorescent signal in the roof of the stomodeum than in the maxillary process at all stages examined. Regional differences in signal intensity and staining patterns were noted within the maxillary process; for example, the lateral surface of the maxillary process displayed a much less intense signal at most stages examined than the inferior and medial surfaces. The signal from sections of the maxillary process stained with laminin was much stronger than the signal from the same tissues stained with collagen. Regional differences in signal intensity within the maxillary process were minimal in sections stained with antibodies to laminin, in contrast to the differences seen in sections stained with antibodies to type IV collagen. Differences in signal intensity between the maxillary process and the roof of the stomodeum with laminin were slight. Sections stained with antibody to fibronectin displayed intense staining throughout the mesenchyme in both the maxillary process and the roof of the stomodeum. From comparison of the data of type IV collagen and laminin, the following hypothesis is proposed. In structures which undergo rapid change in form, such as the facial primordia, collagen distribution and/or organization is altered to a much greater extent than laminin, which is more uniformly distributed and which may be required for structural support of other developmentally regulated macromolecules. Where tissue morphology must be maintained, such as the roof of the stomodeum, the concentration and organization of type IV collagen is maintained in a manner that confers stability to these regions.

Influence of epithelial-mesenchymal interaction on the viability of facial mesenchyme in vitro.

Separation and recombination experiments, employing a variety of tissue configurations in organ culture, were performed to determine the extent to which the epithelium of the maxillary process influences the viability of the underlying mesenchyme during organogenesis. The results of these studies indicated that the viability of mesenchyme of the maxillary process of early stage embryos was severely impaired when separated from the overlying epithelium. The influence of epithelium on the viability of mesenchyme was stage dependent; that is, the requirement for the presence of epithelium for the maintenance of the viability of mesenchyme became progressively less pronounced at older developmental stages. The response of mesenchyme to the presence of recombined epithelium resulted in the appearance of a delimited zone of influence extending, within specific boundaries, from the epithelial-mesenchymal interface. Preliminary data from homotypic (maxillary epithelium-maxillary mesenchyme), heterotypic (limb apical ectodermal ridge-maxillary mesenchyme) and heterochronic (stage 28 epithelium-stage 22 mesenchyme) recombination experiments indicated that viability of mesenchyme could be achieved only through direct epithelial-mesenchymal contact which allowed restoration of normal morphological relationships at the interface of the two tissues.

Relative growth rates of maxillary mesenchyme in the chick embryo.

Chick embryos were injected with [3H]-thymidine at days 3-7 of incubation and were fixed and embedded in plastic. The embryos were divided into three stage groupings of development [Hamburger and Hamilton: J Morphol 88:49-92, 1951], and labeling indices were determined for each of the following delineated regions within the maxillary process at each stage: region 1, subepithelial mesenchyme located at the medial side of the maxillary process adjacent to the roof of the stomodeum; region 2, subepithelial mesenchyme at the ventral tip of the maxillary process (as seen on cross section); region 3, subepithelial mesenchyme at the lateral portion of the maxillary process below the eye; and region 4, interior mesenchyme defined as the central portion of the maxillary process and separated from the epithelium by the three other regions. Results indicated that differences exist among the regions examined and that these differences were stage specific. At stages 19-21 and stages 24-25 1/2, growth rates were higher in subepithelial regions than interiorly. At stages 28-29, however, a statistically significant difference among the regions was not found. These results suggested that there is an association between growth rates in the maxillary process mesenchyme and its proximity to the overlying epithelium and that these effects are related to the stage of development.

Pathogenesis of median facial clefts in mice treated with methotrexate.

Methotrexate (MTX), administered as a single 20 mg/kg intraperitoneal dose to C57Bl/6J mice on their 9th day of pregnancy results in high incidences of median facial clefts in the surviving gestational-day-18 fetuses. We have shown the presence of dilated and congested blood vessels in the frontonasal prominences (FNP) of embryos from treated mothers as early as 3 hours following drug administration. Within 24 hours, large vascular blebs are located in the FNP and the neural tubes appear somewhat distended. By 32 hours after treatment, distention of the neural tube is marked while blebs have become less evident. Subsequent to these changes, FNP mesenchymal deficiency as well as neural tube distention lead to the formation of median facial clefts. It is hypothesized that, as with a number of other teratogenic agents (especially hypoxia), initial fluid imbalance is the primary teratogenic insult.

Morphometric and autoradiographic analysis of frontonasal development in the chick embryo.

Dimensional changes in the nasal processes were measured in chick embryos from Hamburger and Hamilton (1951) stages 20 through 27.5. Transverse measurements in the frontonasal region of freshly fixed embryos were compared to frontal sections of the nasal region of comparably staged embryos. These observations were correlated with autoradiographic studies of cell movement employing an implant labeling technique. Morphometric analysis indicated that between stages 20 and 25 the separation of the nasal pit orifices increased coincidentally with rapid forebrain enlargement. Since the separation of the nasal pit fundi increased more rapidly, the orientation of the nasal pits changed. Autoradiographic studies indicated that lateral movement of medial nasal process mesenchyme into the base of the nasal groove and medial area at the base of the lateral nasal process had occurred. After stage 25, the separation of the nasal orifices declined dramatically, coincidental with rapid orbital enlargement. In contrast, the separation of the nasal pit fundi was maintained. It is proposed that nasal development of the chick embryo may be governed initially by forebrain enlargement and associated lateral movements of mesenchyme in the medial nasal processes, resulting in reorientation of the invaginating nasal placodes; subsequently, orbital enlargement and an associated medial redirection of growth of the lateral nasal processes assumes greater significance to the continued development of the frontonasal region.

An implant labeling technique employing sable hair probes as carriers for 3H-thymidine: applications to the study of facial morphogenesis.

An implant labeling technique is described that utilizes sable hair probes as carriers for a tritiated thymidine marker. The protocol that was developed produced localized labeling of specific embryonic cell populations. This procedure was applied to the analysis of facial process development in chick embryos. Evaluation of the technique demonstrated that the probe preparation procedure was consistently successful in producing labeled probes. Using labeled probes, the procedure was reliable in producing acceptable levels of labeling in chick embryonic tissues and labeling of localized cell populations was possible using the implant labeling technique. Surrounding the center of labeling, a gradient in intensity of labeling was often observed. This pattern presumably reflects declining availability of labeled thymidine as distance from the probe increased. The technique allowed excellent survival rates to be achieved provided that aseptic procedures were followed. Additionally, careful analysis of older embryos failed to reveal any malformations induced by the implant labeling procedure. The localized labeling patterns that were demonstrated during this investigation suggest that the implant labeling technique would provide a useful tool for following cell migration during facial process formation.

Cell cycle analysis of facial mesenchyme in the chick embryo. II. Label dilution studies and developmental fate of slow cycling cells.

The spatial distribution and developmental fate of quiescent and/or slow cycling cell populations of the primary palate were studied employing label-dilution techniques. 3 1/2-day-old chick embryos were labelled sequentially for 12 h with [3H]thymidine and then chased with cold thymidine. The embryos were reincubated to continue development and were subsequently sacrificed at intervals from the end of labelling at 4 days to 14 days of incubation (10 days after labelling) and processed for autoradiography. Retention of label was used as the assay for identification of quiescent and/or slow-cycling cells. Grain density over the nuclei of labelled cells was determined in the maxillary process and the roof of the stomodeum. Cells with a label density at later time points comparable to that found in cells immediately after the labelling period were defined as label-retaining cells, i.e. those which had become quiescent or had significantly altered generation times. The location and developmental fate of these cell populations were confirmed using slides containing adjacent sections stained with Nuclear Fast Red, Alcian Blue, and Tartrazine. The results demonstrated an association between label-retaining cells and chondrogenic differentiation in the roof of the stomodeum. Subpopulations of label-retaining cells (quiescent and/or slow cycling) which we believe to be prechondroblasts appeared in the chondrogenic region of the roof of the stomodeum prior to, or coincident with, cartilage formation. The retention of label, as evidenced by comparison of nuclear grain counts at the end of the labelling period with subsequent time points, indicated that a cell cycle block may have occurred in the prechondroblastic cell population. The block lasted until these cells expressed the chondrogenic phenotype, after which they resumed cell division.

Cell cycle analysis of facial mesenchyme in the chick embryo. I. Labelled mitoses and continuous labelling studies.

Cell cycle parameters were analysed in mesenchyme of the maxillary process and the roof of the stomodeum in the chick embryo from stages 19 through 28. The generation times at stages 24-26 were determined by pulse labelling of embryos with [3H] thymidine, followed by labelled mitosis counts and construction and analysis of percent-labelled mitosis curves employing computer-assisted curve-fitting techniques. The median generation time was approximately 10.6 h in the maxillary process, and 16 h in the roof of the stomodeum; corresponding values for mean generation times were approximately 12.0 and 18.2 h, respectively. Median values for transit times of G1, S, and G2 were 2.0, 5.4, and 2.5 h in the maxillary process and 5.2, 6.7, and 2.7 h in the roof of the stomodeum. The distribution of generation times of cells in the roof of the stomodeum, however, appeared to be more heterogeneous than those of cells in the maxillary process. The percentage of cells which continue to cycle rapidly (i.e. the 'growth fraction') was determined by repeated-labelling experiments with [3H]thymidine in chick embryos from stages 19 through 28. Cumulative labelling of mesenchymal cells in both the maxillary process and roof of the stomodeum approached 100% at stage 19 but dropped markedly from stage 19 to 25 declining to approximately 60-70% in the maxillary process, and to 30% in the roof of the stomodeum. The decline in cell proliferation rates for these regions, determined in previous studies with labelling indices, appears to be a result of the removal of cells from rapidly cycling cell populations into subpopulations which are cycling more slowly and possibly into subpopulations which have become quiescent; the difference in growth rates between these regions could be attributed to the time of appearance and the size of these emerging slow cycling or quiescent subpopulations.

Autoradiographic and cytochemical studies of phagocytic cells in selected fibre tracts of the mouse periodontium.

Proliferative and protein synthetic activities of phagocytic cells of specific fibre tracts of the periodontium of C57Bl mice were employing autoradiographic techniques; these were combined with a histochemical technique for horseradish peroxidase (HRP) as a marker for phagocytic activity. Animals were injected either with [3H]thymidine as a marker for proliferative activity, or with [3H]proline as a marker for protein synthetic activity prior to HRP injection. Blocks from the maxillae of experimental and control animals were fixed, decalcified, and sectioned at 50 micrometers. These were incubated with HRP localization media, dehydrated and flat embedded in Epon 812 wafers. The entire length of the periodontium, including adjacent tooth and bone, were selectively cut from the wafers, mounted on epoxy blocks and serially sectioned at 2 micrometers. Slides containing these sections were then dipped in NTB-3 nuclear track emulsion, and after appropriate exposure times, were developed and post-stained. Sections were examined microscopically, employing an ocular grid, and phagocytic cells within each area examined were delineated as either 'fibroblast-like' (FL cells) or 'endothelial/macrophage-like' (EML cells) according to criteria such as morphology, location, orientation and proximity to a vascular channel. They were then subclassified as labelled or unlabelled with respect to the autoradiographic markers. The thymidine labelling index obtained for non-phagocytic FL cells was 3.09%; this was more than twice that for phagocytic FL cells (1.35%). Similarly phagocytic FL cells in all regions studied incorporated less than half as much [3H]proline as did their non-phagocytic counterparts. This was determined by silver grain counts over HRP-stained and unstained cells using a matched pair system. In addition, the variation of the relative number of phagocytic FL cells in specific fibre tracts suggested a relationship to functional demand. The distribution of these cells was closely related to experimentally determined rates of protein turnover. Phagocytic FL cells have a markedly reduced proliferative rate and synthesize proline-containing proteins at a reduced rate. This may reflect protein production primarily for the purpose of cell maintenance. These findings are consistent with the presence of subpopulations of fibroblasts (or fibrocytes) developmentally or functionally modified for phagocytosis; alternatively, this could signify modulation of fibroblasts from primarily biosynthetic activities to degradative functions in response to varying microenvironmental conditions.