Appearance of a unique cell type in the fusion sites of facial processes.

The contact sites between the medial and lateral nasal processes during the period of facial formation of the mouse embryo were examined by light and electron microscopy. Characteristic superficial cells were observed at the transitional regions between the surface ectoderms and the nasal epithelia at the end of the isthmus, where the initial contact of the opposing nasal processes took place. At the later stage the contact sites extended to the bottom of the ravine formed by the two nasal processes, where the superficial cells always seemed to bridge the area between the nasal processes. These superficial cells had a large, clear nucleus and abundant cytoplasm as well as the common structural features characteristic of the embryonic cells. These cells were also observed on the surface near the contact site in the presumptive fusion area. These observations suggest that these superficial cells play a critical role in the epithelial adhesion of the medial and lateral nasal processes throughout the fusion.

Changes in the subepithelial mesenchymal cell process meshwork in developing facial prominences in mouse embryos.

We examined temporal and spatial changes in the subepithelial mesenchymal cell process meshwork (CPM) in normally developing medial (MNP) and lateral nasal prominences (LNP) in mouse embryos by light and scanning electron microscopy. Marked changes were found only in the MNP during the fusion of the MNP and LNP. The CPM density in the prospective fusion area of the MNP gradually increased as the epithelial surfaces approached each other, attained its maximum just before contact, and decreased after contact. The CPM density in the prospective fusion area of the LNP changed only slightly even when the epithelial surfaces approached each other. The increase in CPM density paralleled that in the density of mesenchymal cell bodies. The LNP grew more actively toward the line of fusion than did the MNP during the progressive fusion of the two prominences. A larger number of fusion-associated epithelial morphological changes--the appearance of superficial protruding cells and cell degeneration--occurred in the MNP than in the LNP. These findings suggest that the increased CPM density is closely related to the growth of the facial prominences and the fusion-associated epithelial morphology and that the CPM plays an important role in the epithelial-mesenchymal interaction during the formation of the upper lip and primary palate.

Light and electron microscopy study of fusion of facial prominences. A distinctive type of superficial cells at the contact sites.

The contact site between the medial nasal prominence (MNP) and the lateral nasal prominence (LNP) during the period of primary palate formation in the mouse embryo was examined by light and electron microscopy. Throughout this period, a distinctive type of superficial cell was observed at the contact site. These superficial cells had a large nucleus and abundant cytoplasm as well as structural features characteristic of embryonic cells. At earlier stages, these cells were seen at the transitional region between the surface ectoderm and the epithelia of the nasal pit at the end of the isthmus, where initial contact of opposing MNP and LNP took place. At later stages, these superficial cells appeared to bridge the gap between MNP and LNP at the contact sites, which extended to the bottom of the valley formed by MNP and LNP. These cells were also observed on the surface near the contact sites, that is, the presumptive fusion area. These superficial cells displayed well-developed junctional complexes (intermediate and gap junctions, and desmosomes). Many filaments were observed subjacent to the plasma membranes of these superficial cells, some of which were associated with junctional complexes. These observations suggest that this kind of distinctive superficial cell may play critical roles in the contact of MNP and LNP throughout the fusion process.

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.

Active role of embryonic facial epithelium: new evidence of cellular events in morphogenesis.

Epithelial cells of the C57B1/6J mouse embryo participate in a temporal sequence of events associated with the approximation, fusion and consolidation of components of the facial primordia into a definitive structure. These cells lose their surface microvilli, and after a brief period of quiescence they begin to fill the grooves separating facial constituents by producing a series of surface projections that increase in size and complexity as the process of fusion nears termination. Cessation of surface activity and the restoration of epithelial microville indicate the end of the temporal sequence. Significantly, the epithelial cells of primary palates of embryos with genetically-and phenytoin-induced cleft lip remain unchanged and do not participate in fusion. This epithelial sequence has not been described previously and we suggest that all of its steps may be critical to the normal development of the mammalian face.