Bmp4 gene is expressed at the putative site of fusion in the midfacial region

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate portion of the craniofacial region are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these primordia leads to the most common craniofacial birth defect in humans (i.e. clefts of the lip and/or palate). Bmp4 plays a critical role during early embryonic development and has previously been shown to play a role in epithelial-mesenchymal interactions in the craniofacial region of chicks. We analyze the expression of bmp4 in mouse as the midfacial processes undergo fusion to form the primary palate. We show that bmp4 is expressed in a very distinct manner in the three midfacial processes (lateral nasal, LNP, medial nasal, MNP, and maxillary processes, MxP) that ultimately fuse to form the midface. Prior to fusion of the midfacial processes, bmp4 is expressed in the ectoderm of the LNP, MNP, and MxP in a distinct spatial and temporal manner near and at the site of fusion of the midface. Bmp4 appears to demarcate the cells in the LNP and MNP that will eventually contact and fuse with each other. As fusion of the three prominences proceeds, some bmp4 expressing cells are trapped in the fusion line. Later, the expression of bmp4 switches to the mesenchyme of the midface underlying its initial expression in the ectoderm. The switch occurs soon after fusion of the three processes. The pattern of expression in the midfacial region implicates the important role of bmp4 in mediating the fusion process, possibly through apoptosis of cells in the putative site of fusion, during midfacial morphogenesis.     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Growth of the secondary palate in the hamster following hydrocortisone treatment: shelf area, cell number, and DNA synthesis

The contribution made by mesenchymal cells during the later stages of palatal development was examined in control and hydrocortisone-treated hamster embryos. Cross-sectional area of the palatal shelf was measured, and the numbers of both epithelial and mesenchymal cells were counted. DNA synthesis was measured by 3H-thymidine incorporation and was used as an index of growth by cell proliferation. The observations in controls indicated that, unlike development during the initial 24 hr, the later period of vertical palate development, followed by reorientation of shelves and their closure, was characterized by a steady level of mesenchymal cell number and palatal shelf area. An absence of corresponding growth in the epithelial cell number suggests that the cells may accommodate the growth either by increasing their size and/or by stretching along the basal lamina. Hydrocortisone treatment did not alter the growth pattern of cell numbers or shelf area. However, it prevented the fusion between the opposing shelves, perhaps by affecting the cytodifferentiation of the palatal tissues. Although a continuous increase in the number of mesenchymal cells during the latter half of vertical shelf development, i.e., between days 11:00 and 12:00 of gestation, is not required for reorientation and fusion of the shelves, it is not clear from the data from the present study whether a critical number of cells and/or cell density is essential for reorientation and fusion of the palate. It was suggested that, for normal palatal development, information on cell cycle and positioning of mesenchymal cells within the shelf during the vertical development may be crucial for further understanding of subsequent events of palatogenesis.     

Effects of embryonic hypoxia on lip formation

The upper lip is formed by the fusion of facial processes, a process in which many genetic and environmental factors are involved. Embryonic hypoxia is induced by uterine anemia and the administration of vasoconstrictors during pregnancy. To define the relationship between hypoxia and upper lip formation, hypoxic conditions were created in a whole embryo culture system. Hypoxic embryos showed a high frequency of impaired fusion, reflecting failure in the growth of the lateral nasal process (LNP). In hypoxic embryos, cell proliferation activity in the LNP mesenchyme was decreased following downregulation of genes that are involved in lip formation. We also observed upregulation of vascular endothelial growth factor expression along with the induction of apoptosis in the LNP. These results suggest that embryonic hypoxia during lip formation induces apoptosis in physiologically hypoxic regions, hypoxia-induced gene expression and downregulation of the genes involved in maxillofacial morphogenesis as immediate responses, followed by reduction of mesenchymal cell proliferation activity, resulting in insufficient growth of the facial processes.     

The development of rat palatal shelves in vitro. An ultrastructural analysis of the inhibition of epithelial cell death and palate fusion by the epidermal growth factor.

Unfused palatal shelves from day-15 rat embryos were cultured with their medial edges in contact. During the next 20 hr epithelial surfaces of apposing shelves adhered, and the epithelial cells in this area degenerated and were replaced by mesenchymal cells in a manner similar to that reported for palate fusion in vivo in the rat and in vitro in the mouse. Four hours after contact the superficial cells of apposing palates adhered with a 10–25-nm distance separating cell membranes while after 20 hr these cells were either eliminated or had lost their flattened appearance and become indistinguishable from the cuboidal-shaped cells adjacent to the basement membrane. The ratio of cytoplasm to nucleoplasm was reduced, and degenerated cells were eliminated by macrophages.Addition of epidermal growth factor (EGF) to the culture medium inhibited epithelial adhesion and degeneration. Basal and superficial cells retained their original appearance, while the ratio of cytoplasm to nucleoplasm was increased. Numerous desmosomes and tonofilaments were evident as well as elaborate membrane foldings. The appearance of these cellular elements is similar to that observed in keratinizing palatal epithelial cells from the oral region. These results suggest that EGF induces the epithelial cells to keratinize, and as a consequence the normal sequence of steps of palate fusion are inhibited.     

Changes in mesenchymal cell-basal lamina relationships preceding palatal shelf reorientation in the mouse

Two specific regions of the future nasal and oral epithelial surfaces of the secondary palatal shelves increase in cell density during shelf reorientation. The relationships of mesenchymal cells to the basal lamina underlying these regions were examined and compared to those of cells underlying adjacent regions which did not change in cell density. CD-1 mouse fetuses were obtained on day 13.5 of gestation. Some palatal shelves were excised immediately and fixed for electron microscopy; other heads were partially dissected and incubated for 4 hr prior to fixation. Although shelf movement is detected only after 6 hr incubation, the shorter time period was selected in order to detect events which precede reorientation. Electron micrographs were taken of the epithelial-mesenchymal interface of nasal and oral regions known to increase in epithelial cell density (active segments) and of nasal and oral regions which did not increase (inactive segments). Several measurements were made in a 500-nm-wide zone delimited on photographic prints. Distinct differences in mesenchymal cell configuration were found between nasal and oral regions. Active and inactive segments of each region also differed. A filamentous layer attached to the undersurface of the lamina densa was observed to vary in thickness and character between regions as well. After 4 hr incubation, differences in mesenchymal cell configuration and ultrastructure of the sublaminar zone were apparent between regions. These results suggest that local epithelial-mesenchymal interactions, possibly mediated by the extracellular matrix, precede shelf reorientation. Whether these changes in mesenchymal cell configuration actually reflect mesenchymal cell activities that are necessary for shelf reorientation remains to be elucidated.     

Epithelial morphogenesis in mouse embryonic submandibular gland: Its relationships to the tissue organization of epithelium and mesenchyme

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force-generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell-cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.     

Developmental Changes of Cell Process Meshwork and Extracellular Space in Subepidermal Mesenchyme in Mouse Forelimb Buds

This study was designed to reveal morphological changes in epithelial-mesenchymal interface during limb development. An electron microscopic and morphometric analysis was done on the cell process meshwork (CPM) and subepidermal extracellular space (SEECS) in mesenchyme in mouse forelimb buds at embryonic ages from day 9.5 to 12.5 (vaginal plug=day 0). At days 9.5 and 10.0, when the apical ectodermal ridge (AER) had not yet appeared, no spatial differences were noted in the CPM density and SEECS width. However, at days 10.5, 11.0 and 11.5, when the AER was distinct, the SEECS was wide and the number of mesenchymal cell processes was small beneath the AER as compared with those at days 9.5 and 10.0; they differed significantly from those beneath the dorsal or ventral non-ridge epidermis. Between days 10.0 and 10.5, the SEECS width decreased and the number of cell processes increased in the dorsal region. At days 12.0 and 12.5, when the AER was regressing, the spatial differences in the CPM density and the SEECS width became less obvious. These findings indicate that spatial and temporal differences of the SEECS width and CPM density exist in mouse limb buds, and these differences are closely associated with the AER development.     

STRUCTURAL FEATURES OF THE EPITHELIO-MESENCHYMAL INTERFACE OF RAT DUODENAL MUCOSA DURING DEVELOPMENT

In fetal rats 5–7 days before birth, the duodenal epithelium is separated from mesenchymal cells by a well-defined basal lamina. By 3–4 days before birth, when small rudimentary villi are first seen, direct contact between epithelial and mesenchymal cells occurs by means of epithelial cell cytoplasmic processes which project through gaps in the basal lamina into the lamina propria. At contact sites, the epithelial and mesenchymal cell plasma membranes were less than 100 A apart but membrane fusion was not seen. In number and size these epithelial cell processes increase strikingly during the last 2 days of gestation, and they persist in large numbers until 7–10 days after birth. Thereafter, they decrease gradually in both number and size until 3–4 wk after birth, when the morphology of the epithelio-mesenchymal interface resembles that seen in adult rats, i.e., there are only rare epithelial cell processes which penetrate deeply into the lamina propria. The presence of a large number of epithelio-mesenchymal contact sites during the period of rapid growth and differentiation of duodenal mucosa may reflect epithelio-mesenchymal cell interactions which may facilitate the maturation of the duodenal mucosa.     

Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection

Normal developmental events during human primary palate formation and alterations associated with cleft lip remain poorly defined. The purpose of this study was to analyze serially sectioned human embryos to identify morphological changes during normal palatal closure and alterations associated with failure of palatal formation. Normal and cleft embryos from the histological collection at the Congenital Anomaly Research Center at the University of Kyoto were studied and photographed for detailed evaluation. Seven serially sectioned cleft lip embryos of stages shortly after primary palate formation (Streeter-O'Rahilly stages 19, 20, and 22) with unilateral or bilateral clefts with varying degrees of clefting were studied. In the normal Kyoto embryos, initial nasal fin (epithelial seam) formation was observed between the medial nasal process and the lateral nasal and maxillary processes at stage 17. During stages 18 and 19, the nasal fin epithelium was replaced by an enlarging mesenchymal bridge, as the maxillary processes united with the medial nasal processes to form the primary palate. The most prominent features observed in the cleft embryos were a reduced thickness of mesenchymal bridging between the medial nasal and maxillary processes, with an excessive amount of epithelium at the junctions between these processes. With ingrowth of the maxillary processes, greater cell dispersion and apparent extracellular matrix accumulation were observed in the medial nasal region. During closure of the primary palate, terminal branches of the maxillary nerve crossed the mesenchymal bridge to the medial nasal region. The partial clefts had reduced maxillary ingrowth and smaller union areas with the medial nasal process. Detailed studies of experimental animal models are required to identify regional growth required for contact between the facial prominences, to clarify the mechanisms of mesenchymal ingrowth and epithelial displacement during palatal formation, and to identify local and/or general factors causing alterations that lead to primary palatal clefting.     

A Critical Role for PDGFRα Signaling in Medial Nasal Process Development

The primitive face is composed of neural crest cell (NCC) derived prominences. The medial nasal processes (MNP) give rise to the upper lip and vomeronasal organ, and are essential for normal craniofacial development, but the mechanism of MNP development remains largely unknown. PDGFRα signaling is known to be critical for NCC development and craniofacial morphogenesis. In this study, we show that PDGFRα is required for MNP development by maintaining the migration of progenitor neural crest cells (NCCs) and the proliferation of MNP cells. Further investigations reveal that PI3K/Akt and Rac1 signaling mediate PDGFRα function during MNP development. We thus establish PDGFRα as a novel regulator of MNP development and elucidate the roles of its downstream signaling pathways at cellular and molecular levels.     

Ultrastructure of initial nasal process cell fusion in spontaneous and 6-aminonicotinamide-induced mouse embryo cleft lip

A search was made for cell ultrastructure differences in the initial fusion process of the medial and lateral nasal processes in mouse embryos of the following types: A/J with 12% cleft lip (CL), CL/Fr with 23% CL--both cleft-lip-predisposed strains, CL/Fr 6-aminonicotinamide (6AN)-treated (94% CL) and controls from the C57BL/6 strain (0% CL) and dancer stock (0% CL). No detectable differences were found between the A/J and CL/Fr strains and the controls in the epithelial cells showing initial contact and fusion. Epithelial surfaces not in contact in controls and where clefts were developing were smooth. Cells approaching or in contact had cell projections, intercellular junctions, desmosomes, and microfilaments demonstrating firm contact between the apposed epithelia. It has been postulated that spontaneous cleft lip was due to a predisposing face shape bringing about a failure of contact in some embryos and in others where contact was achieved fusion was normal. These data support this view. The situation, however, in 6AN-treated embryos is different. A few 6AN-treated embryos showed abnormal contact that appeared malpositioned and tenuous. The teratogen also caused increased cell death and a denser epithelium and mesenchyme. Thus 6AN-induced cleft lip could be due to the epithelial cell changes and/or to the reduction in size of the nasal processes.     

Epithelial bridging of the primary palate: II. In vitro model mimics in vivo behavior

Previously, Forbes et al. [J Craniofac Genet Dev. Biol, 9:271-284, 1989] and Millicovsky et al. [Am J Anat 164:29-44, 1982], demonstrated that some of the epithelial cells of the primary palate formed extensive projections, bridging the medial and lateral nasal prominences. These connections are thought to aide in the fusion process by facilitating union of the prominences, a process known as secondary fusion [Millicovsky et al., 1982]. In order to study the epithelial cell and its behavior more closely an in vitro model was established [Gibson et al.: J Craniofac Genet Dev Biol, 1989], where epithelial cells in culture were shown to produce many of the morphologic characteristics observed in vivo. In the present study, an in vitro model is discussed which reproduces the epithelial projections observed in vivo. Epithelial cells, previously characterized, were obtained from the primary palate of 13-day-old rat embryos and sub-cultured as explants. Comparisons were made with the epithelial bridging observed in vivo of two species of animals. The results indicated sub-cultured epithelium as isolated cells, at either low or high density, rarely formed bridges. Primary cultures of epithelial explants also infrequently formed projections. However, sub-cultures of epithelial explants, plated as small clusters of cells with intervening spaces between cell groups, demonstrated extensive epithelial bridging. Epithelial projections did not form from cells that were directly attached to the plastic culture dish; only superficial, elevated cells formed projections. Significantly, the connections that occurred between explants did not attach to the plastic substratum. Instead, they appeared as line connections suspended by the medium. With time, the number of projections increased and epithelial cells could be seen along the projections forming an epithelial bridge. This study established a model of epithelial bridging in vitro for analysis of a process which has been shown to be an integral part of primary palate fusion.     

Histogenesis of Swiss white mouse secondary palate from nine and one-half days to fifteen and one-half days In utero. I. Epithelial-mesenchymal relationships—light and electron microscopy

Development of the secondary palate in Swiss white mouse embroyos was studied from age nine-and-one-half days in utero to the stage of mesenchymal coalescence in the secondary palate (approximately fifteen-and-one-half days). The greatest changes observed occur in the mesenchyme. At early stages, mesenchymal cells underlying oral ectoderm of the head are few and only occasionally contact the ectoderm. Electron micrographs show large intercellular spaces between the ectodermal cells. As embryogenesis continues, the mesenchymal cells become more numerous, closer to each other and closer to the epithelium. Just prior to horizontal transposition of shelves, the mesenchymal cells spread farther from each other and from the palatal epithelium and epithelium of the palatal tip becomes stretched. Ultrastructurally the intercellular spaces between epithelial cells of the palate tip have become much smaller. Some mitochondria in some epithelial cells are swollen and have clear matrices and distorted cristae. The shelves become horizontal and meet in the midpalate. Cells with degeneration bodies are seen in the epithelial seam. The seam undergoes autolysis and is replaced by mesenchyme. The morphological changes described, particularly in the mesenchyme, may play an important role in determining the effect of various teratogens at different stages of palatal development. The changes in both mesenchyme and epithelial cells in the later stages may constitute part of the process of preparing shelves for fusion as postulated by Pourtois ('66).     

Variations in 5'-nucleotidase activity in established mesenchymal cell lines from syngeneic A/J mice

5'-Nucleotidase activity was analyzed in four different mesenchymal cell lines (F, m, e and SP) established from syngeneic A/J mice. The 5'-nucleotidase activity of fibroblasts was lower in transformed cells (F and m) than in nontransformed cells (e). An increase in cell contact during confluence or during high cell density increased 5'-nucleotidase activity, and a decrease in cell contact caused a decrease in 5'-nucleotidase activity in both fibroblastic (F, m and e) and reticulum (SP) cell lines. These results are evidence that 5'-nucleotidase activity in mesenchymal cells is influenced by intercellular contact as well as transformation.     

Molecular and morphologic changes during the epithelial-mesenchymal transformation of palatal shelf medial edge epithelium in vitro.

The fate of the medial edge epithelial (MEE) cells during palatal fusion has been proposed to be either programmed cell death or epithelial-mesenchymal transformation. Vital cell labeling techniques were used to mark the MEE and observe their fate during palatal fusion in vitro. Fetal mouse palatal shelves were labeled with Dil and allowed to proceed through fusion while maintained in an organ culture system. The tissues were examined at several stages of palatal fusion for the distribution of Dil, presence of specific antigens and ultrastructural appearance of the cells. The MEE labeled with Dil occupied a midline position at all stages of palatal fusion. Initially the cells had keratin intermediate filaments and were separated from the underlying mesenchyme by an intact basement membrane. During the process of fusion the basement membrane was degraded and the Dil-labeled MEE were in contact with the mesenchymal-derived extracellular matrix. In the late stages of fusion the Dil-labeled MEE altered their cellular morphology, had vimentin intermediate filaments, and were not associated with an identifiable basement membrane. Dil-labeled cells, without an epithelial phenotype, remained present in the midline of the completely fused palate. The data indicate that the MEE did not die but underwent a phenotypic transformation to viable mesenchymal cell types, which were retained in the palatal mesenchyme.     

Freeze-fracture features of epithelioid cells,multinucleated giant cells,and phagocytic macrophages

The freeze-fracture morphology of epithelioid cells, multinucleated giant cells (Langhans' type), and phagocytic macrophages was investigated. The intensely folded and interdigitating surface membranes of epithelioid cells and multinucleated giant cells displayed no specialized areas of cell contact. The size of the intramembranous particles (IMP) and the fact that the area density of IMPs was higher in the cytoplasmic (P) faces than in the external (E) faces of the cell membranes agreed with observations in other eukaryotic cells. The area densities of the IMPs suggest lower transport rates of molecules across the cell membranes of granuloma cells than of certain epithelial cells. Small pits were detected in the surface membranes of the granuloma cells but an extrusion of granules was not observed. The cytoplasmic granules displayed very different sizes and shapes ranging from spherical to rod-shaped. The latter type of granules (probably primary lysosomes) dominated in multinucleated giant cells. The granule membranes were studded with IMPs whose area densities increased with the granule size. Multilamellar bodies with smooth (lipid) fracture faces were found only in phagocytic macrophages. The nuclear pores of the granuloma cells were distributed over the entire surfaces of the nuclei and displayed moderate clustering. The values of the area densities of the nuclear pores were in keeping with the values observed in mammalian and human epithelial or mesenchymal cells, indicating similar exchange rates of molecules between the nucleoplasm and the cytoplasm in these different cell types. In a single phagocytic macrophage the E-face of the inner membrane of the nuclear envelope displayed a network of fine filaments whose nature is at present unknown.     

Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.     

Human bone marrow-derived stem cells acquire epithelial characteristics through fusion with gastrointestinal epithelial cells

Bone marrow-derived mesenchymal stem cells (MSC) have the ability to differentiate into a variety of cell types and are a potential source for epithelial tissue repair. Several studies have demonstrated their ability to repopulate the gastrointestinal tract (GIT) in bone marrow transplanted patients or in animal models of gastrointestinal carcinogenesis where they were the source of epithelial cancers. However, mechanism of MSC epithelial differentiation still remains unclear and controversial with trans-differentiation or fusion events being evoked. This study aimed to investigate the ability of MSC to acquire epithelial characteristics in the particular context of the gastrointestinal epithelium and to evaluate the role of cell fusion in this process. In vitro coculture experiments were performed with three gastrointestinal epithelial cell lines and MSC originating from two patients. After an 8 day coculture, MSC expressed epithelial markers. Use of a semi-permeable insert did not reproduce this effect, suggesting importance of cell contacts. Tagged cells coculture or FISH on gender-mismatched cells revealed clearly that epithelial differentiation resulted from cellular fusion events, while expression of mesenchymal markers on fused cells decreased over time. In vivo cell xenograft in immunodeficient mice confirmed fusion of MSC with gastrointestinal epithelial cells and self-renewal abilities of these fused cells. In conclusion, our results indicate that fusion could be the predominant mechanism by which human MSC may acquire epithelial characteristics when in close contact with epithelial cells from gastrointestinal origin . These results could contribute to a better understanding of the cellular and molecular mechanisms allowing MSC engraftment into the GIT epithelium.