Ultrastructural and cytochemical observations on 5-fluorouracil-induced cleft-palate development in hamster

Sequential alterations in 5-fluorouracil-treated hamster fetal palate were studied by light and electron microscopy and by acid phosphatase cytochemistry. At an early stage in 5-fluorouracil-treated fetuses, when the palatal shelves were vertical, lysosomes first appeared in cells of the prospective fusion epithelium and then in the cells of subjacent mesenchyme. In contrast to controls, increasing numbers of both the epithelial and mesenchymal cells of the vertical palate showed lysosomal injury in 5-fluorouracil-treated fetuses as development progressed. Subsequently, the basal lamina in the vertical palate showed alterations, characterized initially by disturbances in lamina lucida, by fingerlike extensions of lamina densa, and ultimately by its complete breakdown. At a later stage, when shelves became horizontal, the lysosomes were absent in both the epithelial and mesenchymal cells, and the basal lamina continuity was restored. Unlike controls, however, 5-fluorouracil-treated horizontal shelves never contacted one another. Instead, the epithelia of the horizontal shelves underwent stratification. It appears that premature formation of lysosomes in palatal epithelial and mesenchymal cells following 5-fluorouracil treatment disrupts normal cytodifferentiation and affects the integrity of the basal lamina; both effects are associated with cleft-palate development.     

Genesis of hadacidin-induced cleft palate in hamster: morphogenesis, electron microscopy, and determination of DNA synthesis, cAMP, and enzyme acid phosphatase.

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.     

Palatogenesis in hamster. II. Ultrastructural observations on the closure of palate

Formation of secondary palate in hamster was studied with electron microscopy. Prior to assuming horizontal position, the palatal shelves were covered by a two to three cell layer thick epithelium which was separated from the underlying mesenchyme by an intact basal lamina. Epithelial cells were attached to each other by desmosomes. Early hemidesmosomes could be identified as thickenings of the cytoplasmic membrane opposing the basal lamina. Epithelial cells, like other embryonic cells, contained only few organelles but were rich in polyribosomes. As the horizontal shelves approached each other towards the midline, lysosomes and tonofilaments appeared in the superficial and basal cells of the epithelia. Superficial cells showed degeneration and eventual lysis. Fusion of the opposing epithelia occurred between the deeper cells by means of newly formed desmosomes. The epithelial seam resulting from fusion of the epithelia was limited on each side by a continuous basal lamina. Its subsequent thining and eventual fragmentation resulted from the loss of cells by autophagy. There was no evidence of mesenchymal invasion of the epithelial seam. Mesenchymal macrophages appeared in the later stage of palatogenesis and were responsible for phagocytosis of cellular debris. Formation of the soft palate was basically similar to that of the secondary hard palate and occurred by fusion of the opposing shelves. Similarly, anterior closure of the palate occurred by fusion of the lower end of the nasal septum to the primary and secondary palates. Hyperplasia of the opposing epithelia, prior to their fusion, was often seen. It is suggested that formation of the palate occurs in predictable and coordinated fashion and that timely appearance of lysosomes causing lysis of intervening epithelia is of great significance in normal palatogenesis.     

Inhibition of Smad signaling is implicated in cleft palate induced by all-trans retinoic acid

The effect of all-trans retinoic acid (atRA) on palatal fusion and the underlying mechanisms were investigated using organ culture. Compared with control group, the atRA-treated group (1 μM and 5 μM) had more medial edge epithelium (ME) remaining within the midline epithelial seam (MES). At 10 μM atRA, the opposing shelves were not in contact at the culture end (72 h). Cell death detection by TUNEL and laminin immunohistochemistry demonstrated that atRA (5 μM) induced apoptosis in mesenchyme and inhibited degradation of basal lamina within MES. Notably, migration and apoptosis of ME cells and degradation of basal lamina within MES markedly represented vehicle control palatal shelves in culture. Additionally, apoptosis was not detected in mesenchyme of control palatal shelves. Immunoblotting analysis revealed that Smad2 and Smad3 were endogenously activated and expression of Smad7 was inhibited during the fusion process. In contrast, atRA treatment abrogated phosphorylation of Smad2 and Smad3 and inducible expression of Smad7 in ME. From these data, it is assumed that inhibition of Smad pathway by atRA in ME may play a critical role in abrogation of the ME cell apoptosis and degradation of the basal laminin, which might contribute to failure of palatal fusion.     

Gross and cellular analysis of 6-mercaptopurine-induced cleft palate in hamster

The present study analyzes the morphological, histochemical, and ultrastructural aspects of the pathogenesis of 6-mercaptopurine (6MP)-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that 6MP stunts the growth of vertical palatal shelves and thus induces cleft palate. Ultrastructural analysis showed that, in contrast to controls, 6MP-induced alterations were first seen in the mesenchymal cells 24 hr after drug administration. The initial alterations were characterized by swelling of the nuclear membrane. During the next 12 hr, lysosomes were seen first in the mesenchymal cells and then in the cells of the medial edge epithelium (MEE) of the developing palatal primordia. The appearance of lysosomes was temporally abnormal and was interpreted as a sublethal response to 6MP treatment. Subsequently, the nuclear alterations and the lysosomes diminished; and 48 hr after 6MP administration, they were absent from the palatal tissues. Ninety hours after 6MP administration, unlike the controls (in which the palatal shelves were already fused), changes were seen at the epithelial-mesenchymal interface in the developing cleft palatal shelves. These changes were characterized by breakdown of the basal lamina and epithelial-mesenchymal contacts. Eventually, at term, the MEE of the vertical shelf stratified. It was suggested that 6MP affected cytodifferentiation in the palatal tissues during the critical phase of early vertical shelf development and thereby induced cleft palate.     

Pathogenesis of bromodeoxyuridine-induced cleft palate in hamster

In the present study, the morphological, histochemical, biochemical, and cellular aspects of the pathogenesis of bromodeoxyuridine (BrdU)-induced cleft palate in hamster fetuses were analyzed. Morphological observations indicated that BrdU interferes with the growth of the vertical shelves and thus induces cleft palate. At an ultrastructural level, BrdU-induced changes were first seen in the mesenchymal cells. Eighteen hours after drug administration, the initial alterations were characterized by swelling of the nuclear membrane and the appearance of lysosomes in the mesenchymal cells of the roof of the oronasal cavity. During the next 6 hr, as the palatal primordia developed, lysosomes were also seen in the overlying epithelial cells. The appearance of lysosomal activity, which was verified by acid phosphatase histochemistry, was temporally abnormal and was interpreted as a sublethal response to BrdU treatment. Later the cellular alterations subsided; 48 hr after BrdU treatment, they were absent in both the epithelial and mesenchymal cells of the vertically developing palatal shelves. Subsequently, unlike controls (in which the palatal shelves undergo reorientation and fusion), the BrdU-treated shelves remained vertical until term. Biochemical determination of DNA synthesis indicated that although there was an inhibition of DNA synthesis at the time of appearance of palatal primordia, a catch-up growth during the ensuing 12 hr may have restored the number of cells available for the formation of a vertical palatal shelf. It was suggested that BrdU affected cytodifferentiation in the palatal tissues during the critical phase of early vertical development to induce a cleft palate.     

Differentiation of cyclophosphamide-treated hamster secondary palate: ultrastructural and biochemical observations

A study was undertaken to analyze the ultrastructural aspects and the enzyme acid phosphatase cytochemistry and biochemistry of the pathogenesis of cyclophosphamide (CP)-induced cleft palate in hamster fetuses. The initial CP-induced alterations were the appearance of lysosomes in the mesenchymal cells of the vertically developing palatal primordia within 8 hr of drug administration. The mesenchymal lysosomal activity, which increased during the next 16 hr, was abnormal and interpreted as a sub-lethal response to CP treatment. Subsequently, the lysosomal activity in the mesenchyme diminished gradually and, 48 hr after CP treatment, was absent. At this time, lysosomes were seen in the epithelial cells of the vertical palate. Fifty-six hours after CP treatment, unlike controls where palatal shelves were already fused, lysosomal activity subsided in the epithelial cells. Changes, however, continued to be seen at the epithelial-mesenchymal interface. These changes were characterized by discontinuity in the basal lamina, and by epithelial-mesenchymal contacts. They persisted for 8 hr but were absent thereafter. Sixty-four hours after CP administration, the vertical shelves became horizontal and remained so until term. Following analysis of data, both from the literature and from the present study, it was suggested that CP first affected mesenchymal cell proliferation, and then its cytodifferentiation, during the critical phase of early vertical development; consequently the reorientation of the shelves to a horizontal plane was delayed, inducing cleft palate.     

Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation

During fusion of the mammalian secondary palate, it has been suggested that palatal medial edge epithelial (MEE) cells disappear by means of apoptosis, epithelial-mesenchymal transformation (EMT) and epithelial cell migration. However, it is widely believed that MEE cells never differentiate unless palatal shelves make contact and the midline epithelial seam is formed. In order to clarify the potential of MEE cells to differentiate, we cultured single (unpaired) palatal shelves of ICR mouse fetuses by using suspension and static culture methods with two kinds of gas-mixtures. We thereby found that MEE cells can disappear throughout the medial edge even without contact and adhesion to the opposing MEE in suspension culture with 95% O2/5% CO2. Careful examination of MEE cell behavior in the culture revealed that apoptosis, EMT, and epithelial cell migration all occurred at various stages of MEE cell disappearance, including the transient formation and disappearance of epithelial triangles and islets. In contrast, MEE cells showed poor differentiation in static culture in a CO2 incubator. Furthermore, mouse and human amniotic fluids were found to prevent MEE cell differentiation in the cultured single palatal shelf, although paired palatal shelves fused successfully even in the presence of amniotic fluid. We therefore conclude that terminal differentiation of MEE cells is not necessarily dependent on palatal shelf contact and midline epithelial seam formation, but such MEE cell differentiation appears to be prevented in utero by amniotic fluid unless palatal shelves make close contact and the midline epithelial seam is formed.     

The fate of the expected fusion zone in rat fetuses with experimentally-induced cleft palate—An ultrastructural study

The ultrastructure of palatal processes from rat fetuses with cleft palate induced by Meclozine or amniotic sac puncture was studied from gestation days 16 to 20. Degenerative changes involving the cytoplasm of epithelial cells of the expected fusion zone began on Day 16, when palatal fusion occurs normally in the midline seam in untreated fetuses. By Day 17 the cells in the same region showed more advanced degeneration, degraded cytoplasm and occasional dead epithelial cells appearing to be removed via intercellular extrusion or by macrophages. In the expected fusion zone, the basal lamina became discontinuous while in the oral and nasal zones of the palatal processes a firm epithelium-connective tissue attachment developed. As gestation progressed, the zone became narrower until, by Day 20, no degenerating cells remained. Replacement of lost epithelium appeared to have occurred from the nasal side, the termination of the keratinizing oral epithelium having the same location throughout the period studied. The results support the concept of programmed cell degeneration in palatal processes but indicate that this event has the potential to extend over a relatively long period of fetal life.     

Epithelial-mesenchymal transformation during palatal fusion: carboxyfluorescein traces cells at light and electron microscopic levels.

During the fusion of rodent embryo palatal shelves, the cells of the outer epithelial layer slough off, allowing the cells of the medial edge basal layer to form a midline seam that undergoes epithelial-mesenchymal transformation, as judged by electron microscopy and immunohistochemistry. In this study, we analyze the fate of the transformed cells using a lipid soluble dye to label the medial edge epithelium in situ. Prefusion E14 mouse palates were exposed in vitro or in vivo to a fluoresceinated lipid soluble marker, carboxydichlorofluorescein diacetate succinimidyl ester (CCFSE), which localizes in epithelia as a lipid insoluble compound that does not pass into the connective tissue compartment. The midline seam that formed after 24 hours contained labelled epithelial cells that were replaced by individually labelled mesenchymal cells where the seam transformed. By light microscopy, the labelled cells were seen to contain intensely fluorescent bodies that do not react for acid phosphatase. We were able for the first time to identify these structures by electron microscopy as CCFSE isolation bodies. The cells with isolation bodies are clearly healthy and able to participate in subsequent development of the palate. At 4 days after labelling, individual CCFSE containing cells present in the palate mesenchyme occupy both midline and lateral areas and can clearly be classified as fibroblasts by electron microscopy. CCFSE is a far more useful marker than another lipid soluble marker, DiI, for following cells, because the cells can be fixed and identified both at the light and electron microscope levels. Interestingly, if labelled palatal shelves are not allowed to fuse in vitro, the basal epithelial cells do not form mesenchyme after sloughing, indicating that formation of the epithelial midline seam is necessary to trigger its epithelial-mesenchymal transformation.     

Human embryonic palatal epithelial differentiation is altered by retinoic acid and epidermal growth factor in organ culture

Reports of adverse human pregnancy outcomes including cleft palate have increased as the clinical use of isotretinoin (13-cis-retinoic acid) and other retinoic acid (RA) derivatives have increased, but the mechanisms by which their effects are exerted are not understood. Research in craniofacial development is generally performed in rodents, and mouse palatal shelves exposed in organ cultures to retinoids and epidermal growth factor (EGF) display altered medial epithelial cell morphology blocking normal union of apposing shelves. In the present study, precontacting human palatal shelves were maintained in organ culture for 2, 3, or 6 days and exposed to labeled thymidine (3H-TdR) during the last 16 hr. Retinoids and EGF were included in the media so that each shelf was exposed to one of the following: control, EGF, trans-RA at 10(-5)M, cis-RA at 10(-7) or 10(-9) M, or RA + EGF. After exposure of cultured human embryonic palatal shelves to 13-cis-RA and trans-RA with or without EGF, medial epithelial cells do not degenerate, cell surface morphology shifts toward a nasal type, glycogen deposits decrease, smooth endoplasmic reticulum (SER) increases, and basal lamina appear altered. In shelves exposed to EGF and trans-RA early in their development, DNA synthesis appears to terminate prematurely as compared to shelves cultured in control media, and this effect is accompanied by excessive mesenchymal extracellular space expansion. Exposure of shelves to EGF alone is sufficient to block degeneration and induce hyperplasia of the medial epithelial cells but does not induce other ultrastructural changes seen with both EGF and RA. The observed alterations in medial cell morphology could interfere with adhesion of the palatal shelves and may play a role in retinoid-induced cleft palate in the human embryo.     

Human prenatal palatal closure related to skeletal maturity of the jaws

The purpose of the present study was to elucidate the stages of skeletal maturation of the maxilla and the mandible at the time of soft tissue palatal closure. Similar studies were not found in the literature. This investigation was based on maxillae and mandibles from 19 human embryos/fetuses selected by visual inspection of palatal structures, eight fetuses "just before palatal closure," and 11 fetuses "just after palatal closure." The findings are related to formerly described skeletal developmental stages in the maxilla, to stages in the symphysis menti region, and to development in the mandibular condylar region. The present study revealed that elevation of the palatal shelves takes place at a specific stage of maxillary skeletal maturity (stage Max III), at a time of mandibular development characterized by absence of condylar cartilage and by constancy in symphysis menti maturity (stage SM I). Knowledge of the normal sequence of prenatal skeletal development is considered essential for understanding the abnormal sequence of skeletal development.     

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.     

Programmed cell death is not a necessary prerequisite for fusion of the fetal mouse palate

It has been widely accepted that programmed cell death (PCD) is an essential event in palatogenesis and that its failure can result in cleft palate, one of the most common birth defects in the human. However, some conflicting results have been reported concerning the timing of cell death occurring in the fusing palate and therefore the role of PCD in palatal fusion is controversial. In order to clarify whether cell death is indispensable for mammalian palatogenesis, we cultivated the palates of day-13 mouse fetuses in vitro and prevented cell death by treating them with the inhibitors of caspases-1 and -3 or with aurintricarboxylic acid which inhibits the activity of caspase-activated DNase. Even when cell death was almost completely inhibited, palatal fusion took place successfully. Histological examination revealed that in the absence of apoptotic cell death, the medial edge epithelia of opposing palatal shelves adhered to each other and subsequently, the midline epithelial seam was disrupted and disappeared to bring about mesenchymal confluence across the palate. It seems that cell death is not a necessary prerequisite for palatal fusion but it may help to efficiently eliminate unnecessary cells which failed to migrate or differentiate properly.     

Palate development after fetal tongue removal in cortisone-treated mice

Morphological studies of cortisone-induced cleft palate have shown retardation in the rotation of palatine shelves from a sagittal to a transverse plane. Cortisone also reduces fetal muscular movements, which may explain why displacement of the tongue from between the palatine shelves is delayed. Previous work with extrauterine development of control fetuses demonstrated that fetal membranes and tongue were major obstacles to shelf rotation. Thus, removal of these obstacles might permit rotation and fusion of palatine shelves in cortisone-treated fetuses. In the present experiment, fetuses from cortisone-treated strain CD-1 mice were released from uterus and membranes and allowed to develop for eight hours in a fluid medium with the umbilical cord left intact. Compared to 4% fusion in utero, there was palatal fusion in 20% of fetuses released from membranes. When the fetal tongue was removed during extrauterine development, the frequency of fusions increased to 61%. Fusion appeared normal by the criteria applicable through light microscopy. Thus, cortisone induces cleft palate primarily through interference with shelf rotation. The palatine shelves of treated fetuses retain their ability to fuse when they can come in contact during the normal time for palate closure.     

Palatal slit: a new spontaneous defect of the palate of C57BL/6 mice

A hitherto undescribed palatal defect, here named "palatal slit," was observed during a teratological study of C57BL/6 fetuses. The defect, involving a failure of fusion of the premaxilla and palatal shelves, corresponds to stage 7 in normal palate closure. Adult C57BL/6 mice have been observed with the defect, so it does not represent a developmental delay that is repaired postnatally. Genetic factors of an unknown nature seem to be involved in the occurrence of palatal slit, which does not appear to be related developmentally to cleft palate. Some preliminary information on the defect is reported here.     

Effects of cyclophosphamide on the secondary palate development in golden Syrian hamster: teratology, morphology, and morphometry

Cyclophosphamide (CP), when injected in hamster mother between days 9 and 11 of pregnancy, was teratogenic in fetuses. On the basis of a morphological study it was deduced that CP delayed the reorientation of hamster palatal shelves by 16-20 h. In a subsequent experiment, in both control and CP-treated palatal shelves, the numbers of epithelial and mesenchymal cells were counted and cross-sectional area was measured. DNA synthesis, measured by 3H-thymidine incorporation, was used as an index of growth by cell proliferation. The results showed that during the vertical development of palatal shelves, the mesenchymal cells reached their peak number during the initial 24 hours, i.e., at the end of the second peak in DNA synthesis, and remained unchanged thereafter throughout reorientation. The shelf area also showed rapid increase during the initial 24 h followed by a spurt 2 h prior to reorientation. Cyclophosphamide prolonged the acquisition of these features by affecting the mesenchymal cells and consequently delayed the reorientation of the vertical shelves until such time that the number of healthy mesenchymal cells and shelf area were restored to the control values. The data lend further support to the hypothesis that the acquisition of a specific number of cells and shelf volume, during vertical palatal development, may be essential for palatal shelf reorientation.     

Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse

The disappearance of palatal medial edge epithelium (MEE) after fusion of secondary palatal shelves is often cited as a classical example of embryonic remodeling by programmed cell death. We reinvestigated this phenomenon in 16-day rat embryos, using light and electron microscopy. We confirm reports that the periderm of the two-layered MEE begins to slough after shelves assume horizontal positions. In vitro, peridermal cells are not able to slough and are trapped during the adhesion process. In vivo, however, surface cells shed before the shelves in the anterior palate adhere, allowing junctions to form between opposing basal epithelial cells. Midline seams so formed consist of two layers of basal cells, all of which appear healthy. Even though its cells are dividing, growth of the seam fails to keep pace with palatal growth and it thins to one layer of cells, and then breaks up into small islands. The basal lamina disappears and elongating MEE cells extend filopodia into adjacent connective tissue. Electron micrographs reveal transitional steps in loss of epithelial characteristics and gain of fibroblast-like features by transforming MEE cells. One such feature, observed with the aid of immunofluorescence, is the turn of the mesenchymal cytoskeletal protein, vimentin. No cell death or macrophages are observed after adhesion and thinning over most of the palate. These data indicate that MEE is an ectoderm that retains the ability to transform into mesenchymal cells. Epithelial-mesenchymal transformation may be expressed in other embryonic remodelings (R.L. Trelstad, A. Hayashi, K. Hayashi, and P.K. Donahue, 1982, Dev. Biol. 92, 27), resulting in heretofore unsuspected conservation of embryonic cell populations.     

Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.     

The effects of chlorcyclizine-induced glycosaminoglycan alterations on palatal mesenchyme-basal lamina relationships in the mouse

The relationships of mesenchymal cells to the basal lamina underlying regions of the palatal-shelf epithelium that are known to increase in cell density during shelf reorientation are quantitatively different from those of cells underlying neighboring regions that do not increase in cell density. Chlorcyclizine-induced alterations of the extracellular matrix were used to investigate the possible contribution of extracellular matrix to these differences. Chlorcyclizine causes hyaluronate and the chondroitin sulfates to be degraded into pieces with smaller molecular weights and lower charge densities, with little or no effect on their synthesis, and also results in cleft palate. Pregnant CD-1 mice were gavaged with chlorcyclizine on days 10.5, 11.5, and 12.5 of gestation, and the fetuses were harvested on day 13.5. Some palatal shelves were excised immediately and fixed for electron microscopy; other heads were partially dissected and incubated for 4 hr prior to fixation. In normal heads differences in mesenchymal cell configurations are detectable after 4 hr in vitro. Electron micrographs were taken of the epithelial-mesenchymal interface in nasal and oral regions that increased in epithelial cell density and in nasal and oral regions which did not. Several variables of mesenchymal cell configuration were measured in a 500-nm-wide zone delimited on photographic prints. Chlorcyclizine-induced glycosaminoglycan alterations resulted in quantifiable, region-specific differences in mesenchymal cell relationships to the basal lamina and in the ultrastructural appearance of the zone immediately subjacent to the basal lamina. These results suggest that the epithelial-mesenchymal interface and sublaminar zone of the nasal and oral regions as well as their active and inactive segments may be constitutively different.