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The ultrastructural development of the surface belonging to the epithelium-connective tissue interface of the hard and soft palatine mucosa of mouse was studied using field emission scanning electron microscopy and high voltage transmission electron microscopy methods. The tissues were analysed at 18 days of gestation, and, 1, 3, 7, 14, 21, 30 and 90 days after birth. The three-dimensional architecture of the connective tissue in the lamina propria showed different shapes. The papillae began to form on days 7 and 14 but continued to growth until the adult period. Different stages of primary and secondary connective tissue papillae were recognized in the hard palatine mucosa whereas the soft palate showed erect connective tissue papillae, small round formations containing taste buds and several openings of the salivary glands ducts.

Objective: The purpose of this study was to investigate, in a normal human fetal sample, the development of the anterior area of the palate. Emphasis has been placed on the bilateral epithelial fusion lines between the soft-tissue palatal shelves and the premaxillary mucosa, and on the relationship between these fusion lines and the locations of the inclusive fissures and of the tooth buds. The goal was to evaluate how the location of clefts in cleft lip and palate (CLP) patients interferes with tooth buds that are often affected in clefting., Design: Nineteen maxillas of human fetuses underwent histologic examination after serial horizontal sectioning. Superimpositions of tracings from photographs of the histologic sections yielded information on the maxillary morphology at different levels., Results: After elevation and fusion of the soft-tissue palatal shelves, the palatal fusions were Y-shaped. During subsequent osseous palate development, including formation of the incisive fissure and intermaxillary suture, the Y-shaped epithelial fusions disintegrated. The locations of the anterior arms of the epithelial Y and of the incisive fissure did not coincide., Conclusions: The incisive fissure is likely not the location of clefts in CLP. The location of anterior palatal clefting in CLP patients follows the original course of the epithelial fusion between the palatal shelves and the premaxillary mucosa found in the region of the lateral incisors.

Five pregnant rats were treated during organogenesis with sc injections of acyclovir (50 mg/kg body weight) on days 9, 10 and 11 of pregnancy. The fetuses (N=62) were evaluated on day 20 of gestation and presented decreased body weight as well as delayed differentiation of fetal rat palate epithelium, with increased nuclear volume, decreased cytoplasmic and cellular volumes, decreased epithelial and keratin thicknesses, and increased cellular numerical density.

The synthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of a palatal shelf-elevating force in mammals. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various stages of palatogenesis. Hyaluronan, heparin sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. The distribution of the GAG differed in the anterior and posterior regions (i.e. the presumptive hard and soft palates) and with the stage of development. At the time of palatal-shelf reorientation, there were no significant differences for either the total amount of GAG or for the percentages of specific GAG types between anterior and posterior regions. Indeed, the most marked differences were detected at the stages of histogenesis after shelf elevation. Nevertheless, the results support the view that hyaluronan is involved in palatal-shelf reorientation, its percentage being initially high and decreasing after shelf elevation. No changes were detected for the other (sulphated) GAG during the time of elevation. The findings point to the need to correlate the events during histogenesis with changes in the ground substance of palatal-shelf mesenchyme and indicate that there are different developmental mechanisms within the presumptive hard and soft palates.

To provide information concerning biosynthetic activities within the mesenchymal cells of rat palatal shelves, the numbers of nucleolar organiser regions (NORs) were detected using an Ag-NOR staining technique within tissues obtained from coronal sections of 30 fetal heads prepared for light microscopy. The study involved comparisons between stages of palatogenesis and between location along the palatal shelves, anterior and posterior regions being analysed to correspond to the presumptive hard and soft palates. For both anterior and posterior regions, the mean Ag-NOR count/cell was initially high (approximately 3.4 anteriorly and approximately 3.0 posteriorly). The count/cell decreased significantly immediately after elevation but increased again after shelf reorientation and complete fusion. When comparisons were made between anterior and posterior regions of the palatal shelves, no differences were discerned at a stage prior to elevation (day 15) and a stage after the shelves had elevated and completely fused (day 17). Immediately post-elevation (day 16.5), however, the mean count/cell was significantly higher anteriorly. Thus the degree of mesenchymal cell activity, as expressed in terms of protein biosynthesis, can change in the developing palate according to both site and stage of development.

Much attention has been focused on the mechanisms responsible for the elevation and fusion of the palatal processes during formation of the secondary palate. In a former publication this closure, defined as fusion of the palatal shelves along their length, was described as occurring at a period in osseous palatal development characterized by incipient ossification of the maxillae and palatine bones, and at a stage in early mandibular development when the mandibular condyle has not developed. The purpose of the present study was to examine the osseous maturation of the cranial base at the time of palatal closure. The investigation was based on 62 human embryos and fetuses, CRL 22-57 mm, and general skeletal hand and foot maturation CNO = 0-0 to CNO = 8-1 (CNO, Composite Number of Ossified bones in hand and foot). From the fetuses, cranial tissue was dissected out and skeletal maturity was determined radiographically according to formerly described skeletal maturity stages in the cranial base. The study revealed that the soft tissue palatal shelves always elevate and fuse along their length at a specific, well-defined stage of maturity in the cranial base. The cranial base development in this stage is characterized by unossified cartilage. This information is essential for understanding abnormal skeletal development of the oral and nasal cavities.

Fetuses were obtained from CD-1 mice at a time estimated to be 12 h prior to vivo secondary palate closure. One of the palatal shelves of each partially dissected fetal head was lesioned in one of five ways, the other left intact to serve as control. Single transverse cuts extending the width of the shelf were made at one of three positions along the longitudinal axis of the shelf: one-third, one-half or two-thirds the shelf length estimated from the rostral edge. Some specimens were cut in two places, dividing the shelf into three equal segments. Another group received a lesion which separated the caudal third of the shelf from its maxillary connections. All specimens were cultured for 18 h. At the end of the culture period the heads were fixed, examined and the degree of elevation of each shelf piece assessed. Intact, control shelves of all preparations were elevated in the rostral two-thirds of the shelf, while the caudal third was partially elevated. Results seen in lesioned shelves depended upon both the size of the segment and the region of the shelf contained in the segment. The rostral two-thirds of the shelf, the presumptive hard palate, whether intact or in segments elevated without physical connections to neighboring shelf tissue. Thus, it is unlikely that this elevation requires a wave of contraction be transmitted from the caudal soft palate region. In contrast, the presumptive soft palate requires continuity with the rostral portions of the shelf both to maintain structural stability and to elevate.

Closure between the secondary palate and the primary palate and between the former and the nasal septum was studied in the Syrian golden hamster at light- and electron-microscopic level. Sequential changes in the epithelia before, during and after the closure of the primary and the secondary palate were described. A characteristic epithelial thickening was observed prior to epithelial fusion between the nasal septum and the secondary palatal shelf. Fusion of the opposing epithelia was characterized by formation of desmosomes. An unilateral or bilateral empty space was observed on each side of the epithelial seam, and it was suggested that this may represent the incisive foramen in the adult.

The intrinsic forces necessary for directing the reorientation of the secondary palate appear to reside in the anterior two thirds of the palate or presumptive hard palate. The hard palate could reorient regardless of whether it was intact or separated from the posterior third or presumptive soft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsic forces, within the hard palate, may be mediated by the mesenchymal cells, their extracellular matrix, or the epithelium surrounding the shelves. This latter possibly was tested by removing the epithelium, from either the presumptive oral or nasal surface followed by measurement of reorientation in vitro. Only after removal of the oral epithelium was a significant inhibition in reorientation observed. The treatment used to remove the epithelium, EDTA and scraping, was shown to remove 41% of the oral epithelium leaving the majority of the basement membrane intact. The observed inhibition of reorientation did not appear to be a consequence of wound healing. Creation of wounds twice the area that was observed after treatment with EDTA and scraping inhibited reorientation minimally. These results suggest that the epithelium and particularly the anterior oral epithelium plays a major role in the reorientation of the murine secondary palate.

Cell release from the palatal shelves and the fusion line prior to and during closure of the secondary palate was studied in Sprague-Dawley rat fetuses of a precise, individually determined age. Fetuses with normal and with experimentally retarded or failing palatal closure were examined by light- and electronmicroscopy. Three different phenomena were observed involving either locally restricted areas or the entire medial shelf edge: (1) desquamation of superficial cells, (2) extrusion of cell blebs, and (3) surface accumulation of basal cells. It is suggested, that desquamation is a process facilitating primary contact and fusion between undifferentiated cells, while extrusion of cell blebs probably represents degradation of the epithelial seam by cellular escape. Basal cells were seen at the surface of unfused shelf edges in fetuses with partial or total palatal clefting, as well as in fetuses with a normal but timewise delayed palatogenesis. It was assumed that, under normal conditions, epithelial surface desquamation is followed or accompanied by basal cell migration and that this may provide an additional mechanism assuring shelf contact between deep-seated cells.

A refined technique of amniotic sac puncturing at day 16.2 (i.e., 16 + 2/10 days) of gestation was employed in order to produce a series of total clefts and rare forms of partial clefts in Sprague-Dawley rat fetuses. From a total of 410 fetuses of a precise, individually determined age, 95 upper jaws were examined in the scanning electron microscope and, in part, in serial Epon sections. All fetal heads were examined macroscopically. Total clefts were found in 48.9% of a total of 184 viable rat fetuses examined at day 17.8 of smear age and in 21.8% of a total of 211 fetuses examined at day 19.3. Partial clefts were observed in 14.1% and 18.5% of fetuses at days 17.8 and 19.3 of smear age, respectively. At day 19.3, 16.1% of the viable fetuses showed a very inconspicuous, small abnormality (with residual clefting and incomplete fusion with the nasal septum) in the region of the palatine foraminae. Morphological observations suggested that under conditions of detained palatal closure (1) fusion of the soft palatal shelves commences independently from and prior to fusion of the hard palate, (2) delayed palatal shelf fusion proceeding in the anterior direction may occur with or without remaining sickle-shaped clefts in the anterior hard palate, and (3) in fetuses with small sickle-shaped clefts, fusion of the palatal shelves with the nasal septum does not occur. The present data imply that an almost total prenatal repair and delayed closure of the secondary palate may occur in rats that, at day 16.2 of multiple analysis age, most certainly had a total palatal cleft resulting from tongue resistance.

The structural relationships of the longitudinal fibromuscular component of the soft palate (musculus uvulae and raphe) were studied using histologic sections from 19 early human fetal specimens. Musculus uvulae arises in association with the palatine aponeurosis near the beginning of the second quadrant of the velum, follows a sigmoid course, and terminates near the base of the uvula. In addition, an occasional muscular loop may arise from the bony palate, arch downwards, and then recur into the uvular muscle. A complex relationship exists between the raphe in the velum and several palatal muscles. With regard to musculus uvulae, small muscular bundles arise from the raphe to embrace the muscle near its crest. These branches may aid in contouring the dorsal surface of the velum in the region of the levator eminence to complement the surface of the posterior pharyngeal wall and thus enhance the efficiency of the velopharyngeal seal.

The patterns of distribution of both total mesenchymal cells and the ratios of [3H]thymidine-labelled to total cells were mapped during secondary palatal shelf reorientation in vivo and in vitro. Smoothed spatial averaging, a computer-assisted method which takes into account the positions of all cells across an entire histological section of the shelf, was employed. Changes in shelf cross-sectional area and cell size were also measured. Three shelf regions, anterior and posterior presumptive hard and presumptive soft palate, were studied at developmental stages which were 30, 24 and 18 h prior to expected in vivo elevation, after in vivo reorientation and during the course of in vitro reorientation. Region-specific patterns of cell distribution change with shelf reorientation. These changes were observable within 6 h. Increases in cell number by cell division may enhance some high local cell densities, but cannot account for decreases in cell density. Increase in cell size is not a factor in decreasing cell density, nor is cell death. Displacement of cells by expansion of the extracellular matrix may be involved.

In order to enhance further knowledge of palatal shelf movement and the factors involved in palate closure, a method was developed for prematurely elevating palatal shelves in utero. Approximately 7 hours before expected shelf elevation, pregnant Sprague-Dawley rats were laparotomized and two medially directed squeezes were applied to the face of some of the embryos through the intact uterine wall. Palates from control (unsqueezed) and experimental animals were obtained immediately after the procedure and 2, 4, 7, 12 and 24 hours later. In the 0, 2 and 4 hours groups, 82% of experimental palates were elevated, whereas only 6% of control palates were elevated. At 0 hours only the hard palate in the experimental group had elevated, but at 2 and 4 hours almost half this group showed elevation of the soft palate as well, and, in addition, contact had been made between the elevated shelves.

Hyaluronate mediated extracellular matrix swelling has been hypothesized to play a major role in reorientation of the secondary palatal shelves. A computer-assisted method utilizing image registration and subtraction was used to visualize the distribution of hyaluronate (HA) during morphogenesis of the secondary palate. Patterns of HA distribution in anterior, posterior and presumptive soft palate were examined in the secondary palatal shelves of CD-1 mouse fetuses that were 30, 24 and 18 h prior to, and at the time of, shelf reorientation. Adjacent serial sections were taken from each shelf region of three to six specimens from a minimum of three litters for each gestational age. One section was incubated in buffer as a control, the other digested with Streptomyces hyaluronidase to specifically remove HA. Both sections were stained with Alcian blue to visualize the extracellular matrix and counterstained with nuclear fast red to visualize cells. Two different videoimages were then digitized for each tissue section, one using wavelengths of light that were at or near the maximum absorbance of the matrix stain, the other using wavelengths that were at the maximum absorbance of the cellular stain. Thus, a matrix image and a cell image of both control and digested sections were produced. Next, the cell image was subtracted from its respective matrix image, resulting in a control matrix-only image and a digested (HA-removed) matrix-only image. These images were mathematically warped to one another, if necessary, and registered with one another. The digested image was then subtracted from the control image. The resultant difference picture displayed the pattern and relative intensities of HA distribution across the tissue section. Prior to and during shelf reorientation, unique region-specific patterns of HA distribution and relative intensity were identified which became homogeneous after reorientation. Presumptive soft palate shows the most extensive and intense patterns of HA distribution, followed by the posterior region. The anterior region has the most sparse pattern of the three regions examined. The results are consistent with the hypothesized role of HA in shelf reorientation.

Normal palatogenesis was studied in the Sprague-Dawley rat, using fetuses of a precise, individually determined age (between 15 and 21 days of pregnancy) and scanning electron microscopy. Individual fetal age deviated markedly from gestational age determined by the vaginal smear method. Palatal closure, ie elevation and fusion of the anterior and remodeling of the middle zones as well as fusion of the posterior zone all occurred between 16.0 and 17.5 days of fetal age, ie earlier than in Wistar rat fetuses. Prior to fusion, the medial edges of palatal shelves revealed two distinct zones of initial surface alterations: one at the maximum anterior shelf convexity, the other at the caudal end of the posterior shelf portion. A similar zone was found at the free edge of the nasal septum. Subsequent oral aspects of palatogenesis including formation and fate of the midpalatal fusion line, the development and fusion of palatine rugae, the union between primary palate and the secondary palatal shelves, the development of palatine foraminae as well as the development of the soft palate following shelf closure are described in detail. It is suggested that (1) cobblestone appearance of surface cells reflects undifferentiated epithelial status rather than marks future fusion zones; (2) closure between primary and secondary palate involves two different mechanisms, ie furrow closure by growth lateral to and epithelial fusion between the palatine foraminae; and (3) fusion of the posterior shelf portions (soft palate) may occur independently from fusion in the anterior region.

Chlorcyclizine (CHLR) enhances the degradation of hyaluronate (HA) into smaller molecular weight pieces with no effect on its synthesis. Administration of CHLR to pregnant CD-1 mice on gestational days 10.5, 11.5 and 12.5 results in 100% cleft palate in the fetuses. The caudal two thirds of the palatal shelves are reduced in size and unable to reorient in vitro, while anterior shelf regions are relatively unaffected. Alcian blue staining combined with specific enzymic digestion was used to identify HA in sections of CHLR-treated shelves. With the aid of computer-assisted image subtraction the patterns of HA distribution across the tissue section were objectively identified. Anterior, posterior and presumptive soft palatal shelf regions were examined at gestational days 13.25, 13.5, 13.75 and 14.5. Acquisition of a normal pattern of HA distribution was delayed by about 24 h, as compared to untreated specimens in all three shelf regions. The posterior and soft regions, comprising the caudal two thirds of the shelf, also showed pronounced shape change. These regions only displayed normal curvature of the nasal surface when a normal pattern of HA distribution was attained. These results suggest that, for the caudal two thirds of the palatal shelf, normal shape and the ability to remodel are linked to the molecular configuration of HA and to a specific pattern of HA distribution.