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The morphology of M. palatoglossus in the 15-week fetus was studied with the aid of 30-micron serial sections. The sample included 26 specimens, 9 each of which were sectioned in the coronal and sagittal planes and 8 which were sectioned transversely. M. Palatoglossus extends within the anterior pillar to attachments in both the soft palate and the tongue. Characteristically, the muscle fibers radiate within the velum, not only along its antero-posterior axis but superiorly as well, to intersect other palatal structures. Within the root of the tongue, some fibers of M. palatoglossus combine in a longitudinal muscular complex to course anteriorly toward the tip, others appear to join the transverse muscular system. These data are compared with the literature describing the morphology of this musculature in the adult.

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.

Cellular aspects of vertical development of the secondary palate were examined in control and 6-mercaptopurine (6MP)-treated hamster embryos. Cross-sectional area of the palatal shelf was measured and the numbers of both epithelial and mesenchymal cells counted. Also, in 6MP-treated palates the damaged mesenchymal cells, characterized by the presence of dense bodies, were counted. DNA synthesis in both control and treated fetuses was measured by 3H-thymidine incorporation. The results indicated that both the shelf area and cell numbers increased with age in control and 6MP-treated palates. However, in controls the mesenchymal cell density and DNA synthesis showed two peaks that were absent following 6MP treatment. Unlike controls, in treated embryos the damage to mesenchymal cells became increasingly pronounced between days 10:00 and 10:12 but subsided by day 11:00 of gestation. It is suggested that a major force in the development of the initial primordia and early vertical development of the palatal shelf may be provided by a spurt of DNA synthesis in the mesenchymal cells resulting in their increased number. After 6MP treatment, depression of DNA synthesis and consequent reduction in the mesenchymal cell number and density followed by cell damage lead to retardation in the vertical development of the palatal shelves.

Although formation of the secondary palate is known to involve a complex sequence of developmental events, current concepts of palatal clefting emphasize alterations in the palatal shelves. The objective of this study was to identify similarities in facial growth and palatal formation in man and in rodent experimental models and to examine mechanisms of experimentally induced cleft palate that might be relevant to human clefting. Morphometric analyses of facial growth changes reveal similar patterns of mandibular prominence, head extension, and increased oronasal cavity vertical dimension during secondary palate development, with more pronounced changes in the human. Experimental studies of induced cleft palate in rats and mice show that interference with growth changes can contribute to cleft palate. Failure of palatal shelves to make contact, often associated with delayed horizontal movement, has been observed with increased tongue obstruction secondarily to mandibular retrognathia after either growth inhibition in Meckel's cartilage or morphologic deformation of Meckel's cartilage. In other experiments, failure of adequate shelf contact has been observed with reduced shelf growth or with altered craniofacial relations associated with abnormally flexed head posture resulting from fetal growth abnormalities or oligohydramnios. The results of these studies show that the etiology of cleft palate malformation can be related to interference with a number of different development events not immediately in the palatal shelves. Similar alterations of craniofacial growth that affect the palate secondarily appear to be associated with etiology of cleft palate in human syndromes such as Pierre Robin syndrome and the oligohydramnios syndrome.

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.

To define the interrelationships of the human levator and tensor veli palatini muscles and the Eustachian tube, fetal heads were serially sectioned and anatomic reconstruction done. Cephalometric points on fetal and adult skulls were compared to evaluate the effect of growth and development on these interrelationships. Based on the results of this study, we propose a mechanism for Eustachian tube function in the normal and in the cleft palate patient. This mechanism offers some explanations for many previously unexplained and paradoxical clinical observations.

Largely because fusion of the lip and the palate are developmental weak points, common facial clefts may arise in a great many ways, both experimentally and in man. However, we believe that the vast majority of human clefts have similar origins with minor variations. One must always question the appropriateness of animal models. Apparently appropriate animal models are giving us considerable insight into the manner in which genetic and environmental factors alter development and how they interact with one another in the developing embryo. These studies help us understand the nature of the multifactorial threshold concept as it applies to cleft lip and cleft palate, and they emphasize the potential importance of even "minor" environmental factors in determining on which side of the threshold for clefting an individual embryo may fall.

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.

The distribution of collagens during development of the palate in the Rabbit embryo is studied using the technique of indirect immunofluorescence. During the phase prior to epithelial contact, only types II and III are found in the mesenchyme. Type I, which is abundant on the free surface of apical epithelial cells, becomes even denser at the time when the palatine projections unite. Type IV fluorescence clearly outlines the basement membrane. When epithelial development regresses, fluorescence in type I and IV decreases in a way such that, in the areas where mesenchymal coalescence is apparent, no immunofluorescent labeling is observed.