Changes in the expression of extracellular matrix (ECM) and matrix metalloproteinases (MMP) of proliferating rat parotid acinar cells.

Tissue morphogenesis, development, and maintenance of function are mediated by signals generated through the composition of the extracellular matrix. The regulation of the composition of matrix is determined by enzymes specific for their degradation, the matrix metalloproteinases. Chronic injections of the beta-adrenergic receptor agonist, isoproterenol, result in a non-neoplastic hypertrophy and hyperplasia of the rat parotid gland. The activity of matrix metalloproteinases, as measured by gelatin zymography and enzymatic digestion of Azocoll substrates by gland lysates, decreased significantly (P < 0.05) following 24 hrs of agonist treatment, and slowly recovered to control values by 6 days of treatment. Daily administration of the broad-spectrum matrix metalloproteinase inhibitor Galardin for 3 days in combination with isoproterenol resulted in enhanced gland hypertrophy compared with that produced by isoproterenol alone. Given alone, Galardin also caused hypertrophy. The relative abundance of mRNA for the extracellular matrix molecules, collagens I and III and fibronectin, declined rapidly following the initiation of beta-agonist treatment in vivo, while laminin B1 and B2 mRNA levels increased initially before declining below control levels. These changes in patterns of mRNA levels also were observed in the concentrations of glandular protein when Western dot blot analysis of collagens I and III and laminin, respectively, was used. The importance of laminin, in vivo, was demonstrated by coinjection of anti-laminin antibody along with isoproterenol, which resulted in the inhibition of beta-agonist-induced parotid gland hypertrophy and hyperplasia. These data suggest that modulation of the ECM is associated with isoproterenol-induced salivary gland hypertrophy and hyperplasia. It is likely that this modulation of the ECM takes place through transcriptional regulation of some ECM genes and regulation of matrix-degrading enzyme activity.

The role of the mesenchyme in mouse neural fold elevation. I. Patterns of mesenchymal cell distribution and proliferation in embryos developing in vitro.

Using the computer-assisted method of smoothed spatial averaging, spatial and temporal patterns of cell distribution and mitotic activity were analyzed in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Total cell density increased in central and medial mesenchymal regions after 12 hr in culture, decreased after 18 hr, and showed a further decrease after 24 hr when the neural folds of the embryos had elevated, converged, and were fusing or fused. Mitotic activity, as measured by the ratio of 3H-thymidine-labeled cells to unlabeled cells, was highest in the central mesenchyme at all culture times. Embryos were also cultured in the presence of diazo-oxo-norleucine (DON), which inhibits glycosaminoglycan and glycoprotein synthesis. After 24 hr in culture, neural folds of DON-treated embryos had failed to elevate. Total cell density increased in central and medial regions of the mesenchyme of DON-treated folds at 12 hr but showed no significant decrease in these regions with further culture. Mitotic activity was highest in the central mesenchyme of these treated embryos. These results suggest that cell distribution patterns observed in the cranial mesenchyme during neural fold elevation in normal cultured embryos are not produced by regional differences in mitotic activity. Rather, we propose that cell distribution patterns in the central and medial regions of the mesenchyme result from expansion of a glycosaminoglycan-rich extracellular matrix that disperses cells from these regions and decreases their density. In DON-treated embryos, in which expansion of the mesenchyme is prohibited by the decreased glycosaminoglycan and glycoprotein content of the extracellular matrix, mitotic activity apparently determines these patterns.

The role of the mesenchyme in mouse neural fold elevation. II. Patterns of hyaluronate synthesis and distribution in embryos developing in vitro.

Hyaluronate (HA) distribution patterns were examined in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Using standard image-processing techniques, the digitized images of Alcian blue-stained or 3H-glucosamine-labeled sections digested with an enzyme specific for HA, were subtracted from adjacent, undigested sections. The resultant difference picture images (DPI) accurately depicted the distribution of stained or labeled HA within the cranial mesenchyme. 3H-glucosamine-labeled HA was distributed uniformly throughout the cranial mesenchyme as 12, 18, and 24 hr of culture. By contrast, the mesenchyme was uniformly stained with Alcian blue at 12 hr, but stain intensity decreased in the central regions of the mesenchyme at 18 and 24 hr. HA distribution patterns were also examined in the cranial mesenchyme of embryos cultured in the presence of diazo-oxo-norleucine (DON), a glutamine analogue that inhibits glycosaminoglycan and glycoprotein synthesis. In DON-treated mesenchyme, Alcian blue staining of HA was decreased from that in controls at 12, 18, and 24 hr. However, incorporation of 3H-glucosamine into HA was increased. The distribution of labeled HA within treated mesenchyme as 12, 18, and 24 hr resembled that in controls at 12 hr. These results indicate that the distribution of HA within the cranial mesenchyme of normal mouse embryos during neural fold elevation and convergence is not determined solely by regional differences in HA synthesis. We propose that HA distribution patterns result from the expansion of the HA-rich extracellular matrix of the central mesenchyme regions. This expansion may play a major role in fold elevation. These results also suggest that DON treatment reversibly inhibits HA synthesis, since treated mesenchymal cells retain the capability of synthesizing HA when provided with a glucosamine substrate. Patterns of 3H-glucosamine incorporation by DON-treated mesenchyme are similar to those observed in control mesenchyme prior to mesenchymal expansion at 12 hr.

Changes in mesenchymal cell and hyaluronate distribution correlate with in vivo elevation of the mouse mesencephalic neural folds.

The mesenchyme of the elevating mesencephalic neural folds of the mouse is composed primarily of mesenchymal cells embedded in an hyaluronate-rich extracellular matrix. In this study we provide evidence that hyaluronate and mesenchymal expansion may play a role in neural fold elevation and closure. Spatial and temporal patterns of mesenchymal cell and hyaluronate distribution were analyzed during neural fold elevation and closure using the computer-assisted method of smoothed spatial averaging and established methods of image processing. Degree of fold elevation and fold shape changes were analyzed using standard morphometric measures. The results of these analyses defined five distinct stages in mesencephalic neural fold elevation and closure. Mesenchymal cells and hyaluronate were found in a non-random distribution within the neural fold and showed distinct patterns of distribution which could be correlated with specific stages in neural fold elevation. The results of these analyses suggested that the elevation of the mesencephalic neural folds is produced by the expansion of an hyaluronate-rich extracellular matrix in the central mesenchyme which under the direction of surrounding tissues pushes the folds mediad towards the dorsal midline.

Effects of chlorcyclizine-induced glycosaminoglycan alterations on patterns of hyaluronate distribution during morphogenesis of the mouse secondary palate.

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.

Computer-assisted analysis of hyaluronate distribution during morphogenesis of the mouse secondary palate.

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.

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.

Cell distribution during mouse secondary palate closure. II. Mesenchymal cells.

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.

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.

Automatic segmentation and quantification of electron micrographs: extracellular components.

Extracellular glycosaminoglycans when precipitated by tannic acid, appear in electron micrographs as amorphous reticulate masses or fragments sometimes finely beaded and often associated with collagen fibrils. An algorithm for automatic classification, segmentation, and quantification of the amount of tannic acid-precipitable material (TAPM) and collagen in electron microscopic images is presented. Small patches of a region are initially located and the patch boundaries are traced using a binary contour tracing algorithm. The patches are then grown out and merged together to form one large area. This area is classified using a two-dimensional feature vector into one of two classes: a region with TAPM and collagen, or one with cell bodies and/or processes. Once these areas are classified and segmented, the distribution of TAPM is measured. The algorithm was tested on several TAPM images displaying varying amounts and configurations of TAPM with good results. It may also be adapted to process other electron microscopic images containing elements of interest which have complex or amorphous form.

Difference picture algorithms for the analysis of extracellular components of histological images.

A computer-assisted method for objectively identifying and displaying the distribution of molecules that can only be positively identified by a combination of staining characteristics and susceptibility to specific enzymatic digestion or chemical degradation is presented. The visual image of an enzymatically digested tissue section is subtracted from that of an adjacent buffer-incubated control section and the distribution of the extracellular molecules removed from the tissue section displayed. Photomicrographs are taken using white light and narrow bandwidth filters of wavelengths at or near the maximum absorbance for the dye products used to visualize the extracellular matrix and cells. Each negative is standardized using reference gray levels. The cell and matrix images of both digested and undigested sections are then registered. The locations of cells in both control and digested sections are identified and set to an undefined gray level value in the matrix images. The cell-removed images of the control and digested sections are then registered and the difference in gray levels between the two images calculated and displayed. The validity of results obtained is primarily dependent on the soundness of the histological visualization and digestion techniques used, but is independent of investigator interpretation.

Changes in cell distribution during mouse secondary palate closure in vivo and in vitro. I. Epithelial cells.

The distribution of epithelial cells around the perimeter of mouse secondary palatal shelves was observed before and after shelf reorientation in vivo and in vitro. Changes in shelf perimeter, cells per micrometer, and cell layering were measured for each of three shelf regions: anterior and posterior presumptive hard and presumptive soft palate at developmental stages which were 30, 24, and 18 hr prior to expected in vivo elevation, after in vivo elevation, and during the course of in vitro elevation. Pronounced increases in numerical cell density and cell layering accompanying shelf reorientation were noted in the superior nasal and mid-oral portions of the shelf perimeter in all three shelf regions with greatest changes noted in the posterior hard palate region. These changes were not attributable to cell division or to perimeter changes. The localized nature of the changes in cell distribution suggest that the underlying mechanisms may also be localized.

The effects of chlorcyclizine-induced alterations of glycosaminoglycans on mouse palatal shelf elevation in vivo and in vitro.

To define whether glycosaminoglycans play a role in palatal shelf movement, we studied the morphology and elevation behaviour of chlorcyclizine-treated mouse palatal shelves. Chlorcyclizine treatment was used because this agent enhances degradation of the palatal glycosaminoglycans, hyaluronate and chondroitin sulphates, with little or no effect on their synthesis. Use of in vitro and in vivo experiments enabled us to control the complicating effects of other factors on elevation. Drug-administration resulted in a reduction in shelf size, as measured by cross-sectional surface area, in the posterior two thirds of the palatal shelf. In vivo shelf reorientation was also inhibited. When elevation behaviour was observed in vitro, pronounced regional variation was noted. The anterior third of the shelf was able to reorient, the posterior two-third was not. This region also showed distinct histological changes as compared to controls. Mesenchymal cells were rounded with prominent nuclei and nucleoli and were more densely packed than in controls. These results suggest that for at least the posterior two-thirds of the palatal shelf, the intrinsic reorientation ability may in large part be linked to the acquisition of a specific temporal and spatial distribution of hyaluronate and possibly other matrix components.

An improved culture system for secondary palatal elevation.

An organ culture system devised for studying the development of the secondary palate was modified so that it retained high partial pressures of oxygen and supported total anterior and posterior palatal elevation. The cultured tissues appeared healthy as judged by histological examination. Medium was continuously recirculated through the culture system in which Day 13 embryonic mouse heads, with the brain and tongue removed, were totally submerged and suspended. The medium was constantly gassed via hollow fiber devices. A motor-driven stirrer, run at a low rate, agitated the medium so that the boundary layer surrounding the tissue was dispersed. Embryonic mouse heads were cultured in each of four media: Eagle's basal medium, Ham's F-12 medium, Fitton-Jackson's modified BGJb medium, and Waymouth's MB 752/1 medium. Elevation of the palate in both anterior and posterior regions with excellent tissue viability was achieved in all heads grown in BGJb medium.

Elevation of lesioned palatal shelves in vitro.

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.