The role of the mesenchyme in cranial neural fold elevation.

The basic aim of this research was to examine the role of the mesenchyme in cranial neural fold elevation. It has been previously postulated that the expansion of an hyaluronate-rich extracellular matrix in the fold mesenchyme is responsible for neural fold elevation. In this study we provide evidence that such expansion may play an important role in cranial neural fold elevation by pushing the folds towards the dorsal midline to assist in their elevation. For mesenchymal expansion to result in fold elevation, hyaluronate (HA) and mesenchymal cells must be non-r and omly distributed within the mesenchyme. Patterns of mesenchymal cell distribution and cell proliferation were analyzed using the computer-assisted method of smoothed spatial averaging. The distribution of Alcian blue-stained and $\\sp3$H-glucosamine-labelled HA was also analyzed during cranial neural fold elevation using established image processing techniques. Our results showed that mesenchymal cells and HA were found in a non-r and om distribution within the mesenchyme and showed distinct temporal and spatial patterns of distribution which could be correlated with stages of neural fold elevation. Elevation was accompanied by significant mesenchymal expansion and with decreased mesenchymal cell density and HA concentration in the central mesenchyme. Analysis of the distribution of $\\sp3$H-thymidine-labelled mesenchymal cells indicated that differential mitotic activity was not responsible for decreased mesenchymal cell density. Likewise, analysis of distribution patterns of $\\sp3$H-glucosamine-labelled HA indicated that decreased HA concentration was not produced by regional differences in HA synthesis. These results suggest that decreases in mesenchymal cell density and HA concentration that occur during neural fold elevation are produced by mesenchymal expansion. When mesenchymal expansion was inhibited by exposure to diazo-oxo-norleucine (DON) which interferes with HA synthesis, the cranial neural folds failed to elevate. Analysis of distribution patterns of labelled and unlabelled mesenchymal cells indicated that in treated folds regional differences in mesenchymal cell density were produced by differential mitotic rates. Analysis of Alcian blue-stained and $\\sp3$H-glucosamine-labelled HA distribution patterns showed that HA synthesis was decreased in treated folds. Patterns resembled those of control folds prior to mesenchymal expansion and fold elevation.Ph.D.MorphologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/162099/1/8907107.pd

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 chlorcylizine 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49691/1/1001760310_ftp.pd

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 3 H-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. 3 H-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 3 H-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 3 H-glucosamine incorporation by DON-treated mesenchyme are similar to those observed in control mesenchyme prior to mesenchymal expansion at 12 hr.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49697/1/1001880204_ftp.pd