Abstract. The patterns of deposition of thrombospondin (TSP), a trimeric extracellular matrix glycoprotein, were determined during the initial establishment of the external granule cell layer and the subsequent inward migration of granule cells forming the molecular and (internal) granule cell layers. The early homogeneous deposition of TSP became restricted to the rhombic lip in the region of granule cell exit from the neuroepithelium, and was present between migrating granule cells.During the later inward migration of granule cells, little TSP was associated with dividing granule cells; it was enriched in premigratory granule cells. With the cessation of migration, TSP was lost except in association with fasciculating axons in the molecular layer where staining persisted briefly. At the EM level, TSP was associated with the leading process of granule cells as they associated with Bergmann glial cells and migrated through the molecular layer. TSP was present within granule cell axons; Purkinje cells and their dendrites, as well as Bergmann glial fibers and endfeet were negative for TSP.When anti-TSP antibodies were added to explant cultures of cerebellar cortex during active granule cell migration, a dose-dependent inhibition of migration was observed. In control cultures, granule cells migrated into the (internal) granule cell layer, while granule cells exposed to anti-TSP antibodies were arrested within the external granule cell layer. These resuits suggest that TSP plays an important role in the histogenesis of the cerebellar cortex by influencing granule cell migration.
The pattern of deposition and the role of laminin, a major glycoprotein constituent of basement membranes, were investigated during lung morphogenesis in the fetal mouse. Lung primordia were removed from Day 13 embryos, right lower lobes were further dissected and placed in filter membrane assemblies. Explants were then cultured at the liquid-air interface for 3 days in the presence of anti-laminin, anti-thrombospondin (another extracellular matrix constituent), preimmune serum, laminin-neutralized anti-laminin, or medium alone. Cultures were monitored by (direct) phase-contrast microscopy, light microscopy, and immunofluorescence. We found that anti-laminin antibodies altered normal lung morphogenesis in a dose-dependent manner. The anti-laminin-treated explants presented a marked inhibition of branching morphogenesis and a distortion of the bronchial tree. A lower rate of growth was also observed in the explants exposed to this antibody. High concentrations of anti-thrombospondin antibodies, normal rabbit serum, or laminin-neutralized anti-laminin antibodies had no effect on lung morphogenesis. These results were not modified by culturing the explants in submersion culture or on Vitrogen 100-coated surfaces.
The neuroepithelial basal lamina (BL) appears to be crucial in controlling cell-cell interactions during the early histogenesis of the nervous system. In this investigation we examined the changes in the BL in a neurological mutant of the rat previously characterized as having BL anomalies which progress to aqueductal stenosis and prenatal hydrocephalus. Embryos were obtained from matings of rats homozygous for the prenatal hydrocephalus gene or from controls, both originally derived from the same Wistar albino stock. On days 12 and 13 of development, embryos were processed for indirect immunofluorescence localization of BL components type IV collagen or laminin. Additional whole litters were processed for ultrastructural analysis of neuroepithelial and BL morphology. In control embryos, neuroepithelial BL components formed a smooth linear boundary to the basal surface of the neuroepithelium. This unbroken border was interrupted only in regions of active neural crest cell migration (day 12), and in areas of imminent vascularization (day 13). In hydrocephalic embryos on the 12th day there were gaps in the continuity of the BL and an apparent reduction in deposition of type IV collagen. By day 13, blood vessels had prematurely colonized the neuroepithelium, few BL breaks were observed, and deposition of type IV collagen appeared similar to that seen in control embryos. Ultrastructurally, a similar pattern of change was observed. The neuroepithelium of control embryos was uniformly bounded by an organized BL consisting of a lamina lucida subjacent to the plasma membrane, and a lamina densa which merged with scattered collagen fibrils in the mesenchymal compartment. On day 12, the BL of hydrocephalic embryos had large gaps through which neuroepithelial cells projected.(ABSTRACT TRUNCATED AT 250 WORDS)
This investigation was carried out to determine the early structural abnormalities of the cephalic region in a genetic mutant of the rat characterized by prenatal aqueductal stenosis and hydrocephalus. The appearance of hydrocephalic and control embryos was examined on days 13-15 of gestation, and the structure and organization of the neuroepithelium and basal lamina were studied using scanning electron microscopy. In addition to some overall developmental delay, hydrocephalic embryos were characterized by abnormalities of forebrain and midbrain development, and eye and external ear anomalies. There were also associated defects of the midfacial region. The lateral cell surface of the neuroepithelium reflected the developmental delay of hydrocephalic embryos, and failed to undergo the morphogenetic cell-shaping changes seen in control embryos. There were also variations in the number of lateral cell-cell specializations as well as regions of neuroepithelial disorganization and occasional herniation into the mesenchymal compartment. The role of the neuroepithelial basal lamina and extracellular matrix in the development of these defects is considered.
In order to examine the deposition of basal lamina components in the developing neuroepithelium, a technique for frozen thin sectioning and immunogold staining of early embryonic tissue was developed. Different fixatives and buffer systems were evaluated to determine which best retained immunoreactivity and satisfactory ultrastructure of day 9 and 10 mouse embryos. Fixation in sodium phosphate and sodium bicarbonate buffers did not retain antigenicity, and incubations in TBS (trishydroxymethyl-aminomethane buffered saline) in an effort to 'restore' immunoreactivity were similarly unsuccessful. Fixation in sodium cacodylate buffer, however, did retain the antigenicity of basal lamina components; the pattern of type IV collagen and laminin distribution was clearly determined. These results represent the first report of on-grid immunocytochemistry of early embryonic material.
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