Interference with the development of early generated neocortex results in disruption of radial glia and abnormal formation of neocortical layers

Early generated layers of neocortex are important factors in forming the su bsequent architecture of the cerebral cortex. To further explore the role o f early generated cortex, we disrupted formation of an early generated coho rt of cells by intraperitoneal injections of the mitotic inhibitor methylaz oxymethanol (MAM) into pregnant ferrets timed to coincide with generation o f subplate neurons in the ventricular zone. Our studies demonstrate that if early development of the neocortex is interrupted by injection of MAM duri ng embryogenesis ton embryonic day 24 or 28; E24 or E28), a distinct lamina r pattern fails to form properly in the parietal cortex. A reduced number o f MAP2-positive cells were observed in the region of the subplate when comp ared with the number of MAP2-positive cells found in normal animals. Interf erence with the superficial neocortical layers that form later during devel opment ton embryonic day 33) by appropriately timed MAM injections does not result in a severely disrupted laminar pattern. The interrupted laminar pa ttern that arises after early MAM injections coincides with distorted radia l glial cells (identified by immunoreactivity to the intermediate filament protein, vimentin), which occur after early, but not late, MAM injections. Further analysis suggests that interference with early development of neoco rtex leads to premature differentiation of radial glial cells into astrocyt es, as demonstrated by the presence of glial fibrillary acidic protein (GFA P). Experiments involving injections of the thymidine analog, bromodeoxyuri dine (BRDU), demonstrated that 4 days after E24 MAM injection cells are gen erated and migrate into the thin cortical plate. By E38, however, cells con tinue to be generated in animals treated with MAM on E24 but do not reach t heir normal positions in the cortical plate. In addition, immunoreactivity using the CR50 antibody, which identifies presumptive Cajal-Retzius cells p resent in layer 1, demonstrates that the CR50-positive cells, normally prec isely located in the outer portion of layer 1, are distributed in disarray throughout the thickness of the neocortex and intermediate zone in early MA M-treated animals, but not in those treated with MAM injections later durin g gestation. These findings are consistent with the idea that early generat ed layers are important in providing factors that maintain the environment necessary for subsequent neuronal migration and formation of neocortical la yers.