Development of layer I of the human cerebral cortex after midgestation: architectonic findings, immunocytochemical identification of neurons and glia, and in situ labeling of apoptotic cells.

The development of layer I was studied in the human frontal cortex from 21 weeks of gestation (GW) to 2.5 postnatal months in series of adjacent sections processed for thionin staining, Bodian silver staining, and immunocytochemical labeling of neurons and glia. In addition, the terminal dUTP nick-end labeling (TUNEL) method was used to label in situ DNA fragmentation. A progressive decrease of cell density and the disappearance of the subpial granular layer (SGL) appeared as distinctive developmental features of human layer I, consistently with previous investigations. The neuronal antigen microtubule-associated protein2 was found to label preferentially Cajal-Retzius cells and dendritic processes extending from the cortical plate. At midgestation, the calcium binding protein calretinin stained in the marginal zone numerous neurons, including the Cajal-Retzius cells and their processes. Calretinin-immunoreactive neurons decreased during the subsequent maturation: such decline was abrupt in the SGL, whereas bipolar calretinin-immunopositive cells accumulated in the inner marginal zone to be presumably incorporated into the cortical plate. Cajal-Retzius cells expressed calretinin throughout the examined developmental stages. The glial antigen vimentin was already expressed at midgestation, and vimentin immunopositivity decreased progressively in cell bodies and fibers of layer I during development. Glial fibrillary acidic protein-positive elements gradually matured, and the positive cell bodies displayed the features of mature astrocytes at the end of gestation. Moreover, a decrease of free glial cells was observed in layer I, suggesting their progressive incorporation into the cortical plate. TUNEL-positive cells were detected at midgestation in the marginal zone, and they were concentrated in the SGL until its disappearance; their number decreased dramatically throughout layer I after 30 gestational weeks. TUNEL-positive nuclei or regressive changes were not detected in Cajal-Retzius cells throughout the examined developmental stages. Thus, our data point out that naturally occurring cell death is an active mechanism contributing to the disappearance of the SGL but not to the subsequent developmental reshaping of human layer I, in which, instead, migratory phenomena should play a major role. In addition, our findings argue against a disappearance of Cajal-Retzius cells due to regressive processes.