The proposed experiments seek to develop a Molecular Map of (1) the G1 cyclins E and D (and the S phase cyclin A), (2) the kinase activity of cdk2-cyclin complexes, and (3) the phosphorylation state of the retinoblastoma protein (Rb) with respect to the cell cycle of the proliferative murine neocortical pseudostratified ventricular epithelium (PVE). The map will be a starting point for formulating and experimentally testing hypotheses relating to histogenetic mechanisms of PVE proliferative behavior. The molecular map will be comparative for the PVE at embryonic days 12 and 15 (E12 and E15) which are near the outset and the termination, respectively, of the period of neocorticaI cytogenesis. The map will be based upon molecular analyses of tissue lysates, immunocytochemistry and quantitative in situ hybridization of tissue from the intact embryo. Cdk2-cyclin complex kinase activity in other proliferative cell systems regulates G1 progression and the G1/S phase transition. Rb in hypophosphorylated state is thought to restrain cells in G1 phase. Our studies in the present funding period have established that the rate of cell production accelerates with progression through the 10 cell cycles that contribute to formation of the murine neocortex. The length of G1 (T-G1) is the only variable phase of the cell cycle and increases 4x between E12 and E15. The emphasis upon TG1 for the proposed experiments is motivated by the hypothesis that TG1 and variation in the rate of cell production are under histogenetic regulation and that the regulatory mechanisms will act via cdk2-cyclin modulations. We propose that mechanisms regulating TG1 could act via modulations in the rate of cyclin synthesis, the latency before synthesis is initiated, or the G1/S phase triggering threshold. In additional experiments with E14 cerebral explants we will seek to modulate the rate of G1 progression by added growth factors: to be increased with beta-FGF or VIP; decreased with TGF- beta. Associated variations in the parameters of cdk2-cyclin complex kinase activity or the phosphorylation state of Rb should reinforce those observed with TG1 prolongation between E12 -E15 in vivo.
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