lsotype During Chick Embryogenesis

Five P-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene cP4 and has been assigned to an isotypic family designated as Class I11 (PIII). In the nervous system of higher vertebrates, PI11 is synthesized exclusively by neurons. A PIII-specific monoclonal antibody was used to determine when during chick embryogenesis cP4 is expressed, the cellular localization of PHI, and the number of charge variants (isoforms) into which PI11 can be resolved by isoelectric focusing. On Western blots, PI11 is first detectable at stages 12-13. Thereafter, the relative abundance of PI11 in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of PI11 isoforms increases from one to three during neural development. This evidence indicates that PI11 is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while cp4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that cP4 expression occurs either during or immediately following terminal mitosis, and suggests that PI11 may have a unique role during early neuronal differentiation and neurite outgrowth.