Expression of unphosphorylated class III beta-tubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation

Neuronal microtubules have unique stability properties achieved through dev elopmental regulation at the expression and posttranslational levels on tub ulins and microtubule associated proteins. One of the most specialized tubu lins specific for neurons is class-III beta-tubulin (also known as beta 6-t ubulin), Both the upregulation and the post-translational processing of cla ss-III beta-tubulin are believed to be essential throughout neuronal differ entiation. The present investigation documents the temporal and spatial pat terns of class-III beta-tubulin expression throughout neurogenesis. For thi s study a novel polyclonal antiserum named U-beta 6, specific to unphosphor ylated class-III beta-tubulin has been developed, characterized and compare d with its commercial homologue TuJ-1. Our experiments indicate that the tw o antibodies recognize different forms of class-ill P-tubulin both in vitro and in vivo. Biochemical data revealed that U-beta 6 bound unphosphorylate d soluble class-III beta-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-beta 6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin an d vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differ entiating neurons. These studies with U-beta 6 illustrate three main develo pmental steps in the neuronal lineage: the commitment of neuroepithelial ce lls to the lineage (U-beta 6 +ve/TuJ-1 -ve cells); a differentiation stage (U-beta 6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlati ng with a drop in unphosphorylated class-III beta-tubulin immunostaining le vels. These investigations also conclude that U-beta 6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest n euronal lineage marker known so far.