Abstract
Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.
