RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1-4 genes in the developing cortex

Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure, Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here we report that a transcriptional factor, RP58, negatively regulates all 4 Id genes (Id1-Id4) in developing cerebral cortex. Consistently, Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild-type cortical progenitors. Furthermore, Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally, we determined p57 as an effector gene of RP58-Id-mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self-renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis. Sample RNAs were obtained from E16.5 Rp58 KO mouse cerebral cortex or control cortex. Two independent total RNA samples from each mouse strain were mixed and purified using the RNeasy Mini Kit (Qiagen). Oligonucleotide microarray analysis was performed using Panorama Micro Array gene expression chips, each containing approx 22000 probe sets (Sigma-Aldrich) according to the manufacturer's instructions.神经干细胞（NSCs）通过调控细胞周期退出及后续分化，可产生数量适宜的神经元与神经胶质细胞。尽管其调控机制尚不明确，但已知分化抑制因子（Id）家族蛋白可通过控制细胞周期，对NSCs的增殖发挥关键调控作用。本研究报道，转录因子RP58可在发育中的大脑皮层内负调控全部4种Id基因（Id1-Id4）。 与此一致的是，Rp58基因敲除（KO）小鼠出现了NSCs过度增殖伴随星形胶质细胞生成增强的表型。该表型可通过在野生型皮质祖细胞中过表达所有Id基因进行模拟。进一步研究发现，在突变型皮质祖细胞中敲低所有Id基因可挽救Rp58 KO小鼠的表型，但仅敲低单个Id基因则无法实现该挽救效果。最后，我们明确了p57是RP58-Id通路介导细胞命运调控的效应基因。上述研究结果确立了RP58作为一种全新的关键调控因子，其在大脑皮层发育过程中通过抑制所有Id基因，调控NSCs的自我更新与分化，并限制星形胶质细胞生成。 实验样本采集自E16.5时期的Rp58 KO小鼠大脑皮层及对照小鼠皮层。将每个品系小鼠的两份独立总RNA样品混合后，使用RNeasy迷你试剂盒（Qiagen）进行纯化。按照制造商说明书，使用包含约22000个探针组的Panorama微阵列基因表达芯片（Sigma-Aldrich）开展寡核苷酸微阵列分析。