Pax6 is required for normal cell cycle exit and the differentiation kinetics of retinal progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 was implicated in both proliferation and differentiation of multiple regions within the CNS, its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6- retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6- precursors. These alterations were accompanied by co-expression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27kip1 and P27kip2) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks which function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages. The data contains 3 control and 3 Pax6 negative samples representing biological repeats. Each sample was prepared from ~1.8 million cells taken from the distal retina of either E12 alpha-Cre or Pax6 floxed alpha-Cre animals. Distally located retinal progenitors expressing Cre and eGFP were separated using FACS.细胞周期退出与分化启动之间的耦合作用是发育神经系统中的普遍特征，但连接这两个过程的分子机制大多尚未阐明。尽管转录因子配对盒基因6（Pax6）已被证实参与中枢神经系统（CNS）多个区域的增殖与分化过程，但其在这两个连续状态间转换的具体作用仍不明确。为阐明Pax6在增殖性祖细胞向分化性前体细胞转换过程中的作用，我们对Pax6缺陷型视网膜祖细胞（RPCs）中的细胞周期变化与转录组改变进行了研究。我们的分析揭示了Pax6缺陷型RPCs独特的细胞周期表型：包括S期细胞数量减少、退出细胞周期的细胞数量增多，以及Pax6缺陷型前体细胞的分化动力学延迟。这些改变伴随有促进细胞周期的因子（Ccnd1、Ccnd2、Ccnd3）与抑制细胞周期的因子（P27kip1、P27kip2）的共表达。进一步对Pax6缺陷型RPCs的转录谱变化进行表征后发现，多种已知参与调控RPCs增殖的因子的表达被消除，其中包括转录因子Vsx2、Nr2e1、Plagl1以及Hedgehog信号通路相关分子。本研究为阐明Pax6在RPCs中多效性活性的分子机制提供了新的见解。研究结果进一步表明，Pax6并非仅对RPCs发挥线性调控作用（例如促进其增殖、抑制其分化），而是同时调控多条转录网络，从而赋予RPCs增殖、获得多种细胞命运以及执行视网膜谱系分化程序的能力。本数据集包含3份对照样本与3份Pax6阴性样本，均为生物学重复样本。每份样本均取自约180万个细胞，这些细胞分离自E12期α-Cre或Pax6 floxed α-Cre小鼠的远端视网膜。通过荧光激活细胞分选术（FACS）分离表达Cre与增强绿色荧光蛋白（eGFP）的远端视网膜祖细胞。