The Transcriptional Regulator SnoN Promotes the Proliferation of Cerebellar Granule Neuron Precursors in the Postnatal Mouse Brain

Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional knockout of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal mouse brain. In laser capture microdissection followed by RNASeq, designed to profile gene expression specifically in the external granule layer (EGL) of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo. Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional knockout of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain. We deployed a laser captured microdissection (lcm) approach to focus our analyses of SnoN specifically in the EGL of the developing mouse cerebellum. The transcriptome of laser captured microdissected EGL from conditional SnoN knockout and control littermate mice were were measured using RNA-seq.神经元前体细胞（neuronal precursor cell）的增殖调控对于正常脑发育至关重要，而这一核心发育事件的失调会引发脑部疾病。通常而言，神经元前体细胞增殖在脑发育进程中持续较长时长，但神经元前体细胞增殖如何受时序特异性调控的机制仍有待阐明。本研究发现，在小鼠出生后脑发育的后期阶段，小脑颗粒神经元前体细胞（cerebellar granule neuron precursors）内转录调控因子SnoN的条件性敲除（conditional knockout）可显著抑制这些细胞的增殖，并促进其退出细胞周期。我们采用激光捕获显微切割（Laser Capture Microdissection, LCM）联合RNA测序（RNA-seq）的方法，针对小鼠小脑外颗粒层（External Granule Layer, EGL）特异性开展基因表达谱分析，结果显示，SnoN在活体状态下可促进颗粒神经元前体细胞中细胞增殖相关基因的表达，同时抑制分化相关基因的表达。值得注意的是，生物信息学分析表明，受SnoN调控的基因包含转录因子（transcription factor）N-myc与Pax6的结合位点，这两类转录因子分别可促进颗粒神经元前体细胞的增殖与分化。据此，我们揭示了SnoN与N-myc、Pax6在细胞内的新型物理相互作用。行为学分析显示，SnoN的条件性敲除会在延迟眨眼条件反射范式（delayed eye-blink conditioning paradigm）中损害小脑依赖性学习（cerebellar-dependent learning）能力，这表明SnoN对颗粒神经元前体细胞增殖的调控具有机体层面的功能意义。本研究明确了核心转录调控因子SnoN在哺乳动物大脑颗粒神经元前体细胞增殖调控中的新型功能与作用机制。我们通过激光捕获显微切割技术，将分析聚焦于发育中小鼠小脑的外颗粒层，利用RNA-seq检测了条件性SnoN敲除小鼠及其同窝对照小鼠的激光捕获显微切割外颗粒层组织的转录组。