During cerebellar development, granule cell progenitors (GCPs) proliferate exponentially for a fixed period, promoted by paracrine mitogenic factor Sonic Hedgehog (Shh) secreted from Purkinje cells (PCs). Dysregulation of Shh signaling leads to uncontrolled GCP proliferation and medulloblastoma. Serendipitously our previous work discovered insulin-like growth factor 1 (IGF1) as another key driver for medulloblastoma, which led to the current investigation into the role of IGF1 in GCPs during normal development. While the IGF1R conditional knockout model revealed GCP defects in anterior cerebellum, the posterior cerebellum was mostly intact, likely owing to incomplete excision of floxed alleles. To circumvent this hurdle, we enlisted a mouse genetic system called Mosaic Analysis of Double Markers (MADM), which sporadically generates homozygous null cells unequivocally labeled with GFP and their wildtype sibling cells labeled with RFP, enabling phenotypic analysis at single-cell resolution. Using MADM, we found that loss of IGF1R resulted in a 10-fold reduction of GCs in both anterior and posterior cerebellum; and that hindered S phase entry and increased cell cycle exit collectively led to this phenotype. Genetic interaction studies showed that IGF1 signaling prevents GCP cell cycle exit at least partially through suppressing the level of p27kip1, a negative regulator of cell cycle. Finally, we found that IGF1 is produced by PCs in a temporally regulated fashion: it is highly expressed early in development when GCPs proliferate exponentially, then gradually decline as GCPs commit to cell cycle exit. Taken together, our studies reveal IGF1 as a paracrine factor that positively regulates GCP cell cycle in cooperation with Shh, through dampening the level of p27 to prevent precocious cell cycle exit. Our work not only showcases the power of phenotypic analysis by the MADM system but also provides an excellent example of multi-factorial regulation of robust developmental programs.

在小脑发育过程中，由浦肯野细胞 (PC) 分泌的旁分泌促有丝分裂因子 Sonic Hedgehog (Shh) 促进颗粒细胞祖细胞 (GCP) 在固定时期内呈指数增殖。Shh 信号的失调导致不受控制的 GCP 增殖和成神经管细胞瘤。偶然地，我们之前的工作发现胰岛素样生长因子 1 (IGF1) 作为髓母细胞瘤的另一个关键驱动因素，这导致了目前对 IGF1 在正常发育过程中 GCP 中作用的研究。虽然 IGF1R 条件敲除模型显示小脑前部存在 GCP 缺陷，但小脑后部大部分完好，可能是由于 floxed 等位基因的不完全切除。为了绕过这个障碍，我们招募了一种称为双标记镶嵌分析 (MADM) 的小鼠遗传系统，它偶尔会产生明确标记有 GFP 的纯合空细胞及其标记有 RFP 的野生型同胞细胞，从而能够以单细胞分辨率进行表型分析。使用 MADM，我们发现 IGF1R 的缺失导致小脑前部和后部的 GC 减少 10 倍；并且阻碍 S 期进入和增加的细胞周期退出共同导致了这种表型。遗传相互作用研究表明，IGF1 信号传导至少部分通过抑制 p27kip1（细胞周期的负调节因子）的水平来阻止 GCP 细胞周期退出。最后，我们发现 IGF1 由 PC 以一种时间调节的方式产生：它在发育早期当 GCP 呈指数增殖时高度表达，然后随着 GCP 进入细胞周期退出而逐渐下降。综合起来，我们的研究表明 IGF1 作为一种旁分泌因子，与 Shh 合作积极调节 GCP 细胞周期，通过抑制 p27 的水平来防止细胞过早退出。我们的工作不仅展示了 MADM 系统表型分析的力量，而且还提供了一个很好的例子，说明了强大的发育程序的多因素调节。