Role of cell cycle dynamics in early cell fate decisions of interneuron precursors in the MGE

抑制性中间神经元是一类数量虽小但却十分重要的神经元，它们在功能、形态和放电模式上表现出极大的多样性。事实上，各种人类病理学，如癫痫、自闭症谱系障碍或精神分裂症，都与中间神经元功能障碍有关。两个主要的中间神经元亚群，小白蛋白（PV）或生长抑素（SST）的表达分类，起源于内侧神经节隆起（MGE）的瞬态结构。细胞成为PV或SST中间神经元的命运决定在早期启动，在这个项目中，我的目标是阐明PV与SST命运决定的分子程序，并探索细胞周期和分裂模式的关键调节因子如何对中间神经元特化起作用。我将使用单细胞RNA测序技术，（scRNAseq）来研究在突变小鼠系的辅助下在胚胎（E）天E11.5、E13.5和E15.5指导MGE祖细胞分化的分子程序。分区缺陷3（Pard 3cKO）的MGE中的条件性敲除增加了成人前脑中的SST中间神经元，而细胞周期蛋白D2敲除（cD 2KO）显示PV中间神经元密度降低。在E13.5的先导scRNAseq实验显示，在野生型（WT）MGE中，不同的细胞类型可以被稳健地鉴定并分类为9个细胞簇，代表放射状神经胶质细胞（RGC）、中间祖细胞（IPC）和早期神经元。与WT相比，cD 2KO小鼠缺乏IPC簇，支持先前的文献表明PV中间神经元起源于室下区的IPC。在Pard 3cKO MGE中，RGC簇缺失，表明在E13.5，祖细胞过早停止分裂以采用SST身份，与SST神经元直接从RGC产生的数据一致。来自E11.5和E15.5的数据将测试这些遗传操作是否改变了MGE的中间神经元输出的时间，并将有助于确定关键的命运determiners.Previous研究表明，在皮层不对称与对称分裂是调节胚胎生发区神经发生和细胞数量输出的关键。然而，很少有人知道这些分裂模式如何在分子水平上驱动细胞周期。PARD 3促进RGCs的不对称分裂，而CD 2主要在IPC中表达，这表明它通过一种未知的机制促进对称分裂。与cD 2相反，cD 1仅在分裂的RGC中表达，表明cD 1对不对称分裂很重要。令人兴奋的试验数据显示PARD 3与cD 1或cD 2的物理相互作用具有重要差异。为了探索这些生化差异的功能，我将研究PARD 3和cD 2的双重失活的影响，PARD 3-cD 1相比，MGE细胞周期特征在不同的胚胎年龄，并确定PV与SST细胞的命运决定结果在成人前脑。

Inhibitory interneurons are a small but crucial neuronal population, which show a great diversity in function, morphology and firing pattern. Indeed, diverse human pathologies like epilepsy, autism spectrum disorders or schizophrenia are associated with interneuron dysfunctions. Two major interneuron subpopulations, classified by the expression of Parvalbumin (PV) or Somatostatin (SST), originate from the transient structure of the medial ganglionic eminence (MGE). The cell fate decision to become a PV or SST interneuron is initiated early, in MGE mitotic progenitors whose specification may be influenced by regulators of cell divisions.In this project I aim to elucidate the molecular program underlying the PV vs. SST fate decision and probe how key regulators of the cell cycle and division mode act toward interneuron specification.I will use single cell RNA sequencing (scRNAseq) to investigate molecular programs directing MGE progenitor differentiation at embryonic (E) days E11.5, E13.5, and E15.5, assisted by mutant mouse lines. The conditional knockout in MGE of partition defective 3 (Pard3cKO) increases SST interneurons in the adult forebrain, while the cyclin D2 knockout (cD2KO) shows decreased PV interneuron densities. Pilot scRNAseq experiments at E13.5 show that different cell types could be robustly identified and classified into nine cell clusters in the wildtype (WT) MGE, representing radial glia cells (RGCs), intermediate progenitor cells (IPCs), and early neurons. In contrast to WT, the cD2KO mice lack an IPC cluster, supporting previous literature suggesting PV interneurons originate in the IPCs of the subventricular zone. In Pard3cKO MGE an RGC cluster is missing, suggesting that at E13.5, progenitors prematurely stop dividing to adopt an SST identity, consistent with data that SST neurons arise directly from RGCs. Data from E11.5 and E15.5 will test whether these genetic manipulations shift the timing of interneuron output from the MGE and will help identify key fate determinants.Previous studies in cortex established that asymmetric vs. symmetric divisions are key to regulation of neurogenesis and cell number output from embryonic germinal zones. However, little is known of how these division modes are connected to driving the cell cycle at the molecular level. PARD3 facilitates asymmetric divisions of RGCs, while cD2 expression mostly in IPCs suggests that it promotes symmetric divisions through an as yet unknown mechanism. In contrast to cD2, cD1 is only expressed in dividing RGCs, suggesting cD1 is important for asymmetric divisions. Exciting pilot data show PARD3 to physically interact with cD1 or cD2 with important differences. To probe the function of these biochemical differences, I will examine the impact of dual inactivation of PARD3 and cD2, compared to PARD3-cD1, on MGE cell cycle characteristics at different embryonic ages and determine the PV vs. SST cell fate decision outcomes in the adult forebrain.