Time-resolved function of cell polarity kinases PRKCZ and PRKCI in CNS myelination [snrna_oligo]

The atypical Protein Kinase C (aPKC) is a critical component of the PAR polarity complex, as well as of cell polarity in general. There are two paralogs of aPKC in the mammalian genome. Their expression pattern and functional role in oligodendrocyte lineage differentiation and development remains heretofore uncharacterized. Here we show that both paralogs of aPKC – PRKCI, the primary transcript of Prkci and PKMz, an alternate transcript of Prkcz – are expressed during oligodendrocyte differentiation, yet are functionally non-redundant. Asymmetric cell division and early specification of the oligodendrocyte fate stage required both paralogs. By contrast, the stage of oligodendrocyte maturation involving membrane polarization and myelination of axons continued to require PKMz, but no longer needed PRKCI. Taken together, our results indicate to a two-stage role of aPKCs and cell polarity in oligodendrocyte differentiation and development. Overall design: To understand the effects of aPKC paralogs in OL lineage development, we performed single nucleus (sn)RNA-seq of cerebral cortices from Control and Prkci f/f and Prkcz f/f mice crossed with Ng2-cre+ or Plp1-creERT+ at various time points, using the 10X Genomics platform (3-4 biological replicates for each genotype) and injected some of them with 4-OH Tamoxfien followed by cuprizone-mediated demyelination.

非典型蛋白激酶C（atypical Protein Kinase C, aPKC）是PAR极性复合物（PAR polarity complex）的核心组分，亦是维持细胞极性的关键分子。哺乳动物基因组中存在aPKC的两个旁系同源基因（paralogs），二者在少突胶质细胞（oligodendrocyte）谱系分化与发育过程中的表达模式及功能角色迄今仍未被阐明。本研究证实，aPKC的两个旁系同源基因——Prkci的主要转录本PRKCI，以及Prkcz的可变剪接转录本PKMz——均在少突胶质细胞分化阶段表达，但二者功能并不冗余。少突胶质细胞命运决定早期的不对称细胞分裂与特化阶段，需要两个同源基因共同发挥作用；而在涉及细胞膜极性建立与轴突髓鞘化的少突胶质细胞成熟阶段，则仅需PKMz发挥功能，不再依赖PRKCI。综上，本研究结果表明，aPKC介导的细胞极性在少突胶质细胞分化与发育中承担双阶段调控作用。实验设计概述：为解析aPKC旁系同源基因在少突胶质细胞谱系发育中的功能效应，本研究采用10X基因组学平台（10X Genomics），对不同时间点下，Ng2-cre+或Plp1-creERT+分别与Prkci^f/f、Prkcz^f/f小鼠杂交获得的对照组及基因敲除小鼠的大脑皮层开展单细胞核RNA测序（single nucleus RNA-seq, snRNA-seq），每组设置3-4个生物学重复；同时对部分小鼠注射给予4-羟基他莫昔芬（4-OH Tamoxifen），随后通过铜螯合剂（cuprizone）诱导脱髓鞘模型。