A non-canonical role for p27Kip1 in restricting proliferation of corneal endothelial cells during development

The cell cycle regulator p27Kip1 is a critical factor controlling cell number in many lineages. While its anti-proliferative effects are well-established, the extent to which this is a result of its function as a cyclin-dependent kinase (CDK) inhibitor or through other known molecular interactions is not clear. To genetically dissect its role in the developing corneal endothelium, we examined mice harboring two loss-of-function alleles, a null allele (p27−) that abrogates all protein function and a knockin allele (p27CK−) that targets only its interaction with cyclins and CDKs. Whole-animal mutants, in which all cells are either homozygous knockout or knockin, exhibit identical proliferative increases (~0.6-fold) compared with wild-type tissues. On the other hand, use of mosaic analysis with double markers (MADM) to produce infrequently-occurring clones of wild-type and mutant cells within the same tissue environment uncovers a roughly three- and six-fold expansion of individual p27CK−/CK− and p27−/− cells, respectively. Mosaicism also reveals distinct migration phenotypes, with p27−/− cells being highly restricted to their site of production and p27CK−/CK− cells more widely scattered within the endothelium. Using a density-based clustering algorithm to quantify dispersal of MADM-generated clones, a four-fold difference in aggregation is seen between the two types of mutant cells. Overall, our analysis reveals that, in developing mouse corneal endothelium, p27 regulates cell number by acting cell autonomously, both through its interactions with cyclins and CDKs and through a cyclin-CDK-independent mechanism(s). Combined with its parallel influence on cell motility, it constitutes a potent multi-functional effector mechanism with major impact on tissue organization.细胞周期调控因子p27Kip1是调控多种谱系细胞数量的关键因子。尽管其抗增殖作用已得到广泛证实，但这一效应究竟源于其作为细胞周期蛋白依赖性激酶（cyclin-dependent kinase, CDK）抑制剂的功能，还是通过其他已知的分子相互作用，目前仍不明确。为从遗传学角度解析其在发育中小鼠角膜内皮中的作用，我们对携带两种功能缺失等位基因的小鼠开展了研究：一种是可完全消除蛋白功能的无效等位基因（null allele，p27⁻），另一种是仅靶向其与细胞周期蛋白及CDK相互作用的敲入等位基因（knockin allele，p27CK⁻）。全动物突变体（即所有细胞均为纯合敲除或纯合敲入）与野生型组织相比，增殖水平均出现约0.6倍的相同程度升高。与之相反，利用双标记嵌合分析（mosaic analysis with double markers, MADM）在同一组织环境中生成罕见的野生型与突变体细胞克隆时，我们发现单个p27CK⁻/CK⁻和p27⁻/⁻细胞的扩增倍数分别约为3倍和6倍。嵌合分析还揭示了不同的迁移表型：p27⁻/⁻细胞高度局限于其产生部位，而p27CK⁻/CK⁻细胞则更广泛地分散在内皮组织中。通过基于密度的聚类算法定量分析MADM生成克隆的扩散情况，我们观察到两种突变细胞的聚集程度存在4倍差异。综上，我们的分析表明，在发育中的小鼠角膜内皮中，p27通过细胞自主方式调控细胞数量：一是通过与细胞周期蛋白及CDK的相互作用，二是通过不依赖细胞周期蛋白-CDK的机制。结合其对细胞运动的并行影响，p27构成了一种多功能的强效效应机制，对组织构建具有重要影响。