Abstract
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.
Highlights
The corneal endothelium is a fluid transporting epithelium that is essential for corneal hydration and transparency
The use of animals for this study was approved by the University Committee on Animal Care (UCAC) at East Tennessee State University (ETSU; Animal Use Protocol P190702) and was in compliance with the National Institutes of Health Guidelines for Care and Use of Animals in Research and the ARVO Statement for Use of Animals in Ophthalmic and Vision Research
Mice were housed in the Division of Laboratory Animal Resources at ETSU, a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC), and maintained in an environment of 12-h light/12-h dark
Summary
The corneal endothelium is a fluid transporting epithelium that is essential for corneal hydration and transparency. Despite the presence under certain circumstances of a corneal wound healing response, in which cell polyploidization and enlargement is triggered [15,16], this and other potential repair mechanisms are apparently insufficient to maintain tissue integrity under severe conditions In these cases, surgical intervention via transplantation is generally required to correct the resulting endothelial dysfunction [17,18,19]. In the normal corneal endothelium, tissue structure and function are largely dependent on the continued maintenance of cells generated during the developmental period For this reason, the mechanisms by which cell number is established and preserved are of fundamental importance. While expansion of p27CK− cells is almost three-fold greater than wild-type cells, that of p27-null cells is increased approximately six-fold These data indicate that p27 exerts its influence on cell division through both cell cycle-dependent and -independent mechanism and that, in both cases, the effect is cell-autonomous. Our evidence showing that the two mutants have opposite effects on cell dispersion within endothelial monolayers confirm previous work documenting the inhibitor’s involvement in cell migration and highlight the fact that p27 is able to regulate proliferation and migration in a coordinate fashion during development
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