Panicker Nikita, Coutman Melody, Lawlor-O'Neill Charley, Kahl Richard G S, Roselli Séverine, Verrills Nicole M
School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, Priority Research Centre for Cancer Research, Innovation and Translation, University of Newcastle, Callaghan, NSW, Australia.
Hunter Cancer Research Alliance, Hunter Medical Research Institute, New Lambton, NSW, Australia.
Front Cell Dev Biol. 2020 Jun 5;8:358. doi: 10.3389/fcell.2020.00358. eCollection 2020.
The serine/threonine protein phosphatase 2A (PP2A) is a master regulator of the complex cellular signaling that occurs during all stages of mammalian development. PP2A is composed of a catalytic, a structural, and regulatory subunit, for which there are multiple isoforms. The association of specific regulatory subunits determines substrate specificity and localization of phosphatase activity, however, the precise role of each regulatory subunit in development is not known. Here we report the generation of the first knockout mouse for the gene, encoding the PP2A-B55α regulatory subunit, using CRISPR/Cas9. Heterozygous animals developed and grew as normal, however, homozygous knockout mice were not viable. Analysis of embryos at different developmental stages found a normal Mendelian ratio of embryos at embryonic day (E) 10.5 (25%), but reduced embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live pups were observed at birth. embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in embryos were highly disorganized, with reduced immunolabeling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Thus PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis.
丝氨酸/苏氨酸蛋白磷酸酶2A（PP2A）是哺乳动物发育各阶段复杂细胞信号传导的主要调节因子。PP2A由一个催化亚基、一个结构亚基和多个调节亚基组成，存在多种亚型。特定调节亚基的结合决定了磷酸酶活性的底物特异性和定位，然而，每个调节亚基在发育中的精确作用尚不清楚。在此，我们报告了利用CRISPR/Cas9技术生成的首个编码PP2A-B55α调节亚基基因的敲除小鼠。杂合子动物发育和生长正常，但纯合敲除小鼠无法存活。对不同发育阶段胚胎的分析发现，在胚胎第10.5天（E10.5），胚胎的孟德尔比率正常（25%），但在E14.5时胚胎数量减少（18%），在E18.5时进一步减少（10%）。出生时未观察到存活的纯合子幼崽。纯合子胚胎明显小于野生型或杂合子同窝幼崽，并表现出多种神经缺陷，如无脑儿、脊柱裂和颅顶塌陷，以及并指和严重的表皮缺陷；所有这些过程均由外胚层的生长和分化驱动。纯合子胚胎的表皮屏障形成不完全，与薄的、分化不良的复层上皮有关，且与下方真皮的附着较弱。纯合子胚胎的基底角质形成细胞高度紊乱，整合素和基底膜蛋白的免疫标记减少，提示粘着斑和半桥粒组装受损。纯合子胚胎的棘层和颗粒层较薄，粘着连接和紧密连接相关蛋白表达异常。上方的角质层要么缺失要么不完整。因此，PP2A-B55α是表皮分层的重要调节因子，对胚胎发育过程中的外胚层发育至关重要。
