Daley William P, Gulfo Kathryn M, Sequeira Sharon J, Larsen Melinda
Graduate program in Molecular, Cellular, Developmental, and Neural Biology, Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, USA.
Dev Biol. 2009 Dec 15;336(2):169-82. doi: 10.1016/j.ydbio.2009.09.037. Epub 2009 Oct 3.
Cleft formation is the initial step in submandibular salivary gland (SMG) branching morphogenesis, and may result from localized actomyosin-mediated cellular contraction. Since ROCK regulates cytoskeletal contraction, we investigated the effects of ROCK inhibition on mouse SMG ex vivo organ cultures. Pharmacological inhibitors of ROCK, isoform-specific ROCK I but not ROCK II siRNAs, as well as inhibitors of myosin II activity stalled clefts at initiation. This finding implies the existence of a mechanochemical checkpoint regulating the transition of initiated clefts into progression-competent clefts. Downstream of the checkpoint, clefts are rendered competent through localized assembly of fibronectin promoted by ROCK I/myosin II. Cleft progression is primarily mediated by ROCK I/myosin II-stimulated cell proliferation with a contribution from cellular contraction. Furthermore, we demonstrate that FN assembly itself promotes epithelial proliferation and cleft progression in a ROCK-dependent manner. ROCK also stimulates a proliferation-independent negative feedback loop to prevent further cleft initiations. These results reveal that cleft initiation and progression are two physically and biochemically distinct processes.
裂沟形成是下颌下唾液腺（SMG）分支形态发生的起始步骤，可能由局部肌动球蛋白介导的细胞收缩引起。由于ROCK调节细胞骨架收缩，我们研究了ROCK抑制对小鼠SMG体外器官培养的影响。ROCK的药理学抑制剂、异构体特异性的ROCK I而非ROCK II的小干扰RNA（siRNAs），以及肌球蛋白II活性抑制剂均在起始阶段使裂沟停滞。这一发现意味着存在一个机械化学检查点，调节起始裂沟向具备进展能力的裂沟的转变。在该检查点的下游，裂沟通过由ROCK I/肌球蛋白II促进的纤连蛋白的局部组装而具备进展能力。裂沟进展主要由ROCK I/肌球蛋白II刺激的细胞增殖介导，细胞收缩也有一定作用。此外，我们证明纤连蛋白组装本身以ROCK依赖的方式促进上皮细胞增殖和裂沟进展。ROCK还刺激一个不依赖增殖的负反馈环以防止进一步的裂沟起始。这些结果表明裂沟起始和进展是两个在物理和生化方面截然不同的过程。
