We show here that Arhgap28 is differentially regulated during mouse embryonic development. The functions of Arhgap28 have not been reported but its differential expression has been listed in a variety of cDNA microarray studies, as summarized in Table S1. Based on amino acid sequence similarities Arhgap28 is closely related to Arhgap6. Some of these RhoGAPs have been shown to regulate actin reorganization. Knockout of DLC1 is embryonic lethal at E10.5 of mouse development and examination of fibroblasts isolated from E9.5 mouse embryos revealed disrupted stress fibers and focal adhesions. Mice lacking functional Arhgap6 protein are phenotypically normal, despite the fact that Arhgap6 is a RhoGAP for RhoA and causes the loss of actin stress fibers in cultured cells. Arhgap18 also has specificity for RhoA and disrupts actin stress fibers, where knockdown of Arhgap18 can enhance stress fiber formation. On the basis of these studies, we hypothesized that Arhgap28 regulates actin stress fiber assembly. To further explore the possibility that Arhgap28 is involved in the assembly of ECM, we examined the expression of Arhgap28 in a 3D cell culture model of tissue assembly in which embryonic fibroblasts deposit and tension a collagen fibril-rich ECM. In this system, fibroblasts are moved from conventional 2D culture to medium containing fibrinogen and thrombin. The formation of a fibrin gel occurs Troxipide within 5 minutes to produce a loose gel in which the fibroblasts find themselves suspended. The mechanical stiffness of musculoskeletal tissues is directly related to the organization of collagen fibrils in the ECM. For examples, the strongest tensile tissues such as tendon and ligament have collagen fibrils arranged in parallel register, which presumably is the best organization to resist uniaxial force; in bone the collagen fibrils provide a template for mineralization. Furthermore, the collagen fibrils are pre-stressed by cells to ensure that tissues can respond directly to applied Sulfacetamide Sodium forces. It is poorly understood how the tissue-specific arrangement and pre-stressing of collagen fibrils is achieved. Tissue stiffening requires cellular contraction via actin stress fibers, which are regulated by Rho GTPases.