ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity

Abstract

SUMMARYThe basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin 1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

Sp6 regulation of <i>Rock1 </i>promoter activity in dental epithelial cells

“…Rock1 plays pivotal roles in epithelial cell polarity by regulating PAR-1b activity and restricting basement membrane position (25), supporting that ROCK1 contributes to the polarity, proliferation, and differentiation of ameloblasts (9). In agreement with this, we found that Sp6 regulates ameloblast morphology and polarity by restoring polarization, alignment, and height of ameloblasts in Ami/Ami Tg rats with an Sp6 transgene (5).…”

Region‐specific epithelial cell dynamics during branching morphogenesis

“…3B and Movie 2). In fact, polarity of such peripheral cells in direct contact with the basement membrane has been described previously (Daley et al, 2012). The outer bud cells periodically left the basement membrane and moved towards the bud interior accompanied by loss of their elongated, columnar shape, particularly when undergoing cell division (Movie 3).…”

Genetic Modification and Recombination of Salivary Gland Organ Cultures

“…We recently used this adenoviral transfection/tissue recombination method to identify a role for the polarity protein, PAR-1b, in the control of the placement of basement membrane by the epithelium in developing salivary glands. Kinase-dead PAR-1b adenovirus and wild-type PAR-1b adenovirus were both used in this study 13 to infect epithelial rudiments and recombined with E13 mesenchymes. The ability to use mutant viruses to interrogate functions of specific molecules greatly enhances the utility of this method.…”

“…To directly assess effects of inhibitors on the epithelium, epithelial rudiments must be cultured in the absence of mesenchyme in either Matrigel or laminin-111 gels 6,7 . Small inhibitory RNAs (siRNAs) are an effective method to down-regulate epithelial gene expression since siRNAs are preferentially taken up by the epithelial cells in the presence of most lipid carriers 13,14,17,18 . However, neither of these methods for interfering with protein levels or function allow overexpression of a wild-type or mutant gene.…”

“…Unlike 4% PFA, 2% PFA will preserve a significant amount of the GFP signal if the fixative is completely removed after fixation. Glands can be stored for up to 1 week in 1X PBS in the dark at 4 °C until whole mount immunocytochemistry and confocal imaging is performed, as reported previously 3,6,[13][14][15] . Ideally, immunocytochemistry and imaging should be performed soon after fixation to obtain the best quality images.…”