(b) Fluorescence staining of F-actin with rhodamine-phalloidin following treatment of E12.5 SMGs with Y-27632 for 24?h revealed interrupted and condensed F-actin organization (? ) with an accompanying loss of extending ductal DNMT1 paths and formation of structures resembling micro-lumens (?). Acetone/methanol-fixed tissues prior to immunofluorescence staining revealed that a single bud with three clefts displayed asymmetric distribution of ZO-1 which marked future paths for duct extension. (c) Immunofluorescence of paraformaldehyde-fixed E13.5 SMG explants showed that distal regions of buds, defined by the newly formed clefts, displayed ductal paths enriched for F-actin and ZO-1 (Size bars, 20?m. (d) Schematic representation of K5?+?cells with ZO-1 asymmetries in the developing ductal regions of the salivary gland in (aCd, red, green). Results show representative images of 5 experiments (n?=?10/experiment). ZO-1 is required for ductal extension into the bud To confirm the early role of ZO-1 in setting up cellular asymmetries for the formation of new ductal structures, we inhibited ZO-1 expression with siRNA in E12.5 SMGs and examined the consequences on duct extension after 24?h in culture. Quantitative RT-PCR revealed a 90% knockdown of ZO-1 mRNA levels in siRNA-treated glands compared to controls (Fig.?3a, bar graph). This was confirmed by a statistically significant loss of ZO-1 labeling by immunofluorescence (Fig.?3a, arrow). SMGs treated with ZO-1 siRNA at E12.5 exhibited morphological aberrations within 24?h involving a flattening of the proximal duct-forming region with shallow initial clefts (Fig.?3b and schematic Fig.?3b), indicating perturbation of the branching process in the absence of ZO-1. Quantification of changes in morphologies of ZO-1-inhibited glands confirmed an increased number of clefts per bud that coincided with a greater bud circumference and substantial reduction in bud numbers (n?=?16/group) (Fig.?3c & schematic Fig.?3b). To identify cells targeted by the ZO-1 siRNA and to determine the transfection efficiency, we co-transfected SMGs with BLOCK-iT?, a FITC-conjugated oligo reagent. Transfection-competent cells take up the siRNA (or scrambled control, Non-Silenced) and the BLOCK-iT reagent. The results confirmed that BLOCK-iT was effectively taken up by the inner bud cells including the subpopulation of ductal progenitors (Fig.?3d). Interestingly, there K145 hydrochloride was a much lower level of uptake in the columnar cells of the outer cell layer in contact with the basement membrane, a population shown previously by us to be acinar progenitors20, and in mature ducts (Fig.?3d, block arrows). This preferential uptake allowed us to perturb expression of ZO-1 in immature duct cells without significantly affecting either acinar progenitors or differentiated duct cells. To determine how silencing of ZO-1 impacted F-actin organization along the ductal paths, we quantified areas of F-actin distribution and found that in the absence of ZO-1 F-actin organization was significantly constrained to ~?50% of the proximal bud area with concomitant loss of extensions (Fig.?3d, arrowheads and dotted lines, and ?and3e,3e, bar graph). Partial loss of ZO-1 resulted in a failure of F-actin to delineate and extend ducts into the more K145 hydrochloride proximal regions of the inner bud (Fig.?3d). SMGs co-transfected with BLOCK-iT and non-silencing control siRNA had normal ductal structures delineated by F-actin that extended into the buds even in regions where BLOCK-iT was prominent (Fig.?3d, Non-Silenced, dotted lines). Open in a separate window Figure 3 ZO-1 maintains F-actin at the sites of asymmetry and is required for duct extension early in SMG development. (a) Immunofluorescence staining of ZO-1 () in E12.5 SMG following ZO-1 knockdown with siRNA revealed loss of ZO-1-specific staining (n?=?16). Bar graph of K145 hydrochloride siRNA-mediated inhibition of ZO-1 in E12.5 SMG showed a significant, more than 80%, knockdown of the ZO-1 transcript levels (p??0.01). (b) Bright field images in Non-silenced (scrambled sequences) and ZO-1 siRNA-treated SMGs show inhibition of cleft progression and gland expansion. (b) A schematic of observed changes in bud morphologies used for quantification of ZO-1-inhibited glands in (c), with marked () clefts and maturing buds. (c) Quantification of defects in branching morphogenesis following ZO-1 knockdown in E12.5 SMGs. Bar graphs and a box plot of non-silenced and ZO-1 siRNA-treated E12.5 SMGs revealed significant changes in gland morphologies, K145 hydrochloride including increased number.