Wnt Signaling and Cdc42 Regulate Junctional Localization of Alcama in Developing Pouches(A–D) Immunohistochemistry shows progressive junctional localization of Alcama in the wild-type fourth pouch. Below each image, the normalized intensity of fluorescent signal is plotted along the 35 μM red lines. Asterisks indicate clear cell-cell boundaries.(E–H) Still images from a time-lapse confocal recording of fourth pouch development in nkx2.3:Alcama-GFP; her5:mCherryCAAX transgenic embryos (see Movie S6). As with endogenous Alcama protein, plots of fluorescence intensity through the red lines show progressive junctional localization of Alcama-GFP (green) within her5:mCherryCAAX-positive pouch cells (red).(I–X) Immunohistochemistry shows Alcama localization at the level of the fourth pouch in wild-type, mutant, and fzd8a-MO embryos, as well as UAS transgenic embryos (capitalized) doubly positive for nkx2.3:Gal4VP16. Alcama is partially mislocalized from pouch cell membranes to the cytoplasm in wnt4a-, wnt11r-wnt4a-, and fzd8a-MO embryos but not in wnt11r- embryos. In contrast, misexpression of Wnt11r results in disorganized endoderm (n = 28/32, data not shown) and occasionally rosette-like structures (n = 3/32), with rosette-like structures never observed in fzd8a-MO-injected siblings (n = 0/23, p = 0.003). Similarly, the inappropriate Alcama junctional localization induced by Wnt4a misexpression (n = 26/114) was never seen in fzd8a-MO-injected siblings (n = 0/74, p = 0.004) or doubly transgenic Wnt4a; DN-Cdc42 embryos (n = 0/31, p = 0.005). Rosette-like structures were observed in both CA-Rac1 embryos (n = 104/112) and their wnt11r mutant siblings (n = 11/17), and hyperelongated pouches were observed in both CA-Cdc42 embryos (n = 85/122) and their wnt4a-MO-injected siblings (n = 72/101). Plots of fluorescence intensity through the red lines are shown, with asterisks indicating clear cell-cell junctions.(Y–AB) Immunohistochemistry and normalized line plots show junctional localization of E-cadherin throughout wild-type pouch development, with pronounced apical enrichment (arrow in AA) as pouches mature. Inhibition of Dvl in nkx2.3:Gal4VP16; UAS:DvlΔDEP embryos does not significantly disrupt E-cadherin localization.See also Figure S3.
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