Fig. S3

Alcama localization in rescued fzd8a-MO and Rac-inhibited embryos and quantification of cell shape, related to Figure 4.

(A-E) Immunohistochemistry shows Alcama localization in 34 hpf pouch-forming endoderm in fzd8a-MO-injected nkx2.3:Gal4VP16; UAS:CA-Rac1 embryos (A), fzd8a-MO-injected nkx2.3:Gal4VP16; UAS:CA-Cdc42 embryos (B), nkx2.3:Gal4VP16; UAS:DN-Rac1 embryos (C), control embryos incubated in 6% DMSO from 18-26 hpf (D), and embryos incubated in the Rac inhibitor NSC23766 in 6% DMSO from 18-26 hpf (E). The normalized intensity of fluorescent signal is plotted along the 35 μM red lines. Asterisks indicate identifiable cell-cell boundaries. Activated Rac1 resulted in rosette-like structures in 59/81 fzd8a-MO-injected embryos, and activated Cdc42 rescued Alcama membrane localization and induced hyperelongated cell shape in 24/63 fzd8a-MO-injected embryos. (F) Quantification of 34 hpf pouch cell shape in wild type, mutants, fzd8a-MO, and embryos doubly transgenic for UAS transgenes (capitalized) and nkx2.3:Gal4VP16. The ratios of the longest versus shortest cell axes are plotted. Asterisks indicate significant differences compared to wild type (p<0.05 in a student’s t-test). Data represent mean ± SEM. Total cells analyzed: Wild type (96), wnt11r-; wnt4a- (100), fzd8a-MO (97), nkx2.3:Gal4VP16; UAS:DvlΔDEP (100), nkx2.3:Gal4VP16; UAS:DN-Rac1 (96), nkx2.3:Gal4VP16; UAS:DNCdc42 (92), nkx2.3:Gal4VP16; UAS:Wnt11r (45), nkx2.3:Gal4VP16; UAS:Wnt4a (100), nkx2.3:Gal4VP16; UAS:CA-Rac1 (92), fzd8a-MO; nkx2.3:Gal4VP16; UAS:CA-Rac1 (81), wnt11r-; nkx2.3:Gal4VP16; UAS:CA-Rac1 (48), nkx2.3:Gal4VP16; UAS:CA-Cdc42 (88), wnt4a-MO; nkx2.3:Gal4VP16; UAS:CA-Cdc42 (53), fzd8a-MO; nkx2.3:Gal4VP16; UAS:CACdc42 (57), alcama-MO; nkx2.3:Gal4VP16; UAS:CA-Cdc42 (60), and alcama-MO (95).