Figure 4.—

rhomboid1 overexpression disrupts salivary gland morphogenesis, but is not sufficient to induce salivary duct fate. (A) Scheme of the rho locus indicating the position of the EP identified in the screen. (B) Scheme depicting the known involvement of EGF signaling in salivary gland morphogenesis. EGF is released from the midline (red line) and induces, in the cells close to the midline (light green), the repression of fkh, which in turn leads to suppression of secretory fate in these cells, inducing them to adopt duct cell fate. Fkh expression remains high in the remaining salivary gland primordium (dark green), thus inducing these cells to form the secretory part of the gland (Kuo et al. 1996). (C–C″) Overexpression of rhomboid1 in the salivary glands using EP(3)3704 led to glands that, at stage 15 of embryogenesis, were located too far anterior with secretory cells that appeared cuboidal instead of columnar, no proper duct connecting the secretory portions to the outside, and an aberrantly shaped lumen. The SrcGFP marker is green in C and a single channel in C′; phalloidin is red in C and a single channel in C″. (D–D″) The same phenotype as in C is observed when a UAS-rhomboid1 construct is expressed in the glands using fkhGal4. The GFP-EFGas2 marker is green in D and a single channel in D′; Crumbs is red in D and a single channel in D″. (E–F′) Already at stage 13 the invaginated portion of the gland shows aberrant morphology (ectopic lumen indicated by the arrow in E′), and the amount of cells remaining at the surface appears too large (bracket in E′ and F′). The SrcGFP marker is green in E and F (and a single channel in E′ and F′); crumbs is in red. (G–P″) Analysis of dCreb-A and Eyegone expression: markers of secretory and duct fate, respectively. (G–I″) Control glands expressing only the GFP-EFGas2 marker labeled with antibodies against dCreb-A and Eyg at stage 11 (G–G″), stage 14 (H–H″), and stage 15 (I–I″). (K–M″) Glands expressing UAS-rhomboid1 in the salivary glands using fkhGal4 labeled for dCreb-A and Eyg at stage 11 (K–K″), stage 14 (L–L″), and stage 15 (M–M″). Note that, despite the irregular shape and ectopic cells (bracket in L), dCreb-A is strongly expressed in the early invaginated part of the glands (L′ and M′). Eyg is expressed in the most anterior cells of the invaginated glands (L″ and M″), as in the control, and also in the ectopic cell bulge on the surface of the embryo (L″; bracket in L denotes the bulge, and the dashed line indicates the ventral midline). (N–P″) Glands expressing UAS-rhomboid1 using armGal4. (N–N″) dCreb-A and Eyg expression in the placode at stage 11. (O and O′) Ventral view of the remaining placode (O) and invaginated glands (O′) at stage 13. More ectopic cells expressing Eyg are found on the ventral surface (bracket in O). Small stubby glands have invaginated and expressed dCreb-A (arrows in O′). (P–P″) Lateral view of glands at stage 14. More cells have invaginated and expressed dCreb-A (arrow in P), and the most ventral cells on the surface still express Eyg (P″). (G–P) GFP makers, green; dCreb-A, red; and Eyg, blue. (G′–N′ and P′) dCreb-A as a single channel. (G″–N″ and P″) Eyg as a single channel. All panels are projections of confocal stacks that cover the entire thickness either of the invaginated glands or of the placode at earlier stages.