FIGURE 8:

ZEN-4 activity is required at arcade cell birth. (A) The temperature-shift strategy. Rectangles denote developmental time (scaled to 20°C). Blue represents permissive temperature (15°C), and red denotes restrictive temperature (26°C). The control sample was incubated at permissive temperature only. The early shift started ∼50 min before arcade cell birth, the late shift started ∼50 min before arcade cell polarization, and the mid shift is in between. Representative DIC (B–E) and DLG-1 (F–I) images of embryos after the foregoing incubations. The foregut-associated basement membrane (BM) is highlighted by a red dotted line. The control, mid, and late embryos all have a polarized arcade cell epithelium spanning the epidermis and foregut. Note that the BM surrounds an elongated foregut. The early shift results in an unattached foregut because the arcade cells (arrowhead) failed to polarize (C, G). Note that the BM completely surrounds the foregut primordium, which is unattached to the anterior epidermis (C), and that DLG-1 is not expressed in the arcade cells (G). (J) The epidermal temperature-shift strategy. The epidermal cells are born over a longer period of time than the arcade cells (gray), and this period encompasses the time at the restrictive temperature (red). Embryos were shifted back to permissive temperature before the onset of epidermal polarization. (K, L) Representative maximum intensity projections through the epidermis of embryos at the end of the shift. In the control embryo (K), DLG-1 (white) is found at the cell junctions of epidermal cells that have formed two rows on the dorsal surface. These cells are mononucleate. In the shifted embryo (L), the epidermal cells generate and localize DLG-1 properly, even though many cells are obviously multinucleate (arrowhead) due to cytokinesis defects. Anterior is to the left. Scale bars, 5 μm.