Globally induced en activates the endogenous en gene in novel cells. Embryos (115×) are stained with an anti-en antibody. b–e, Embryos between 6 and 7 h AEL. a, Schematic diagram of hs–en. The en sequences consist of a 2-kb EcoRI–SnaBI fragment of the en cDNA clone c-2.4 (). Transcription of en sequences (stippled box) is driven by the hsp70 heat-shock promoter (hsp), contained in a 1.3-kb Sphl/Pstl fragment from pUCHSP 70 (provided by E. Gavis). The polyadenylation sequences (pA+) are from SV40. For transformation, the above sequences are inserted into the pCaSpeR vector containing P element ends (ovals) and the white gene (w+) as a selectable marker. b, An hs–en embryo, heat shocked at ∼6 h, then immediately fixed and stained. The en expression induced by heat shock is detectable in all cells. c, An hs–en embryo, heat shocked at ∼3 h AEL, aged 3.5 h, then fixed and stained. The en stripes are abnormally broad in the ventral and lateral regions compared with wild type (compare distance between arrowheads in c and d). Inset, 326× magnification of part of one stripe showing that the en antigen is confined to the nucleus. d, Wild-type embryo, showing the normal pattern of en expression. e, An enCX1/+;hs–en embryo, heat shocked at ∼3 h AEL, aged 3.5 h, then fixed and stained. The pattern of cytoplasmic enCXI antigen is similar to the pattern of en in c. Inset is a 326× magnification of part of one stripe showing that the cytoplasmic antigen, characteristic of enCX1, is produced in novel cells after heat shock. Both the wild-type nuclear and mutant cytoplasmic en antigens are expressed in these cells, although the nuclear signal is weak relative to the cytoplasmic signal. f, An hs–en embryo, heat shocked at ∼5 h then fixed after 3 h recovery at 25 °C. Striped en staining is nearly wild type. There are isolated cells in the anterior compartment that express en (small arrowheads), as well as staining in the amnioserosa (large arrowhead), neither of which is seen in wild type.
METHODS. Transgenic lines were established using the p-wings clipped helper and Df(1)w67c2,y as host. For heat-shock experiments, embryos were collected and aged at 25 °C on grape agar plates. At the appropriate stage, plates were put in moist chambers at 37 °C for 35 min. They were then returned to 25 °C for recovery. The novel expression pattern was detectable as soon as the en expressed from the heat-shock promoter decayed to background levels, ∼45 min after heat shock. Similar results were seen with three different hs–en lines, one on chromosome III and two on chromosome II.