Fig. 2. Embryonic expression pattern of Dorsocross. Nuclear Dorsocross proteins were detected by immunostaining with anti-Doc3+2 antibody (see Materials and Methods) and visualized either with DAB (brown in A-D,F-I,L) or fluorescent secondary antibodies (J). Dorsocross mRNA was detected by in situ hybridization with specific probes for Doc3 (E) or Doc1 (K). Images from fluorescent staining are combined ectodermal (E), subepidermal (J) or mesodermal (K) confocal sections. Views are lateral (B,D,E,G,I-K) or dorsolateral (C,F) with dorsal upwards and anterior towards the left, or dorsal with anterior towards the left (A,H,L). Embryos are oriented the same way in all figures and all non-flourescent images are taken using Nomarski optics. (A) Blastoderm stage embryo, (B) stage 7 embryo and (C) stage 8 embryo showing Dorsocross protein in the anlagen of the amnioserosa (as) and the procephalic neuroectoderm (pne). Amnioserosa nuclei are distinguishable by their larger size. (D) At stage 10, a metameric expression pattern (PS4, parasegment 4) has emerged in addition to continued expression in the amnioserosa. (E) Patches of Dorsocross expression (green) alternate with the tracheal placodes labeled by anti-Cf1a antibody staining (red) in the dorsolateral ectoderm of the embryonic trunk. (F,G) At stage 14, epidermal stripes are visible dorsally and ventrolaterally. (H) Enlarged view of stage15-16 embryo that has completed dorsal closure, showing expression in dorsally fused epidermal stripes (de) and in the dorsal pouch (dp), and two pairs of cardioblasts per segment (cb). (I) Stage 16 embryo focussed on Doc expression in Malpighian tubules (arrowhead). (J) Dorsocross expression in the pentascolopidial chordotonal organs (arrowheads, green), which are marked by their staining with mab 22C10 (red). Dorsocross-positive cells are identified as ligament cells based on their ventral juxtaposition to 22C10-labeled LCh5 neurons (arrowheads). Some epidermal Doc staining is also present in this image. (K) The metameric expression as seen in D includes the dorsal mesoderm, where Dorsocross-expressing cells (green) alternate with visceral mesoderm precursors that express bap (red). The combined sections include ectodermal expression seen in more radial areas [see broken line between mesoderm (ms) and ectoderm (ec)]. (L) Stage 11 embryo focussed on the bilateral caudal visceral mesoderm anlagen (cvm) close to the posterior tip of the germ band.

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