Identification of regulatory elements responsible for hand expression in the visceral mesoderm. A) hand-GFP expression in the wildtype. The third intron of the hand gene harbors all cis-regulatory sequences sufficient to control GFP reporter gene expression in circular visceral muscles. (a) Double labeling was performed using an anti-GFP (green channel) and an anti-FasciclinIII (red channel) antibody. Fasciclin III stains the progenitors as well as the fusion competent myoblasts (double arrows) that form the circular muscles. Solely the progenitor cells coexpress hand driven GFP at the stage shown here (arrows). (b) is an enlargement of a (boxed). (c) hand driven GFP expression is maintained through embryonic and larval development. The stage 16/17 embryo shown (lateral view) reveals hand driven GFP expression in the heart and the circular visceral midgut muscles. (d) shows a 2nd larvae exhibiting direct GFP fluorescence in the nuclei of the circular visceral muscles, the heart and the garland cells. The diffuse yellow color inside the gut is caused by autofluorescence from yeast. B) eShadow analysis for the hand gene. The alignment of multiple sequences was performed with the ClustalW algorithm implemented in the eShadow web application . Sequences from D. melanogaster, D. erecta, D. yakuba, D. simulans and D. virilis were used. Exons and conserved intron sequences, identified to harbor enhancer elements driving hand in the heart and the visceral mesoderm, are marked. The x-axis corresponds to size in base pairs. The y-axis corresponds to percentage of variation in a window of 50 bp. C) Highly conserved regions in the 3rd intron of the hand gene are responsible for driving expression in cardiac tissue and within the visceral mesoderm. All embryos were stained with an anti-GFP antibody. a, c, e and g show embryos at stage 13, lateral view. b, d, f and h show stage 16/17 embryos in a dorsal view. (a and b) Transgenic embryos carrying the whole 3rd intron of the hand gene reveal expression of the reporter gene in the developing heart and circular visceral mesoderm. Garland cells, which express hand-GFP as well, are out of focus. (c and d) Transgenic embryos with a mutated 3rd intron, lacking the first highly conserved element (region 395–824), show a total loss of reporter gene expression in the heart, whereas expression in the visceral mesoderm is unaffected. (e and f) Transgenic embryos with a mutated 3rd intron, lacking the second highly conserved element (region 1074–1374) show a total loss of reporter gene expression in the visceral mesoderm, whereas expression in the cardiac mesoderm is unaffected. (vm) circular visceral muscles. (g and h) Transgenes carrying the visceral mesoderm enhancer (region 1054–1400) fused to GFP show expression in the visceral mesoderm but not in garland cells, lymph glands and the heart.