Various heart phenotypes generated by RNAi. Examples of X-Gal-stained embryos at late stage 16, representing abnormal heart phenotypes caused by RNAi of individual dsRNAs, are shown. Computed gene (CG) number corresponds to individual genes that caused mutant heart phenotypes by RNAi. Dorsal views of embryos with anterior on the left are shown. WT, control embryo showing the normal heart phenotype.

Host embryo and strategy of the screen for cardiogenic genes by RNAi in the developing Drosophila embryo. (Left) The WT host embryo for microinjection of dsRNAs stained with X-Gal, showing the expression pattern of the D-mef2-lacZ reporter gene and normal heart morphology during embryogenesis. Sn, embryonic stage n; CC, cardial precursor cells; SC, a subset of somatic muscle founder cells; DV, dorsal vessel (heart). (Right) A schematic of the screen for cardiac genes. Embryos were initially injected with dsRNAs from a pool of three different genes, incubated at 18°C overnight for development, and subjected to X-Gal staining. Stained embryos were examined under a microscope to select a positive pool that caused abnormal heart development. Each dsRNA from the positive pool was injected again into embryos for the identification of individual genes that affect heart development. N, negative; P, positive.

simj gene function is required for the specification of a subset of cardiac cells. (A) Embryo injected with the simj dsRNA is stained for Mef2. Arrowheads indicate missing cardial cells. (Inset) Embryo stained with X-Gal. (B) Schematic diagram showing loss of cardiac cells in the simj mutant embryo. CC, cardial cell; PC, pericardial cell; T, Tin-positive cardial or pericardial cell; S, SVP-positive cardial cell; E, Eve- and Tin-positive pericardial cell. Only Tin-positive pericardial cells are depicted. (C–F) Embryos collected from either WT or simj mutant flies are subjected to immunostaining with the indicated antibody. Dorsal view of stage 16 WT (C and E) and simj mutant (D and F) embryos stained for Mef2 (C and D) or Tin (E and F). Arrowheads indicate Tin-positive cardial cells, and stars indicate SVP-positive cardial cells. In simj mutant embryos, only four cardial cells within each segment are stained for Mef2. Two are stained for Tin (arrows). Lateral view of stage 14 WT (G) and simj mutant (H) embryo fluorescently stained for Tin (green), showing that two Tin-positive cardial cells in each hemisegment are missing in the simj mutant embryo. (I and J) Forced expression of simj resulted in overspecification of cardial cells. Embryos were collected from either WT or transgenic flies ectopically expressing simj driven by dpp-gal4 (UAS-simj) and subjected to immunostaining with an anti-Mef2 antibody. Lateral view of stage 14 embryos is shown. Arrowhead indicates supernumerary Mef2-expressing cardial cells.

Axial patterning genes play roles in Drosophila heart development. Embryos were injected with control dsRNA (A–C), the ftz-f1 dsRNA (D–F), and short dsRNA (21-mer) of prd (G–I) and abd-A (J and K) and subjected to X-Gal staining (A–J) followed by immunostaining with an anti-Mef2 antibody (B, E, and H). The half-size heart was generated by loss of either prd or ftz-f1 gene function (D, E, G, and H). The shape of the heart is maintained, but its size is reduced by half. At late stage 11 (C, F, and I), the embryo injected with the ftz-f1 (F) or the prd (I) dsRNA shows segmental deletion of both ventral muscle founder cells (arrowheads or stars) and cardiac precursor cells (arrow). A subset of muscle founder cells at the abdominal segments A1 and A3 is marked with an arrowhead, and stars indicate either muscle founder cells at segment A2 (C) or corresponding cells missing in the injected embryo (F and I). The embryo injected with the short dsRNA of abd-A shows transformation of the heart portion into the aorta, which is demonstrated by heart morphology (J) and cardial cell staining (K). Note that in the embryo injected with abd-A dsRNA (K), the posterior heart region (bracket) has the same narrower width as the aorta.