Cdc42 is necessary for cardiac alignment and lumen formation. (A and B) Wild-type Drosophila heart at the end of heart morphogenesis, st17. Cardiac nuclei (Nmr1, blue) are aligned at the dorsal midline. Basement membrane (Dg, red) and heart lumen (Slit, green) are properly localized to the respective domains (see schematic in A). (B and C) CB cell shape in wild-type (B–B″) and Cdc42³ mutant (C–C″) hearts at embryonic early stage 17, stained with anti-Nmr1 (B and C), anti-Dg (B′ and C′), and anti-Slit (B″ and C″). Transverse cross sections (insets) were taken from the indicated positions (broken line in A–D). Wild-type hearts show aligned cardioblasts enclosing a single, central lumen (open arrowheads in B–B″ and inset), with Slit enriched at the luminal surface (arrowheads). CBs in Cdc42³ zygotic mutant hearts fail to form a central heart lumen (C′ and C″) or are mispositioned (curved arrowheads), and Slit protein is located at the CB interface (C″). (D) Cardiac-specific expression of dominant-negative Cdc42^N17 in mid- to late-stage developing hearts (using tinCΔ4-Gal4) causes multiple heart lumina (arrows in d). Dg (red) indicates the ECM. (E) Expression of Cdc42^N17 in the somatic and cardiac mesoderm causes severe alignment defects (e) or collapsed heart lumina at the point of CB alignment (f). Arrows point to the luminal domain marked by Dg. The arrowhead shows lack of Dg at contralateral CB. (F) Expression of activated Cdc42^V12 causes ectopic, ipsilateral “lumen” (enriched for Dg protein; arrowhead in F) but does not interfere with overall CB alignment (F′). (G and H) CB cell shapes are changed upon Cdc42^V12 misexpression. Wild-type CBs are columnar (G), whereas CBs expressing activated Cdc42^V12 are more rounded (H, arrow) and form round structures resembling ectopic lumina (H, arrowheads). Cell outlines are visualized by expression of Dlg1^GFP. Broken lines indicate the midline. (I and J) Further characterization of Cdc42^V12-induced lumina. Slit (I) and Trol (I′) are additional markers that localize to the luminal domain in wild-type hearts (open arrowheads). Upon cardiac overexpression of activated Cdc42^V12, Slit is found between ipsilateral CBs (J, arrowheads), some of which also colocalize Trol (J′ and J”). Bars in D and E (insets), J′, and J”: 6 µm.