DEV02586 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Mutant embryos that lack tin expression in the dorsal vessel express Doc and Homothorax in the anterior region of the aorta. Dorsal views of stage 16 embryos stained for Homothorax (Hth) and Doc2+3 proteins. (A) Wild type: in the anterior aorta, which lacks the segmental Doc expression seen in the remainder of the dorsal vessel, Hth is detected in 11 pairs of cardioblasts (arrow). (B) tin-ABD; tin³⁴⁶ embryo: Doc expression has expanded into the Hth-positive anterior aorta at variable levels (arrow).

• Supplemental Figure 2 -

  Fig. S2. Analysis of pericardial cell markers in embryos lacking cardiac tin expression. (A,B) Detection of Ladybird (Lbe), Doc2+3 and Mef2 proteins in stage 13-14 embryos (lateral view). (A) Wild type: in addition to its expression in two cardioblasts per hemisegment (green arrowheads), Lbe is present in a subset of pericardial cells (arrows). (B) tin-ABD; tin³⁴⁶ mutant embryo: very few pericardial cells (and cardioblasts) expressing Lbe are present at this stage (green arrows and arrowheads). Asterisk: area lacking Lbe⁺ pericardial cells. Red arrowheads: Doc-expressing cardioblasts. (C) Stage 15 wild-type embryo (dorsal view) stained for Zfh1, Odd and Eve proteins. Eve⁺ Zfh1⁺ pericardial cells appear purple-white, Odd⁺ Zfh1⁺ pericardial and lymph gland cells appear yellow-orange. Odd/Eve-negative Zfh1⁺ pericardial cells (red arrows), which also express tin, align at a position ventromedially to the other cardial cells. (D) tin-ABD, tin³⁴⁶ embryo stained as in C. Eve⁺ Zfh1⁺ and Odd⁺ Zfh1⁺ pericardial cells develop normally, although their alignment is slightly aberrant. The number of Zfh1-expressing ventromedial pericardial cells appears to be reduced. Asterisk indicates an area lacking ventromedial Zfh1⁺ pericardial cells.

• Supplemental Figure 3 -

  Fig. S3. Doc-mediated repression of tin does not require svp. Dorsal views of stage 16 embryos stained for svp-lacZ (β-Gal, green), Tin (blue) and Doc2+3 (red) proteins. (A) Homozygous svp^AE127 mutant: Tin expands into all svp-lacZ-positive cardioblast that have become Doc-negative (turquoise arrows; see Fig. 5A for wild-type pattern). (B) Homozygous svp^AE127 mutant embryo with S59-Mef2-HtΔD-Gal4-driven misexpression of Doc2 in the dorsal vessel: tin expression becomes repressed in most Doc-expressing cardioblasts, including both svp-lacZ⁺ (yellow arrows) and svp-lacZ⁻ (red arrows) cells. Residual ectopic Tin is only observed in some Svp-cardioblasts that failed to express Doc at high levels (turquoise arrows).

• Supplemental Figure 4 -

  Fig. S4. F-actin distribution in third instar wild type (A) and tin-ABD, tin³⁴⁶/tin^EC40 mutant (B) larval dorsal vessels. Phalloidin staining of the entire dorsal vessels as in Fig. 7A,B are shown.

• Movie 1 -

  Movie 1. A 10-second clip of a beating heart taken from dissected open adult abdomens (abdominal segments 2-4) of 1-week-old flies using a Hamamatsu EM-CCD digital camera. tin heterozygotes have normal heart contractions.

• Movie 2 -

  Movie 2. A 10-second clip of a beating heart taken from dissected open adult abdomens (abdominal segments 2-4) of 1-week-old flies using a Hamamatsu EM-CCD digital camera. In flies with ‘cardiac-specific knockout’ of tin the heart contractions are weak, almost failing and arrhythmic (compare with Movie 1).