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First published online 8 February 2006
doi: 10.1242/dev.02285

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Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis

Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75390, USA.

View larger version (23K): [in a new window]   Fig. 1. Hand functions as a potent transcription activator. (A) Schematic drawing of the Hand mutant constructs for structure/function studies. (B) Activation of the L8E6 promoter by Hand and deletion mutants. (C) Mapping the transcription activation domain of Hand with Gal4-Hand fusions. (D) Hand-EnR blocks the transcriptional activity of wild-type Hand on the L8E6 promoter in a dosage-dependent fashion. All experiments were carried out in Drosophila S2 cells.

View larger version (115K): [in a new window]   Fig. 2. Hand functions as a transcription activator in heart and lymph gland formation. (A,B) Stage 13 embryos; (C-H) stage 16 embryos. Embryos with pan-mesodermal expression of Hand developed normal heart and lymph gland (A,C), whereas in embryos with pan-mesodermal expression of Hand-EnR (B,D), formation of heart and lymph gland were severely affected. Targeted expression of Hand using Hand-Gal4 did not cause any defects in the heart and lymph gland, whereas targeted expression of Hand-EnR disrupted heart and lymph gland formation. The heart was labeled by Mef2 antibody (cardioblasts: green in A,B; blue in C,D; red in E,F), Even-skipped antibody (pericardial cells: red in A,B; green in C,D), Odd-skipped antibody (pericardial cells: red in C,D) and Hand-GFP (green in E-H). The lymph gland is labeled by Odd-skipped antibody (red in C,D), Serpent antibody (red in G,H) or Hand-GFP (green in E-H). Arrowheads indicate the positions of the lymph gland.

View larger version (37K): [in a new window]   Fig. 3. Generating the Hand-null mutant by homologous recombination. (A) Strategy for replacing the Hand-coding region with the mini-white gene by ends-out homologous recombination. (B) RT-PCR of the four mutant lines obtained from the screen showing no detectable Hand transcript in homozygous mutant larvae. (C) In situ hybridization using Hand probe (green) did not detect any Hand transcripts in homozygous Hand mutants (lower panel), which were distinguished from heterozygous Hand mutant embryos (upper panel) by using anti-ß-Gal antibody (red) to detect the presence of the balancer chromosome.

View larger version (69K): [in a new window]   Fig. 4. Cardiac and lymph gland defects in Hand mutant embryos. (A-I) Stage 16 embryos labeled with cardiac and lymph gland markers. About 20% of the Hand mutant embryos showed a range of cardiac morphological defects (D-F), including misalignment of Mef2-expressing cardioblasts, reduced Odd-expressing pericardial cells and lymph gland progenitors. Embryos with more severe defects (about 3%) showed a significant reduction of all three cell types (G-I). The ring gland, which is adjacent to the lymph gland anteriorly and is labeled by Pericardin, is not affected (E,F), nor are somatic muscles, labeled by Mef2 (D,F). Arrowheads indicate the positions of the lymph gland.

View larger version (76K): [in a new window]   Fig. 5. Cardiac and lymph gland defects in Hand mutant larvae. (A-J) Three-dimensional reconstruction of confocal scans of Drosophila embryos and larvae carrying Hand-GFP as different stages. (A-C,G,H) Wild-type embryos and larvae; (D-F,I,J) Hand mutant embryos and larvae. Arrowheads indicate the positions of the lymph gland. (A-F) Hand mutant embryos and larvae display cardiac morphological defects compared with wild type. (G,I) Lymph gland hematopoietic progenitors are almost completely abolished in 24 hour AEL Hand mutant larvae (indicated by an arrowhead). (H,J) Three dimensional chamber-like structure is abolished in Hand mutant larvae at 26 hours AEL, accompanied by hypoplastic heart tube and dramatically reduced pericardial nephrocytes.

View larger version (116K): [in a new window]   Fig. 6. Rescue of larval cardiac and hematopoietic defects by P35 or human HAND2. (A-F) Dorsal views of stage 16 embryos labeled with TUNEL (red) and Hand-GFP (green). Compared to wild type (A,B), ectopic apoptosis (shown by TUNEL staining) was observed in Hand mutants in the regions normally occupied by lymph gland hematopoietic progenitors (indicated by an arrowhead) and pericardial cells, with a few TUNEL positive cells found among the cardioblasts (C,D). The ectopic apoptosis in Hand mutants could be rescued by targeted expression of P35 (E,F). Targeted expression of P35 also effectively rescued the cardiac and lymph gland defects in Hand mutant embryos, as shown by Hand-GFP (F). In C-F, arrowheads indicate the position of the lymph gland. At larval stages, Hand mutant larvae rescued by targeted expression of P35 displayed cardiac and lymph gland defects at 18 hours AEL (G). These defects become more severe at 24 hours AEL (H). The cardiac and hematopoietic defects of Hand mutants were more effectively rescued by targeted expression of human HAND2 at 16 hours (I) and 24 hours AEL (J), and completely rescued by targeted expression of Drosophila Hand (data not shown).

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