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First published online 23 June 2005
doi: 10.1242/dev.01899

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Hand is a direct target of Tinman and GATA factors during Drosophila cardiogenesis and hematopoiesis

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

View larger version (93K): [in a new window]   Fig. 1. Expression pattern of Hand is fully recapitulated by the Hand enhancer driven reporter gene in Drosophila heart. (A-F) Hand is strongly expressed in the developing cardioblasts that express Mef2 (blue) and a subset of pericardial nephrocytes that express Even-skipped (red). Hand expression is also detected in the visceral mesoderm (arrowhead in A) and the lymph gland (arrowhead in D). (G,H) All the Tinman-positive cardioblasts and pericardial cells (red) express Hand. (I) All pericardial cells labeled by Zfh1 (red) express Hand. (J,K) Hand is also expressed in all pericardial cells that express Odd-skipped (red), including the lymph gland pre-hemocytes and the pericardial nephrocytes. (L) All lymph gland hematopoietic progenitor cells that express Serpent (red) also express Hand. (M-O) The extracellular protein Pericardin (red), expressed by pericardial nephrocytes, encloses Hand-expressing pericardial cells (N); Hand-expressing lymph gland hematopoietic progenitor cells (arrowhead) do not express Pericardin and Pericardin-positive ring gland cells (arrow) do not express Hand. In all panels, Hand transcripts were detected by in situ hybridization and labeled in green. Other cardiac and hematopoietic markers are labeled in red as indicated. (A-C) Lateral views of stage 13 embryos; (D-O) dorsal views of stage 15-16 embryos. Anterior is towards the left in all panels.

View larger version (42K): [in a new window]   Fig. 2. Identification of the minimal Hand cardiac and hematopoietic (HCH) enhancer. (A) The Hand gene located on chromosome 2 contains four exons. The 13 kb genomic region containing Hand-coding sequence was screened for expression in the embryonic heart. A 517 bp minimal cardiac enhancer (called HCH) was identified between exons 3 and 4 of Hand-coding sequence. The top eight genomic regions were assayed using a lacZ reporter, and the bottom three genomic regions were assayed using a GFP reporter. B, BamHI; R, EcoRI; S, SalI; X, XhoI. (B) DNA sequence of the HCH enhancer with Tinman- and GATA-binding sites highlighted in yellow and blue, respectively. (C) Cardiac and hematopoietic expression pattern driven by the HCH enhancer, shown by lacZ staining (red in b, yellow in c), is identical to that of the endogenous Hand transcripts (green in a, yellow in c). The HCH enhancer can also drive GFP expression in the same pattern in embryos (d) and in lymph gland (arrow) and heart (arrowhead) in larva (e). (C, parts a-d) Dorsal views of stage 16 embryos. (C, part e) A living first instar larva. Anterior is towards the left in all panels.

View larger version (59K): [in a new window]   Fig. 3. The Hand cardiac enhancer is conserved among distinct Drosophila species. (A) Schematic diagrams of Hand enhancers identified in distinct Drosophila species and the positions of Tinman and GATA consensus binding sites. (B, parts a-d) The HCH enhancer from D. virilis can drive GFP expression (green) in the same pattern as that of the D. melanogaster HCH enhancer. Lateral view of a stage 13 embryo (B, parts a,b) and dorsal view of a stage 15 embryo (B, parts c,d) are shown. (B, parts a-d) vir-HCH-GFP is in green, Mef2 is shown in blue and Even-skipped in red. Anterior is towards the left in all panels.

View larger version (55K): [in a new window]   Fig. 4. Binding to and activation of the HCH enhancer by Tinman, Pannier and Serpent. (A) Gel shift assays were performed using GST-Tinman protein and a radiolabeled probe corresponding to the Tin1 site in the HCH enhancer. Competition assays were performed using a 50-fold molar excess of unlabeled oligonucleotide corresponding to the wild-type Tin1, Tin2 or Tin3 sites or mutant sites, as indicated. (B) Gel shift assays were performed using GST-Pannier protein and a radiolabeled probe corresponding to the second GATA site (G2) in the HCH enhancer. Competition assays were performed using a 10- or 50-fold molar excess of unlabeled oligonucleotide corresponding to the wild-type GATA sites or a 50-fold excess of unlabeled mutant GATA sites, as indicated. Similar results were obtained for all the GATA sites. An experiment with the G2, G3 and G4 sites is shown. (C) Gel shift assays were performed using in vitro translated Serpent protein and a radiolabeled probe corresponding to the second GATA site (G2) in the HCH enhancer. Competition assays were performed using 50-fold molar excess of unlabeled oligonucleotide corresponding to the wild-type or mutant GATA sites. (D) S2 cells were transfected with luciferase reporters controlled by the wild-type or mutated HCH enhancers. As indicated, Tinman activates the HCH enhancer over 100-fold, whereas Pannier or Serpent activates the HCH enhancer approximately sixfold. The three factors do not show significant synergy when added simultaneously. Mutation of the Tinman sites (HCH-4T) specifically abolishes the activation by Tinman, whereas mutation of the GATA sites specifically abolishes the activation by Pannier or Serpent. The HCH enhancer with both Tinman and GATA sites mutated (HCH-4T5G) cannot be activated by any of these three transcription factors.

View larger version (88K): [in a new window]   Fig. 5. Ectopic Tinman, Pannier or Serpent induces ectopic Hand expression in somatic muscles, cardioblasts/pericardial nephrocytes or hematopoietic progenitors. (A-C) Wild-type HCH-GFP is expressed in all the Mef2-expressing cardioblasts and Eve-expressing pericardial cells. (D-F) Overexpression of Tinman using twi-Gal4; 24B-Gal4 induces HCH-GFP expression in the somatic muscle cells that express Dmef2 (arrows indicate Ectopic HCH-GFP in the muscle cells). (G-I) Overexpression of pannier in the mesoderm using twi-Gal4; 24B-Gal4 induces the formation of extra cardioblasts (indicated by arrows) and pericardial nephrocytes (indicated by arrowheads), and produces an expanded heart. Expression of HCH-GFP is detected in all the ectopic heart cells. (J-L) Overexpression of Serpent using twi-Gal4; 24B-Gal4 reduces the number of cardioblasts and pericardial nephrocytes (indicated by arrows), but induces ectopic hematopoietic progenitor cells, as shown by the expanded lymph gland (indicated by arrowheads). HCH-GFP expression is detected in all the cells in the expanded lymph gland. HCH-GFP also shows the clustering of the pericardial nephrocytes, which normally form a line, indicating a cell fate transformation from pericardial nephrocytes to hematopoietic progenitor cells. All panels show dorsal views of stage 16 embryos carrying HCH-GFP reporter (green) and are labeled with Mef2 antibody in red and Eve antibody in blue. Anterior is towards the left.

View larger version (75K): [in a new window]   Fig. 6. Tinman, Pannier and Serpent are required for HCH enhancer activity during development. (A) The HCH-GFP is expressed in the lymph gland (arrow) and the heart (arrowhead) in the first-instar larva. (B) In homozygous tin mutant larvae, the lymph gland and heart fail to form and no HCH-GFP is detected. (C) Only residual activity of the HCH enhancer is detected in homozygous pannier mutant larvae, in which no lymph gland is formed (indicated by arrow) and the few surviving cardiac cells fail to fuse at the dorsal midline (indicated by arrowheads). (D) In homozygous serpent mutant larvae, the lymph gland does not form (indicated by the arrow), but most cardiac cells form and express HCH-GFP (indicated by arrowhead). (E) In first-instar larvae that express a dominant-negative form of Tinman in the Hand-expressing cells using HCH-Gal4 driver, HCH-GFP expression is dramatically suppressed in the heart (arrowhead), and less dramatically suppressed in the lymph gland (arrow). (F) Overexpression of a dominant-negative form of Pannier in the Hand-expressing cells using the HCH-Gal4 driver dramatically suppressed the HCH-GFP expression in both the heart (arrowhead) and the lymph gland (arrow). All panels are dorsal view of embryos/larvae carrying the HCH-GFP with anterior towards the left.

View larger version (96K): [in a new window]   Fig. 7. Requirement of Tinman and GATA sites for activity of the HCH enhancer during development. (A-C) The wild-type HCH enhancer drives GFP expression strongly in the lymph gland hematopoietic progenitors (indicated by arrowhead in A), cardioblasts (indicated by arrowheads in B and C, labeled in C by Mef2 antibody in blue) and the pericardial nephrocytes (arrows in B and C indicate a subset of pericardial nephrocytes labeled in C by Eve antibody in red). (D-F) The HCH enhancer with all four Tinman binding sites mutated (HCH-4T) drives GFP expression in a similar pattern to the wild-type HCH, but at a lower level. The activity of this enhancer is not affected in the lymph gland (indicated by the arrowhead in D), but is frequently missing from the Tinman-positive cardioblasts (parallel arrows in D-F, the four Tinman-positive cardioblasts in each hemisegment come from a common progenitor cell), as well as Tin/Eve-positive pericardial nephrocytes (joined arrows in D-F; two Tin/Eve-positive pericardial cells are formed in each hemisegment). (G-I) The HCH enhancer with all five GATA-binding sites mutated (HCH-5G) fails to drive GFP expression in the lymph gland (indicated by arrowhead in G). In contrast to HCH-4T-GFP, HCH-5G-GFP is expressed in an identical pattern to that of Tinman (shown by Tinman antibody in red, which totally overlaps with the HCH-5G-GFP pattern in green). (H,I) Higher magnified panels co-labeled with Mef2 in blue and Eve in red show that HCH-5G-GFP is only expressed in four out of six cardioblasts (parallel arrows) and two Eve pericardial cells (joined arrows) in each hemisegment. (J-L) Mutation of both Tinman and GATA-binding sites totally abolishes HCH enhancer activity in the lymph gland, cardioblasts and pericardial nephrocytes. Each row of panels shows a different enhancer activity in green as indicated. The left column (A,D,G,J) shows dorsal views of stage 15 embryos carrying the enhancer-GFP (green) and labeled by Tinman antibody in red. The right two columns are dorsal/lateral views of three hemisegments of stage 14 embryos carrying different enhancer GFP (green) and co-labeled with DMef2 antibody (blue) and Eve antibody (red). Anterior is towards the left in all panels.

View larger version (183K): [in a new window]   Fig. 8. A model for the position of Hand in the transcriptional networks that control cardiogenesis and hematopoiesis. Both cardiogenesis and hematopoiesis occur in the cardiac mesoderm, which is specified by signaling pathways (Dpp, Wg and Fgf) from the overlaying ectoderm, through direct or indirect transcription activation of the genes encoding Tinman, Pannier and Serpent, which also affect one another at the transcriptional level. Tinman and Pannier directly activate the genes encoding Hand and other transcription factors such as Mef2 and Eve in cardioblasts and pericardial nephrocytes in the transcriptional network that controls cardiogenesis. Serpent activates Hand and probably genes encoding other transcription factors such as Lz (Lozenge) and Gcm (Glial cells missing) in the transcriptional network that controls hematopoiesis. Notch is required for the specification of both cardiogenic and hematopoietic progenitors during its early phase of mesodermal expression, and for inhibiting myocardial cell fate while promoting pericardial and hematopoietic cell fate during its late phase of mesodermal expression. Ush (U-shaped) cooperates with Pannier and Serpent in cardiogenesis and hematopoiesis. Solid arrows indicate verified direct transcription activation if pointing to a gene, or verified requirement for a certain cell type formation if pointing to a cell type; broken arrows indicate unverified or indirect gene activation if pointing to a gene, or proposed requirement for certain cell type formation if pointing to a cell type; broken lines indicate different cell types in which a transcription factor is expressed and may have functions.