The highly conserved basic helix-loop-helix transcription factor Hand plays a crucial role in cardiogenesis, limb formation and other developmental processes of vertebrates. Humans, mice and other higher vertebrates have two related genes, dHand (also known as Hand2, Hed, Thing2) and eHand (also known as Hand1, Hxt, Thing1), whereas fish and Drosophila have only a single hand gene. Drosophila hand has been cloned and its embryonic expression examined in detail by using various tissue-specific markers that allowed the identity of hand-expressing cells to be analyzed. hand was found to be expressed in the entire heart, including all cardioblasts and pericardial cells, in the progenitors of the circular visceral muscles, the lymph gland and garland cells, and in a few cells in the CNS. The expression of Drosophila hand starts after the inductive activity of the early regulators in these tissues, e.g., Tinman and Bagpipe, suggesting a role for Hand in differentiation rather than in tissue determination. In many aspects the expression pattern of Drosophila hand resembles the patterns of its vertebrates orthologues, for instance in cardiac tissues. It is assumed that Hand proteins might play a highly conserved role throughout evolution (Kolsch, 2002).
Using the isolated hand cDNA as probe for whole-mount in situ hybridization experiments, the expression of Drosophila hand was examined in embryos with emphasis on cardiogenesis and visceral mesoderm development. Expression starts at stage 11 and is restricted to 11 bilateral segmentally arranged cell clusters in the dorsal mesoderm. Shortly after these cells form a continuous one- to two-cell-wide stripe that will give rise to part of the primordium of the circular visceral muscles. The circular and longitudinal visceral muscles consist of syncytia. Two different cell populations contribute to the formation of gut muscles: muscle progenitors and fusion-competent cells that form binucleated circular muscle fibers at stage 12. At this time, hand is expressed exclusively in the visceral progenitor cells but not in the fusion-competent myoblasts of the circular visceral mesoderm. When fusion in the circular visceral muscle tissue starts at stage 12, hand expression appears broader and more diffuse, indicating that hand transcripts are present in the syncytial visceral myofibers (Kolsch, 2002).
At stage 12 hand expression is initiated in the heart primordium in segmentally arranged groups of two to five cells. Shortly thereafter they form a continuous row that gives rise to the embryonic heart. At the same time a small group of cells located anterior of the trunk mesoderm starts to express hand. At later stages these cells are associated with the proventriculus and give rise to the garland cells. At stage 13, hand expression is also detectable in the lymph gland cells. These cells are already associated with the developing heart and, as development proceeds, are arranged bilaterally near the anterior end of the heart tube. Expression of hand in the lymph gland cells continues until the end of embryogenesis. Beyond stage 12 hand is also expressed in yet unidentified cells within the CNS. At the end of embryogenesis, hand expression in the visceral mesoderm becomes most prominent in myofibers located at the midgut constrictions. The strongest expression is seen in the heart, the lymph glands and the garland cells (Kolsch, 2002).
The spatial and temporal dynamics of hand expression in the developing heart prompted an analysis to identify hand-expressing cells in more detail. In situ hybridization of whole-mount embryos with cDNA as probe indicates that hand is expressed in the majority of heart cells. Since different cell types contribute to the formation of the embryonic heart, it was of interest to know in detail the identity of hand-expressing cells. To verify when during cardiogenesis hand expression starts, Mef2 and Eve were used as markers. At stage 11, Mef2 expression in the dorsal mesoderm becomes prominent in heart precursors. At this stage hand was not detected in heart cells. A short time later during development, Mef2 and hand are coexpressed in the developing heart. Similar results were obtained with Eve as marker. At early stage 11 Eve is expressed in one somatic muscle progenitor (which gives rise to dorsal muscle 1) and in one pericardial cell that divides into two daughter cells, both of which express Eve and give rise to two pericardial cells (EPC cells). Double staining for hand transcripts and Eve protein reveals that hand is neither coexpressed with Eve in the EPC cells at this stage, nor is there a detectable level of hand expression in neighboring cells (other progenitors of pericardial cells or cardioblasts). From stage 12 onwards, Eve and hand are coexpressed in the developing heart. These results indicate that hand expression starts after the early determination and specification of cardioblasts and pericardioblasts. To identify individual subsets of heart cells at later stages, a set of specific antibodies was used for double-staining experiments and confocal microscopic analysis. Expression of hand was found in all cardioblasts per hemisegment until the end of embryogenesis as indicated by colocalization with Mef2 (all cardioblasts) and Tinman (four out of six cardioblasts). Furthermore, confocal microscopic analysis revealed that hand is expressed in all pericardial cells, as indicated by colocalization with Mab3, Zfh1, Tin and Eve (Kolsch, 2002).
Recently it was shown that the visceral mesoderm originates from at least two different cell types: progenitor cells and fusion-competent myoblasts. Both cell types contribute to the formation of syncytial visceral myofibers and are distinguishable by the expression of specific marker genes. Advantage was taken of a reporter line carrying a lacZ gene under the control of a bagpipe enhancer and an antibody against Tinman that stains a subset of visceral cells. Thus, the spatial and temporal expression of hand was analyzed during visceral mesoderm differentiation. At early stage 11, when bagpipe/lacZ-positive cells of the visceral mesoderm are arranged in segmental groups, hand transcripts are not present at a detectable level, neither in the primordium of the circular visceral muscles nor in the caudal mesoderm. At mid stage 11, when bagpipe/lacZ-expressing progenitors of the circular visceral muscles have formed a continuous stripe, hand is strongly expressed in the trunk mesoderm. Coexpression with bagpipe/lacZ is exclusively observed in the distally located visceral progenitors (lower level of bagpipe/lacZ expression), but not in the fusion-competent cells. In the caudal mesoderm, giving rise to the progenitors of the longitudinal visceral muscles, hand is not expressed, neither at stage 11 nor later. hand and bagpipe/lacZ are coexpressed in all circular visceral muscle progenitors at mid stage 11. At this stage, tinman shows a transient and segmentally interrupted expression in a subset of visceral cells. Recently it was shown that the segmented expression pattern of connectin in the visceral mesoderm is dependent on the intersecting influence of Wingless and Decapentaplegic. The circular visceral muscle progenitor population consists of two distinct cell types at this time. All progenitor cells coexpress bagpipe and hand, but only a subset of cells coexpresses bagpipe, tinman and hand, suggesting specific functional properties of both cell types. When syncytial circular muscles start to form at stage 12, hand expression is still strong. At stage 13 and 14 hand is found in all circular myofibers, as shown by colocalization with bagpipe-lacZ. At stage 16 hand transcripts are still detectable in circular visceral myofibers with highest concentration in fibers near the midgut constrictions (Kolsch, 2002).
In the Drosophila larvae, hematopoiesis takes place in the lymph glands that consist of five pairs of lobes associated with the heart. Precursors of the larval lymph gland are first seen during embryogenesis where they are located in close proximity to the heart in two clusters of about 20 cells in the second thoracic segment. The lymph gland cells start to express hand after their association with the developing heart. As development proceeds, the number of hand-expressing lymph gland cells increases to about 20 on each side of the heart. The garland cells have a so far unidentified function in the fly. They appear during embryogenesis in close connection to the foregut-midgut transition and later during development form a U-shaped cluster around the proventriculus. Pericardial cells and garlands cells show a morphologically and ultrastructurally similar appearance and it was speculated that both cell types might function as nephrocytes. Expression of hand is clearly detectable in garland cells at stage 11/12. The expression continuous until the end of embryogenesis when hand-positive cells are clustered around the proventriculus (Kolsch, 2002).
Drosophila Hand is expressed in a specific pattern in the cardiogenic mesoderm. Hand expression is initiated in the cardiogenic region at late stage 12, immediately following the differentiation of Even-skipped (Eve)-positive mesodermal progenitors into segmentally repeated Eve pericardial cells (EPCs) and DA1 muscles; this differentiaion marks the completion of progenitor cell divisions that give rise to the cardioblasts and pericardial nephrocytes (Han, 2003). Cardiac expression of Hand is initially weak and segmental, but soon becomes strong in most cardioblasts and pericardial cells from stage 13. At the end of embryogenesis, when the heart is completely formed, Hand is expressed in all the cardioblasts that also express Dmef2 and in all the pericardial nephrocytes that express even-skipped (eve) (Han, 2005).
At stage 15, tin is expressed in four of the six cardioblasts in each hemisegment from segment A1 to A5, and all the Eve-positive pericardial cells, as well as all cardioblasts from segment T2 to T3, but not in the lymph gland. Hand expression is detected in all the Tinman-positive cardiac cells. Hand is likely to be expressed in all the pericardial nephrocytes since all Zfh-1-positive pericardial cells express Hand. odd-skipped (odd) is expressed in both the lymph gland hematopoietic progenitor cells and a subset of pericardial nephrocytes. Hand expression is also detected in all the Odd-skipped-positive hematopoietic progenitors and pericardial nephrocytes. In addition, Hand is co-expressed with Serpent in all the lymph gland progenitors. The secreted extracellular protein Pericardin (Prc) labels the ring gland and the extracellular matrix surrounding the pericardial nephrocytes. Hand expression is not detected in the ring gland, but Hand-expressing cells are surrounded by Prc from segment T2-A6. Hand expression also appears in the visceral mesoderm, the garland cells and in a subset of central nervous system cells (Han, 2005).
To examine the functions of Hand in vivo, a null mutant of the gene was generated by replacing it with a mini-white gene using the ends-out homologous recombination technology. Five independent homozygous lethal lines were generated with a trans-location of the mini-white gene from the 3rd chromosome where it was originally located to the 2nd chromosome where the Hand gene resides. Four out of these five lines failed to complement a deficiency line that deletes the Hand locus (BL-7819). RT-PCR from homozygous mutant larvae from these four independent lines, identified by the absence of a GFP-positive balancer chromosome, showed a loss of Hand transcripts. Hand transcripts were also undetectable by in situ hybridization of homozygous Hand mutant embryos, identified by the absence of a ß-Gal-positive balancer chromosome, further demonstrating that the Hand mutation results in a null allele. Sequencing of genomic PCR products demonstrated that expected homologous recombination occurred identically in these four independent mutant lines (Han, 2006).
Most homozygous Hand mutants, identified by the absence of a GFP-positive balancer chromosome, died during late embryonic and early larval stages. About 40% of the homozygous mutant embryos failed to hatch. The remaining 60% of mutant embryos hatched as 1st-instar larvae, but the majority died within 24 hours of hatching. All Hand mutant larvae were less active and smaller than normal. A small number of escapers (~3%) survived for a few days after hatching, but they were sluggish and remained as small as 1st-instar larvae (Han, 2006).
Approximately 20% of Hand mutant embryos showed a range of cardiac morphological defects that included discontinuities and irregularities in the architecture of the heart tube, shown by the misalignment of Mef2-expressing cardioblasts, reduced numbers of pericardial nephrocytes, shown by Odd-skipped (Odd) expression, and random gaps in expression of the secreted extracellular matrix protein Pericardin. A small subset of mutant embryos (~3%) showed more severe cardiac defects characterized by a significant reduction of Mef2-expressing cardioblasts, Odd-expressing pericardial cells and Pericardin expression. In addition, the number of lymph gland hematopoietic cells was reduced in more than half of Hand mutant embryos. In many of these mutants, the lymph gland cell clusters labeled by Odd antibody were completely absent, whereas the ring gland, which is located anterior to the lymph gland and is labeled by the Pericardin antibody, was intact (Han, 2006).
About 80% of Hand mutant embryos showed normal embryonic heart development and 60% of Hand mutants hatched to become 1st-instar larvae. In order to examine for possible abnormalities in larval cardiac morphology, the Hand-GFP transgene was crossed into the Hand mutant background. Recent work has shown that the Drosophila heart undergoes dramatic cardiac remodeling during late larva and early pupa development. However, little is known about the cardiac morphological changes during the early larval stages because of the lack of markers of the living heart and the inaccessibility of antibodies at larval stages. The Hand-GFP transgene strongly labels the entire heart from embryos to adults, providing an opportunity to examine the cardiac morphological changes during the late embryo and early larva transition by confocal microscopy. At 18 hours after egg laying (AEL), cardioblasts and pericardial cells are well aligned at the dorsal midline in wild type and a majority of Hand mutants. The number of lymph gland hematopoietic cells flanking the anterior aorta is largely reduced in most Hand mutants. At around 20 hours AEL, cardioblasts and pericardial cells in wild-type larvae no longer align in perfect rows, as the cardioblasts start to form the heart tube and the pericardial nephrocytes start to migrate to their final positions around the heart tube. A subset of Hand mutants start to show defects around this time with a reduced number of pericardial cells and thinner heart tube. The cardiac morphological defects of Hand mutants become more significant around 24 hours AEL, when 1st-instar larvae hatch from the cuticle. In wild-type 1st-instar larvae, a chamber-like structure is seen in the posterior heart and the size of the pericardial nephrocytes is significantly enlarged. By contrast, most newly hatched Hand mutant 1st-instar larvae display a hypoplastic heart with an abnormally thin heart tube and further reduced numbers of pericardial cells, as well as gaps in the posterior heart tube. Higher magnitude confocal scans show the lymph gland cell clusters flanking the anterior opening of the aorta, and the three-dimensional structures of the posterior heart. In wild-type 1st-instar larvae, the posterior heart tube forms two chamber-like structures flanked by two pairs of ostias and the highly organized posterior heart tip. By contrast, the lymph gland is completely absent or largely reduced in most Hand mutant 1st-instar larvae. The three-dimensional chamber-like structure of the posterior heart is also dramatically disrupted in Hand mutant larvae. Most pericardial nephrocytes were also missing at 26 hours AEL (Han, 2006).
To determine whether ectopic cell death might account for the loss of lymph gland hematopoietic progenitors and pericardial nephrocytes in Hand mutants, apoptosis was examined in Hand mutant embryos by TUNEL labeling. Occasional TUNEL-positive cells could be observed around the heart in 16 hour AEL wild-type embryos. By contrast, ectopic apoptotic cells were found in regions normally occupied by lymph gland hematopoietic progenitors and pericardial cells in more than 30% of Hand mutant embryos. TUNEL-positive cells were also found among the cardioblasts in a subset of Hand mutant embryos. These data suggest that Hand is required for the survival of cardioblasts, pericardial cells and lymph gland hematopoietic progenitors (Han, 2006).
To test whether inhibiting apoptosis in the lymph gland and hearts of Hand mutants might rescue the Hand mutant phenotypes, the apoptosis inhibitor P35, which prevents cell death by inactivating effector caspases, was overexpressed in the heart using Hand-Gal4. P35 has been shown to be an efficient caspase suppressor in Drosophila cells. Targeted expression of P35 in Hand-expressing cells alone did not evoke any phenotypes, whereas targeted expression of P35 in Hand mutant embryos prevented ectopic apoptosis, as well as the phenotype of reduced lymph gland hematopoietic progenitors and pericardial nephrocytes in late stage embryos. Targeted overexpression of P35 also delayed but did not prevent the larval lethality in Hand mutants. At 18 hours AEL, Hand mutant larvae with targeted P35 expression start to display an abnormal appearance. At 24 hours AEL, these larvae develop thin hypoplastic heart and reduced lymph gland hematopoietic progenitors similar to, but less severe than, that of Hand mutant larvae (Han, 2006).
To confirm that the phenotypes of the Hand null mutant are due solely to the absence of Hand, wild-type Hand was specifically overexpressed in Hand mutants using Hand-Gal4. Wild type Hand was able to completely rescue the phenotype and lethality of Hand mutants. Human HAND2 was overexpressed in Drosophila Hand mutants using Hand-Gal4. Control experiments showed that transgenic expression of human HAND2 in wild-type flies caused no abnormalities. Remarkably, expression of human HAND2 in the Hand mutant background effectively rescued the cardiac and lymph gland defects, such that almost all mutant embryos hatched and developed to 1st-instar larvae with nearly normal hearts and lymph glands. Hand mutant larvae rescued by targeted expression of human HAND2 survived up to 6 days and developed a fairly normal heart and lymph gland at 24 hours AEL, suggesting an evolutionary conserved role of HAND factors in cardiogenesis and hematopoiesis (Han, 2006).
Reference names in red indicate recommended papers.
Aiyer, A. R., Honarpour, N., Herz, J. and Srivastava, D. (2005). Loss of Apaf-1 leads to partial rescue of the HAND2-null phenotype. Dev. Biol. 278: 155-162. 15649468
Angelo, S., Lohr, J., Lee, K. H., Ticho, B. S., Breitbart, R. E., Hill, S., Yost, H. J. and Srivastava, D. (2000). Conservation of sequence and expression of Xenopus and zebrafish dHand during cardiac, branchial arch and lateral mesoderm development. Mech. Dev. 95: 231-237. 10906469
Biben, C., et al. (1997). Homeodomain factor Nkx2-5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development. Genes Dev. 11(11): 1357-1369.
Bounpheng, M. A., Morrish, T. A., Dodds, S. G. and Christy. B. A. (2000). Negative regulation of selected bHLH proteins by eHAND. Exp. Cell Res. 257(2): 320-31. 10837146
Bruneau, B. G., et al. (2000). Cardiac expression of the ventricle-specific homeobox gene Irx4 is modulated by Nkx2-5 and dHand. Dev. Biol. 217: 266-277. 10625552
Cserjesi, P., Brown, D., Lyons, G. E. and Olson, E. N. (1995). Expression of the novel basic helix-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development. Dev. Biol. 170(2): 664-78. 7649392
Dai, Y. S. and Cserjesi. P. (2002). The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer. J. Biol. Chem. 277(15): 12604-12. 11812799
D'Autreaux, F., Morikawa, Y., Cserjesi, P. and Gershon, M. D. (2007). Hand2 is necessary for terminal differentiation of enteric neurons from crest-derived precursors but not for their migration into the gut or for formation of glia. Development 134(12): 2237-49. Medline abstract: 17507395
Davidson, B. and Levine, M. (2003). Evolutionary origins of the vertebrate heart: Specification of the cardiac lineage in Ciona intestinalis. Proc. Natl. Acad. Sci. 100: 11469-11473. 14500781
Firulli, A. B., McFadden, D. G., Lin, Q., Srivastava, D. and Olson, E. N. (1998). Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1. Nat. Genet. 18: 266-270. 9500550
Firulli, B. A., Hadzic, D. B., McDaid, J. R. and Firulli, A. B. (2000). The basic helix-loop-helix transcription factors dHAND and eHAND exhibit dimerization characteristics that suggest complex regulation of function. J. Biol. Chem. 275(43): 33567-73. 10924525
Fukushige, T., Brodigan, T. M., Schriefer, L. A., Waterston, R. H. and Krause, M. (2006). Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development. Genes Dev. 20: 3395-3406. Medline abstract: 17142668
Han, Z. and Bodmer, R. (2003). Myogenic cells fates are antagonized by Notch only in asymmetric lineages of the Drosophila heart, with or without cell division. Development 130: 3039-3051. 12756185
Han, Z. and Olson, E. N. (2005). Hand is a direct target of Tinman and GATA factors during Drosophila cardiogenesis and hematopoiesis. Development 132: 3525-3536. 15975941
Han, Z., Yi, P., Li, X. and Olson, E. N. (2006). Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis. Development 133(6): 1175-82. 16467358
Hill, A. A. and Riley, P. R. (2004), Differential regulation of Hand1 homodimer and Hand1-E12 heterodimer activity by the cofactor FHL2. Mol. Cell. Biol. 24(22): 9835-47. 15509787
Kolsch, V. and Paululat, A. (2002). The highly conserved cardiogenic bHLH factor Hand is specifically expressed in circular visceral muscle progenitor cells and in all cell types of the dorsal vessel during Drosophila embryogenesis. Dev. Genes Evol. 212: 473-485. 12424518
Mandal, L., Banerjee, U. and Hartenstein, V. (2004). Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm. Nat. Genet. 36: 1019-1023. 15286786
McFadden, D. G., Charite, J., Richardson, J. A., Srivastava, D., Firulli, A. B. and Olson, E. N. (2000). A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart. Development 127: 5331-5341. 12070084
McFadden, D. G., Barbosa, A. C., Richardson, J. A., Schneider, M. D., Srivastava, D. and Olson, E. N. (2005). The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development 132: 189-201. 15576406
Murakami, M., Kataoka, K., Fukuhara, S., Nakagawa, O. and Kurihara, H. (2004a). Akt-dependent phosphorylation negatively regulates the transcriptional activity of dHAND by inhibiting the DNA binding activity. Eur. J. Biochem. 271(16): 3330-9. 15291810
Murakami, M., Kataoka, K., Tominaga, J., Nakagawa, O. and Kurihara, H. (2004b). Differential cooperation between dHAND and three different E-proteins. Biochem. Biophys. Res. Commun. 323(1): 168-74. 15351717
Popichenko, D., Sellin, J., Bartkuhn, M. and Paululat, A. (2007). Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation. BMC Dev. Biol. 7: 49. Medline abstract: 17511863
Srivastava, D., Cserjesi, P. and Olson, E. N. (1995). A subclass of bHLH proteins required for cardiac morphogenesis. Science 270: 1995-1999. 8533092
Srivastava, D., Thomas, T., Lin, Q., Kirby, M. L., Brown, D. and Olson, E. N. (1997). Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat. Genet. 16: 154-160. 9171826
Tao, Y., Wang, J., Tokusumi, T., Gajewski, K. and Schulz, R. A. (2007). Requirement of the LIM homeodomain transcription factor tailup for normal heart and hematopoietic organ formation in Drosophila melanogaster. Mol. Cell. Biol. 27(11): 3962-9. Medline abstract: 17371844
Thomas, T., Yamagishi, H., Overbeek, P. A., Olson, E. N. and Srivastava, D. (1998). The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196: 228-236. 9576835
Yamagishi, H., et al. (2001). The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev. Biol. 239(2): 190-203. 11784028
Yelon, D., Ticho, B., Halpern, M. E., Ruvinsky, I., Ho, R. K., Silver, L. M. and Stainier, D. Y. (2000). The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development 127: 2573-2582. 10821756
Zang, M. X., Li, Y., Xue, L. X., Jia, H. T. and Jing, H. (2004). Cooperative activation of atrial naturetic peptide promoter by dHAND and MEF2C. J. Cell Biochem. 93(6): 1255-66. 15486975
date revised: 5 October 2007
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