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).
In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).
Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).
A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).
In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).
A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. Movies are provided to demonstrate the location and the beating of wing hearts (Tögel, 2008).
The hand-C-GFP reporter was tested for expression in earlier stages of wing heart development and it was found to be active throughout the entire organogenesis. This enabled identification of the embryonic anlagen of the wing hearts, which consist of eight progenitor cells located dorsally and anterior to the heart, in two pairs in the second and third thoracic segment from stage 16/17 onward. The progenitors exhibit a flattened triangular shape and are interconnected by thin cytoplasmic extensions. In addition, the second and the fourth pair of the progenitors are closely associated with the dorsal tracheal branches at their interconnection in the second and third thoracic segments. The characteristic pairwise arrangement and the connection to the tracheae are retained during the subsequent three larval stages. Proliferation starts at about the transition from the second to the third larval instar, leading to eight clusters of cells that remain arranged in four pairs in the anterior region until 1h after puparium formation (APF). Between 1 and 10h APF, the cell number increases significantly and the anterior three pairs of cell clusters are retracted to join the last pair of clusters, eventually forming one large median cluster. Between 13 and 50h APF, the single large cluster splits along its anterior-posterior axis into two groups of cells that migrate laterally in the forming scutellum, thereby adopting the characteristic arched appearance of the adult wing hearts. During this process some of the cells on either side form the underlying thin layer while the remaining cells arrange in horizontal rows along that layer. First contractions of the mature organs were observed at about 45-50h APF (Tögel, 2008).
The expression of the bHLH transcription factor Hand in the wing heart progenitors, which serves as a general marker for all classes of heart cells in Drosophila, prompted a to screen for the expression of genes known to be active in cardiac lineages. Analysis of Even-skipped (Eve) expression revealed that the embryonic wing heart progenitors arise through the same lineage as the well described Eve expressing pericardial cells (EPCs). At stage 10 in embryogenesis, 12 Eve clusters are present on either side of the embryo, located in parasegments (PS) 2 to 12. Each cluster gives rise to a pair of EPCs, except for the most posterior cluster in PS 14, which generates only one EPC. During subsequent development, the first and the second pair of EPCs, located in parasegment 2 and 3, turn toward the midline of the embryo to accompany the tip of the heart, which later bends ventrally into the embryo. The third and the fourth pair of EPCs in PS 4 and 5 are shifted anteriorly in relation to the heart. This step is not based on migration but on the remodeling of the embryo during head involution, since the cells remain in their PS close to the likewise Eve positive anlagen of the DA1 muscle. The EPCs in PS 4 and 5 subsequently differentiate into the later wing heart progenitors, while all others become the classical EPCs and accompany the heart in a loosely associated fashion. At least from PS 4 to 12, all pairs of Eve positive cells (wing heart progenitors and classical EPCs) are interconnected by cytoplasmic extensions forming a rope ladder-like strand above the heart after dorsal closure at stage 16/17. This mode of contact between the cells persists in the wing heart progenitors in postembryonic stages and might be essential for proper relocation in the prepupae (Tögel, 2008).
Although the Drosophila wing hearts have been known for many years, their origin and development have remained unknown. This study provides the first developmental approach on these organs using in vivo time lapse imaging as well as genetic and immunohistochemical methods. It was found that the wing hearts develop from embryonic anlagen that consist of eight progenitor cells located anterior to the heart. Analysis of gene expression in these progenitors confirmed the hypothesis that the wing hearts originate from the cardiac mesoderm, but not from the contractile cardioblast lineage, as has been suggested based on anatomical data. Surprisingly, the embryonic anlagen derive from a particular subset of the well-known EPCs. EPCs arise in pairs in PS 2 to 12 from the dorsal progenitor P2, which divides asymmetrically into the founder of the dorsal oblique muscle 2 and the founder of the EPCs in a numb-dependent lineage decision. Additionally, a single EPC arises in PS 14. The subsequent differentiation of the founders into EPCs requires the activity of the transcription factors Zfh1 and Eve. This study shows that the EPCs located in PS 4 and 5 are relocated in relation to the heart during head involution at stage 14/15 of embryogenesis and subsequently differentiate into the wing heart progenitors. Until this step, no difference to the EPCs in the anterior and posterior PS could be detected. Like the classical EPCs, which remain close to the heart, the EPCs that give rise to the wing heart progenitors depend on factors involved in asymmetric cell division, e.g. Insc or Numb, and fail to differentiate in embryos mutant for zfh1 as well as in animals lacking mesodermal Eve. Loss of tinman expression is the only event that could be identified that discriminates between a classical EPC fate and the specification of wing heart progenitors. Consistently, ectopic expression of Tinman in the wing heart progenitors effectively represses their specification, probably by committing them to a classical EPC fate, indicating that Tinman plays a crucial role in the involved regulatory pathway (Tögel, 2008).
So far, the biological role of pericardial cells (PCs), and EPCs in particular, is not well understood. In the embryo, three populations of PCs arise in each segment, which are characterized by the expression of different combinations of genes (Odd positive PCs, Eve positive PCs, and Tinman positive PCs). During postembryonic stages, the number of PCs decreases, raising the question which population contributes to the final set of PCs in the adult and whether all PCs have the same function throughout development. Recent studies have shown that postembryonic PCs express Odd and Eve, a combination which is not observed in the embryo, and are dispensable for cardiac function. Genetic ablation of all larval PCs had no effect on heart rate, but increased sensitivity to toxic stress. In contrast, the specification of the correct number of embryonic PCs is crucial for normal heart function. Loss of mesodermal Eve during embryogenesis results in fewer larval pericardial cells, which causes a reduction in heart rate and lifespan. Conversely, hyperplasia of embryonic PCs has no effect on heart rate but causes decreased cardiac output. This was explained by an excess of Pericardin secreted by the PCs into the extracellular matrix enveloping the heart (Johnson, 2007). Taken together, embryonic PCs seem to influence cardiac development by e.g., secreting substances whereas postembryonic PCs function as nephrocytes. However, in this study, functional data is provided on a subset of embryonic EPCs, which differentiate into adult progenitors giving rise to a myogenic lineage. This represents a completely new function of PCs, raising the question whether EPCs might in general have myogenic potential and rather represent a population of adult progenitors, than PCs in a functional sense (Tögel, 2008).
The organogenesis of the wing hearts is a highly dynamic process, which includes distinct cellular interactions. At first, adjacent EPCs (including the wing heart progenitors) on either side of the embryo establish contact via cytoplasmic extensions. After dorsal closure of the embryo, interconnections are also formed between opposing EPCs resulting in a rope ladder-like strand above the heart. These interconnections are assumed to be needed to retain contact between the wing heart progenitors during the subsequent development. During larval stages, some of the wing heart progenitors establish a second contact to specific tracheal branches and proliferation starts. In the prepupa, a relocation event joins all wing heart progenitors in one large cluster. During this step, the progenitors are probably passively relocated in conjunction with the tracheal branches to which they are connected. Finally, the wing heart progenitors initiate active migration and form the mature wing hearts in the pupa. Considering the complexity of their development, it is proposed that wing hearts provide an ideal model for studying organogenesis on several different levels such as signaling, cell polarity, or path finding (Tögel, 2008).
Elimination of the embryonic progenitors by ectopic expression of tinman or by laser ablation causes the loss of wing hearts, which results in a specific wing phenotype in conjunction with flightlessness. In the identified phenotype, the delaminated epidermal cells are not cleared from the wings during wing maturation and bonding of the dorsal and ventral wing surfaces is omitted. Recently, it was reported that the epidermal cells transform into mobile fibroblasts and actively migrate out of the wings. However, in in vivo time-lapse studies migration of epidermal cells could not be observed during wing clearance. Conversely, their movements correlated with the periods of wing heart beating, indicating that they are passively transported by the hemolymph flow. One-sided ablation of mature wing hearts in pupae, confirms that wing hearts play a crucial physiological role in wing maturation, since the wing phenotype occurs only on the treated side, but in the same genetic background. In contrast, mutations in genes coding for proteins involved in cell adhesion, e.g. integrins, or in adhesion to the extra cellular matrix, cause a blistered wing phenotype. In the latter phenotype, the epidermal cells of the immature wings are not attached to their opposing cells or to the cuticle and the wing surfaces are separated during unfolding by the sudden influx of hemolymph. In contrast, in animals lacking wing hearts the wings resemble those of the wild-type shortly after unfolding. The epidermal cells also delaminate later from the cuticle, as indicated by their disarrayed pattern, but are not removed from the wings due to the missing hemolymph circulation and probably impede spatially the bonding of the dorsal and the ventral cuticle. Thus, the wings remain in their immature state and do not acquire aerodynamic properties, which accounts for the flightlessness. It is concluded that wing hearts are crucial for establishing proper wing morphology and functionality in Drosophila (Tögel, 2008).
Wing hearts occur in all winged insects, but differ considerably in their morphology. However, their function is highly conserved, since they all function as suction pumps that draw hemolymph from the wings. In the basal condition, the heart itself is directly connected to the scutellum and constitutes the pump. This connection was lost several times during evolution and other muscles, e.g. the separate wing hearts in Drosophila, were recruited to retain the function indicating a high selection pressure on wing circulation. It is suggested that this is due to the crucial role of wing hearts during wing maturation. Since proper wing morphogenesis is essential for flight ability, insect flight might not have been possible before the evolution of wing hearts (Tögel, 2008).
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).
The Hand proteins of the bHLH family of transcriptional factors play critical roles in vertebrate cardiogenesis. In Drosophila, the single orthologous Hand gene is expressed in the developing embryonic dorsal vessel (heart), lymph glands, circular visceral musculature, and a subset of CNS cells. The absence of Hand activity causes semilethality during the early larval instars. The dorsal vessel and midgut musculature are unaffected in null mutant embryos, but in a large fraction the lymph glands are missing. However, homozygous adult flies lacking Hand possess morphologically abnormal dorsal vessels characterized by a disorganized myofibrillar structure, reduced systolic and diastolic diameter, and abnormal heartbeat contractions, and suffer from premature lethality. In addition, their midguts are highly deformed; in the most severe cases, there is midgut blockage and a massive excess of ectopic peritrophic membrane tubules exiting a rupture in an anterior midgut bulge. Nevertheless, the visceral musculature appears to be relatively normal. Based on these phenotypes, it is concluded that the expression of the Drosophila Hand gene in the dorsal vessel and circular visceral muscles is mainly required during pupal stages, when Hand participates in the proper hormone-dependent remodeling of the larval aorta into the adult heart and in the normal morphogenesis of the adult midgut endoderm during metamorphosis (Lo, 2007).
Of the embryonic tissues that express Hand, only a moderately penetrant loss of the lymph glands was observed in Hand mutant embryos. Neither the dorsal vessel (heart) nor the circular visceral musculature is morphologically affected in these embryos, indicating that Hand is not necessary for the proper embryonic morphogenesis of these tissues, though it is possible that it is required for their normal physiological function and that the loss or reduction of this function may result in the early larval semilethality. The apparent correlation of the fact that a large fraction of late stage Hand embryos lack lymph glands with the high rate of semilethality for early instar larvae, coupled with the observation that surviving 3rd instar Hand173 larvae all appear to possess lymph glands, would seem to imply that the loss of lymph glands is causing larval death due to a deficit in immune function. However, this is unlikely since the hemocytes of the larva arise from the head mesoderm and not the lymph gland, which supplies the hemocytes of the adult. It may be possible that the early larval semilethality is caused by the loss or functional disruption of the subset of CNS cells that express Hand in the late embryo, which was not examined, or by the loss of an unknown function of the lymph gland (Lo, 2007).
The observed adult phenotypes demonstrate a requirement for Hand in the proper morphogenesis of the adult heart and midgut. Adult Hand mutant dorsal vessels are clearly morphologically abnormal, which would reduce cardiac output, and the initiation of heartbeat contraction is also abnormal, which may additionally affect cardiac function. The resulting reduced cardiac output of the Hand adult dorsal vessel is presumed to contribute to the high rate of premature adult mortality seen in Hand mutants. This is supported by the recent demonstration that flies lacking Tinman expression in the dorsal vessel possess morphologically and functionally abnormal adult dorsal vessels and also suffer from a reduced adult lifespan (Lo, 2007).
Since 3rd instar larval dorsal vessels are morphologically and functionally normal, it appears that the morphological defects seen in Hand mutant adult dorsal vessels only arise during metamorphosis, when the adult heart is remodeled from the larval aorta. Hence, it is concluded that pre-adult Hand expression is required for the proper morphogenesis of the adult heart during metamorphosis but not of the late embryonic/early larval dorsal vessel. No evidence is seen for a function of Hand in regulating the morphogenesis of the late embryonic/early larval dorsal vessel (Lo, 2007).
The other major phenotype in Hand adults is malformations of the endoderm of the midgut. Virtually all Hand mutant adults exhibit an anterior midgut bulge, with the remainder of the midgut exhibiting differing degrees of shrinkage and occlusion of the midgut lumen. This occlusion is most relevant to the premature adult mortality since it would prevent the passage of food through the gut and severely reduce or totally prevent the absorption of nutrients from ingested food, resulting in starvation. This is probably a major factor in the high rate of premature Hand adult mortality (Lo, 2007).
Hand is not expressed in the embryonic endoderm and based on the expression of Hand-GFP, which recapitulates the embryonic expression of Hand and is present in the circular visceral musculature through adulthood, it appears most likely that Hand is also not expressed in the endoderm post-embryonically. As the endoderm of the adult gut is reconstituted during metamorphosis from imaginal precursor cells whereas the larval gut musculature persists and appears normal in Hand mutants, it would follow that normal morphogenesis of the imaginal endoderm is dependent on pre-adult Hand expression in the circular visceral musculature, perhaps through Hand-dependent inductive signals from the muscle to the endoderm (Lo, 2007).
Genetic analysis of the function of the Drosophila Hand gene has demonstrated that it is necessary for the proper morphogenesis of the adult dorsal vessel and midgut, which occurs through the remodeling of the corresponding larval organs during metamorphosis. The morphological defects in adult Hand dorsal vessels and the hearts of Hand1 knockout/Hand2 conditional double mutant mice suggest that the role of the Hand gene in regulating cardiac morphogenesis has been conserved to some degree during evolution. Therefore, further analysis of how the Drosophila Hand gene functions molecularly and genetically should prove useful in elucidating the possibly equivalent, evolutionarily conserved functions of the vertebrate Hand genes in cardiac morphogenesis. If the adult midgut phenotype of Drosophila Hand mutants is due to an indirect effect of the loss of Hand function in the morphologically normal circular visceral musculature, this is a phenotype without an equivalent in the vertebrate Hand mutant mice. Like Drosophila Hand, mouse Hand1 is also expressed only in the intestinal smooth muscle layer of the gut during mouse embryogenesis; however, any effect of the loss of Hand1 function in the mouse embryonic smooth muscle layer on endoderm development in Hand1 knockout mice may be masked by the early embryonic lethality that is the result of extra-embryonic defects. It would therefore be interesting to observe if tissue-specific loss of Hand1 function in mouse embryonic intestinal smooth muscle utilizing the Hand1 conditional knockout allele would have an effect on mouse embryonic endodermal development (Lo, 2007).
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. PubMed Citation: 9192865
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
Funato, N., et al. (2009). Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA binding of Runx2. Development 136(4): 615-25. PubMed Citation: 19144722
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
Johnson, A. N., et al. (2007). Defective decapentaplegic signaling results in heart overgrowth and reduced cardiac output in Drosophila. Genetics 176: 1609-1624. PubMed Citation: 17507674
Kimura, K., Kodama, A., Hayasaka, Y. and Ohta, T. (2004). Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster. Development 131: 1597-1606. PubMed Citation: 14998927
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
Krenn, H. W. and Pass, G. (2005). Morphological diversity and phylogenetic analysis of wing circulatory organs in insects, part II: Holometabola. Zoology 98: 147-164.
Lei, J. and Howard, M. J. (2011). Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis. Development 138(21): 4789-800. PubMed Citation: 21989918
Liu, N., et al. (2009). DNA binding-dependent and -independent functions of the Hand2 transcription factor during mouse embryogenesis. Development 136(6): 933-42. PubMed Citation: 19211672
Lo, P. C. H., et al. (2007). The Drosophila Hand gene is required for remodeling of the developing adult heart and midgut during metamorphosis. Dev. Biol. 311: 287-296. PubMed Citation: 17904115
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
Pass, G. (2000). Accessory pulsatile organs: evolutionary innovations in insects. Annu. Rev. Entomol. 45: 495-518. PubMed Citation: 10761587
Pass, G., et al. (2006). Phylogenetic relationships of the orders of Hexapoda: contributions from the circulatory organs for a morphological data matrix. Arthropod. Syst. Phylogeny 64: 165-203.
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
Sellin, J., Albrecht, S., Kölsch, V. and Paululat, A. (2006). Dynamics of heart differentiation, visualized utilizing heart enhancer elements of the Drosophila melanogaster bHLH transcription factor Hand. Gene Expression Patterns 6: 360-375. PubMed Citation: 16455308
Shin, M., Nagai, H. and Sheng, G. (2009). Notch mediates Wnt and BMP signals in the early separation of smooth muscle progenitors and blood/endothelial common progenitors. Development 136(4): 595-603. PubMed Citation: 19168675
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
Talbot, J. C., Johnson, S. L. and Kimmel, C. B. (2010). hand2 and Dlx genes specify dorsal, intermediate and ventral domains within zebrafish pharyngeal arches. Development 137(15): 2507-17. PubMed Citation: 20573696
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
Tögel, M., Pass, G. and Paululat, A. (2008). The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation. Dev. Biol. 318(1): 29-37. PubMed Citation: 18430414
Vokes, S. A., Ji, H., Wong, W. H. and McMahon, A. P. (2008). A genome-scale analysis of the cis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb. Genes Dev. 22(19): 2651-63. PubMed Citation: 18832070
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
Yin, C., Kikuchi, K., Hochgreb, T., Poss, K. D. and Stainier, D. Y. (2010). Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish. Dev. Cell 18(6): 973-84. PubMed Citation: 20627079
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: 15 December 2011
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