tinman is specifically expressed in mesodermal primordia during a short time period early in development. It first appears at blastoderm stage just before the ventral invagination of the mesoderm and shortly after twist is expressed. tinman is also expressed in a group of cells at the anterior tip of the embryo. During germband elongation [Images] all the mesodermal cells in the segmented part of the embryo express tinman, but soon afterwards tinman becomes restricted to the dorsal mesoderm, which includes the primordia for the visceral musculature and the heart. Prior to muscle differentiation, tinman expression ceases, except for two rows of cells that will be included in the dorsal vessel (Bodmer, 1990).

Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm

The Drosophila lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. An essential genetic switch differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. These findings suggest that there is a close parallel between the molecular mechanisms functioning in the Drosophila cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm (Mandal, 2004).

Blood and vascular cells in the vertebrate embryo are thought to derive from oligopotent progenitor cells, called hemangioblasts, that arise in the yolk sac and in the aorta-gonadal-mesonephros (AGM) mesenchyme. A close relationship between blood and vascular progenitors is well established, but in vivo evidence that a single cell can divide to produce a blood cell and an endothelial cell is lacking in vertebrate systems. Similarly, the molecular mechanism that distinguishes between the two lineages is not well understood. To address these issues in a simple, genetically amenable system, the genetic control of hematopoiesis was analyzed in Drosophila. The results show that there are close lineage relationships between hematopoietic and vascular cells, similar to those present in the AGM of mammalian systems. Evidence is provided for conserved cassettes of transcription factors and signaling cascades that limit the pool of hemangioblastic cells and promote the blood versus vascular fate (Mandal, 2004).

In the mature Drosophila embryo, the lymph gland is formed by a paired cluster of ~20 cells flanking the aorta. The aorta and heart represent a contractile tube lined by a layer of myoepithelial vascular cells called cardioblasts. The cells flanking the aorta and heart posterior to the lymph gland are the pericardial cells, which function as excretory cells (nephrocytes). Lymph gland progenitors express the prohemocyte marker Srp and ultrastructurally resemble prohemocytes that develop at an earlier stage from the head mesoderm. Monitoring expression of the zinc-finger protein Odd-skipped (Odd) shows that the lymph gland originates from the dorsal thoracic mesoderm. Odd is expressed in segmental clusters in the dorsal mesoderm of segments T1-A6. The three thoracic Odd-positive clusters coalesce to form the lymph gland, whereas the abdominal clusters formed the pericardial nephrocytes (Mandal, 2004).

Lymph-gland progenitors, cardioblasts and pericardial cells are closely related by lineage. Labeled 'flipout' (FLP/FRT) clones were induced in embryos aged 3-4 h such that the clones contained only 2-4 cells. Of the two-cell clones, ~50% contained cardioblast and lymph-gland cells; the other clones comprised either cardioblasts or lymph-gland cells alone. Mixed clones were recovered at the late third larval stage. The finding of mixed clones indicates that the cardiogenic mesoderm of D. melanogaster contains oligopotent progenitors that, up to the final division, can give rise both to Srp-positive blood-cell progenitors that form the lymph gland and to vascular cells (Mandal, 2004).

The cardiogenic mesoderm forms part of the dorsal mesoderm, which requires the homeobox protein Tin and the GATA factor Pnr. In embryos with mutations in tin or pnr, the lymph gland was absent. Maintenance of Tin expression in the dorsal mesoderm requires the activity of at least two signaling pathways regulated by Dpp (the Drosophila homolog of transforming growth factor-ß) and Heartless (Htl; one of the D. melanogaster homologs of the FGF receptor); the dependence of cardioblast and pericardial nephrocyte development on these signaling pathways has been documented. Lymph-gland progenitors did not develop in loss-of-function dpp and htl mutants (Mandal, 2004).

Between 6 h and 8 h of development, the dorsal mesoderm splits into the cardiogenic mesoderm and the visceral mesoderm. The cardiogenic mesoderm is regulated positively by Wg and negatively by Notch. Lack of Wg signaling results in the absence of all cardiogenic lineages including lymph gland. Notch signaling has the opposite effect and restricts cardiogenic mesodermal fate. Notch is active in the dorsal mesoderm from 6 h to 10 h of development. Eliminating Notch during the first half of this interval by raising embryos homozygous with respect to the temperature-sensitive allele Nts1 at the restrictive temperature resulted in substantially more cardioblasts, pericardial cells and lymph-gland progenitors (Mandal, 2004).

Lymph-gland progenitors, cardioblasts and pericardial nephrocytes are specified in the cardiogenic mesoderm around the phase of germband retraction 8-10 h after fertilization. At this stage, Tin, which was initially expressed in the whole cardiogenic mesoderm, becomes restricted to a narrow medial compartment containing the cardioblasts. Pnr follows the same restriction. Cells located at a more lateral level in the cardiogenic mesoderm give rise to lymph-gland progenitors (in the thoracic domain) and pericardial nephrocytes (in the abdominal domain) and activate the gene odd. Slightly later, Srp is expressed in lymph-gland progenitors. As reported for the early hemocytes derived from the embryonic head, srp is centrally involved in lymph-gland specification. In srp-null embryos, Odd-expressing cells still formed a lymph gland-shaped cluster flanking the aorta, but these cells also express the pericardial marker Pericardin (Prc), suggesting that they lose some aspects of hemocyte precursor identity or gain properties of nephrocytes. As a countercorrelate, ectopic expression of Srp in the whole cardiogenic mesoderm directed by mef2-Gal4 induces pericardial cells to adopt lymph-gland fate (Mandal, 2004).

Downregulation of tin and pnr in cells in the lateral domain of the cardiogenic mesoderm is essential for lymph-gland specification. Ectopic expression of tin or pnr by twist-Gal4 (or mef2-Gal4) causes a marked reduction in the number of lymph-gland and pericardial cells. The antagonistic effect of tin on lymph-gland progenitors resembles its earlier role in the head mesoderm that gives rise to the larval blood cells; here too, ectopic expression of tin causes a reduction in the number of hemocytes (Mandal, 2004).

Inhibiting tin and upregulating odd and srp requires input from the Notch signaling pathway. A function of Notch at 6-8 h in specification of the cardiogenic mesoderm is described. Reducing Notch function between 8 h and 10 h causes an increase in the number of cardioblasts and a concomitant loss of pericardial and lymph-gland cells. Overexpressing an activated Notch construct causes a marked increase in lymph-gland size. This late requirement for Notch signaling is separable from the earlier role of Notch in restricting the overall size of the cardiogenic mesoderm. Thus, the sum total of cardioblasts and pericardial or lymph-gland cells in Nts1 embryos shifts between 8 h and 10 h and does not differ substantially from that in wild type, whereas a combined effect on cell number and cell fate is seen in embryos with a Notch deletion. In these embryos, the cardiogenic mesoderm is hyperplasic and develops as cardioblasts at the expense of lymph-gland progenitors and pericardial nephrocytes. The dual role of Notch in restricting the numbers of a pluripotent progenitor pool and in distinguishing between the progeny of these progenitors is reminiscent of the function of Notch in sense-organ development (Mandal, 2004).

Lymph-gland formation is restricted to the thoracic region by positional cues that are provided by expression of the homeobox proteins of the Antennapedia and Bithorax complex. Specifically, Ultrabithorax (Ubx), which is expressed in segments A2-A5 of the cardiogenic mesoderm, inhibits lymph-gland formation. Loss of Ubx results in the expansion of the lymph-gland fate into the abdominal segments. Conversely, overexpression of Ubx driven by mef2-Gal4 causes the transformation of lymph-gland progenitors into pericardial nephrocytes (Mandal, 2004).

These findings are suggestive of a model of lymph-gland development in Drosophila that is similar to mammalian hematopoiesis. Lymph-gland progenitors develop as part of the cardiogenic mesoderm that also gives rise to the vascular cells (aorta and heart) and to excretory cells. Similarly, progenitor cells of the blood, aorta and excretory system are closely related both molecularly and developmentally in mammals, where they form part of the AGM. Specification of the cardiogenic mesoderm requires the input of FGF and Wg signaling, as in vertebrate hematopoiesis, where the AGM region is induced in response to several converging signaling pathways including FGF, BMP and Wnt (Mandal, 2004).

The cardiogenic mesoderm in Drosophila evolves from the dorsal mesoderm and requires input from the Htl, Dpp, Wg and Notch (N) signaling pathways. The cardiogenic mesoderm then differentiates into lymph gland, vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes). A subpopulation of cardioblasts and lymph-gland cells is derived from one progenitor (hemangioblast; HB). Essential for the differentiation of the cardiogenic mesoderm is the Notch-Delta (Dl)-dependent restriction of Tin and Pnr to cardioblasts and the expression of Srp in the lymph gland. In vertebrates, similar cell types are derived from a mesodermal domain called the AGM, which also requires the input of FGF, BMP and Wnt signaling. A subset of AGM-derived cells has been proposed to constitute hemangioblasts, which produce blood progenitors and endothelial cells (Mandal, 2004).

These findings show that in Drosophila, the cardiovascular and blood-cell lineages are differentiated by an antagonistic relationship between Tin or Pnr expression in the cardioblasts and Srp expression in the lymph-gland progenitors. In vertebrates, GATA factors also have a pivotal role in specifying different lineages among blood-cell progenitors, although not much is known about what differentiates between blood progenitors as a group and endothelial progenitors. The results indicate that this step is driven by input from the Notch signaling pathway. In the thoracic cardiogenic mesoderm, Notch antagonizes tin and pnr expression and aortic cardioblast formation, and promotes srp expression and the development of lymph-gland progenitors. In vertebrates, Notch signaling is also involved in both blood and vascular development. The role of Notch during AGM morphogenesis remains to be investigated (Mandal, 2004).

Cardioblasts and lymph-gland cells can arise from the division of a single cardiogenic mesodermal cell, which should be called a hemangioblast. A previous study induced clones in the cardiogenic mesoderm but used only Tin as a marker. This study also yielded mixed two-cell clones comprising a cardioblast and a nonlabeled cell, which, in light of the current findings, must be interpreted as a lymph-gland cell. Hemangioblasts have been proposed in vertebrates, although the definitive experiment in which a precursor is marked and its lineage is tracked has not been done. Blast colony-forming cells that give rise to both lineages in vitro and common markers that belong to both cell types in vivo have been identified, but direct evidence for the existence of a common precursor has not yet been found. This study, using genetic analysis of two-cell clones, establishes the existence of such a population in Drosophila. On the basis of these results, and given the conservation of the signaling and transcriptional components described here, the prediction is that many cells of the AGM in vertebrates may give rise to only blood or only vascular cells, but a number of intermixed hemangioblasts may give rise to mixed lineages. Future genetic screens aimed at finding components in early lymph-gland development will probably identify additional pathways and strategies important for vertebrate hematopoiesis (Mandal, 2004).

Subdivision and developmental fate of the head mesoderm in Drosophila

This paper defines temporal and spatial subdivisions of the embryonic head mesoderm and describes the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the'T-bar' of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. This study distinguishes between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that is interpreted as a rudimentary cephalic aorta ('cephalic vascular rudiment'). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells (de Velasco, 2005).

The mesoderm is a morphologically distinct cell layer that can be recognized in early embryos of most bilaterian phyla and that gives rise to tissues interposed between ectodermal and endodermal epithelia, including muscle, connective, blood, vascular, and excretory tissue. Besides the differentiative fate of tissues derived from it, the mesoderm shares several common properties in regard to its formation during gastrulation. The anlage of the mesoderm is sandwiched in between the anlage of the endoderm and the neurectoderm. This has been documented in most detail in anamniote vertebrates, where signals from the vegetal blastomeres (the anlage of the endoderm) act on the adjacent marginal zone of the future ectoderm to induce mesoderm. Although gastrulation proceeds quite differently in arthropods from the way it does in chordates, the proximity of the mesodermal anlage to future endoderm and neurectoderm is conserved, and numerous signaling pathways and transcriptional regulators that share similar function and expression patterns in arthropods and chordates have been identified (de Velasco, 2005 and references therein).

Following gastrulation, the mesoderm is subdivided along the dorso-ventral axis into several subdivisions laid out in a distinct dorso-ventral order. In vertebrates, cells located in the dorsal part of the mesoderm anlage give rise to notochord and somites, which in turn produce muscular, skeletal, and connective tissue. Next to the somitic mesoderm is the intermediate mesoderm that will form the excretory and reproductive system. The ventral mesoderm (lateral plate) gives rise to blood, vascular system, visceral musculature, and coelomic cavity. In arthropods, fundamentally similar mesodermal subdivisions can be recognized, and similarities extend to the relative positions these domains obtain relative to each other and relative to the adjacent neurectoderm. For example, precursors of visceral muscles, vascular system, and blood are at the edge of the mesoderm facing away from the neural primordium (ventral in vertebrates, dorsal in arthropods (de Velasco, 2005 and references therein).

The subdivision of the vertebrate mesoderm into distinct longitudinal tissue columns with different fates is seen throughout the trunk and head of the embryo. However, several significant differences between the head and the trunk are immediately apparent. For example, cells derived from the anterior neurectoderm form the neural crest that migrates laterally and gives rise to many of the tissues that are produced by mesoderm in the trunk. As a result, the fates taken over by the head mesoderm are more limited than those of the trunk mesoderm. In contrast, the head mesoderm produces several unique lineages, such as the heart (cardiac mesoderm) and a population of early differentiating macrophages. Moreover, some of the signaling pathways responsible for inducing different mesodermal fates in the trunk appear to operate in a different manner in the head. A recently described example is the Wnt signal that induces somatic musculature in the trunk, but inhibits the same fate in the head (de Velasco, 2005 and references therein).

The head mesoderm of arthropods, like that of vertebrates, also appears to deviate in many ways from the trunk mesoderm. For example, specialized lineages like embryonic blood cells and nephrocytes forming the subesophageal body (also called garland cells) arise exclusively in the head. That being said, very little is known about how the arthropod head mesoderm arises and what types of tissues derive from it. The existing literature mainly uses histology, which severely limits the possibilities of following different cell types forward or backward in time. In this paper, several molecular markers have been used to initiate more detailed studies of the head mesoderm in Drosophila. The goal was to establish temporal and spatial subdivisions of the head mesoderm and, using molecular markers expressed from early stages onward, to follow the fate of the lineages derived from this embryonic tissue. Besides hemocytes and pharyngeal muscles described earlier, the head mesoderm also gives rise to several other lineages, including visceral muscle, putative vascular cells, nephrocytes, and neuroendocrine cells. The development of the head mesoderm is discussed in comparison with the trunk mesoderm and in the broader context of insect embryology (de Velasco, 2005).

The Drosophila head mesoderm, as traditionally defined, includes all mesoderm cells originating anterior to the cephalic furrow. The formation of the head mesoderm is complicated by the fact that (unlike the mesoderm of the trunk) only part of it invaginates with the ventral furrow; by far, the majority of head mesoderm cells, recognizable in a stage 10 or 11 embryo, segregate from the surface epithelium of the head after the ventral furrow has formed. Another complicating factor is that head mesoderm cells derived from different antero-posterior levels adopt very different fates, unlike the situation in the trunk where mesodermal fates within different segments along the AP axis are fairly homogenous, with obvious exceptions such as the gonadal mesoderm that is derived exclusively from a subset of abdominal segments. Using several different markers, this study has followed the origin, migration pathways, and later, fates of head mesoderm cells (de Velasco, 2005).

The anterior part of the ventral furrow, called primary head mesoderm (PHM) in the following, includes cells that will contribute to diverse tissues, including muscle, hemocytes, endoderm, and several ill-defined cell populations closely associated with the brain and neuroendocrine system. For clarification, the anterior ventral furrow will be divided into the following domains:

The anterior lip of the T-bar (PHMA) is the source of the corpus cardiacum, as well as other gt-positive cells that at least in part end up as nerve cells flanking the frontal connective and frontal ganglion. These cells continue the expression of giant throughout late embryonic development; they represent a hitherto unknown class of nonneuroblast-derived neurons (de Velasco, 2005).

The posterior lip of the T-bar (PHMB) can be followed towards later stages by its continued expression of htl. These cells, called the procephalic somatic mesoderm, form a bilateral cluster that moves dorso-anteriorly into the labrum and becomes the dorsal pharyngeal musculature. Htl expression almost disappears in these cells around late stage 11, but is reinitiated at stage 12 and stays strong until stage 14, when the dorsal pharyngeal muscles differentiate. Many of the genes expressed in the somatic musculature of the trunk and its precursors (Dmef2, beta-3-tubulin) are also expressed in the procephalic somatic mesoderm (de Velasco, 2005).

The part of the ventral furrow posteriorly adjacent to the T-bar (PHMC) expresses srp, forkhead (fkh), and other endoderm/hemocyte markers. After the ventral furrow closes in the ventral midline (stage 7/8), these cells form a compact median mass, most of which represents part of the anterior endoderm that gives rise to the midgut epithelium. Starting at around this stage, the lateral part of the hemocyte-forming 'secondary head mesoderm' ingresses in between the endoderm and the surface ectoderm. It is likely that some of the PHMC cells invaginating already with the ventral furrow, along with the cells that form the anterior endoderm, also give rise to hemocytes. Precursors of hemocytes and midgut are difficult to distinguish during and shortly after ventral furrow invagination since both express srp and other markers shared between hemocytes and midgut precursors. At around stage 9, the two populations of precursors disengage. The endoderm remains a compact mesenchyme attached to the invaginating stomodeum; hemocyte precursors move dorsally and take on the shape of expanding vertical plates interposed in between endoderm and ectoderm (de Velasco, 2005).

Domain PHMD, the short portion of the ventral furrow situated posterior to the endoderm, along with a considerable portion of the mesoderm behind the cephalic furrow, forms the mesoderm of the three gnathal segments (mandible, maxilla, labium). The gnathal mesoderm in many ways behaves like the mesoderm of thoracic and abdominal segments. It gives rise to somatic muscle (the lateral pharyngeal muscles), visceral muscle, and fat body. Unlike trunk mesoderm, gnathal mesoderm does not produce cardioblasts and pericardial cells. Instead, a large proportion of gnathal mesoderm cells, joining the anteriorly adjacent secondary procephalic mesoderm, adopt the fate of hemocytes (de Velasco, 2005).

Besides the ventral furrow, other parts of the ventral procephalon produce head mesoderm in a complex succession of delamination and ingression events. The head mesoderm that forms from outside the ventral furrow will be called 'secondary mesoderm' (SHM) in the following. Based on the time of formation and the position relative to the stomodeum, the following phases and domains of secondary head mesoderm development can be distinguished.

Following the obliteration of the ventral furrow at stage 8, the eSHM delaminates from the ventral surface 'meso-ectoderm' (considering that this epithelium still contains mesodermal progenitors!) flanking the endodermal mass. The eSHM forms two monolayered sheets that gradually move dorsally and posteriorly; by stage 9, the eSHM cells line the basal surface of the emerging head neuroblasts. An undefined number of primary head mesoderm cells derived from domain PHMC of the ventral furrow are mingled together with the eSHM cells. The ultimate fate of the eSHM is that of hemocytes: they express srp, followed slightly later by other blood cell markers (e.g., peroxidasin and asrij). A subset of hemocytes, called crystal cells, derive from precursors that form a morphologically conspicuous cluster at the dorsal edge of the eSHM, identifiable from early stage 10 onward by the expression of lz. The mechanism by which at least part of the eSHM delaminates is unique. Thus, it is formed by the vertically oriented division of the surface epithelium, whereby the inner daughters will become eSHMe and the outer ones ectoderm. The focus of vertical mitosis has named the procephalic domain in which it occurs 'mitotic domain #9' (de Velasco, 2005).

From late stage 9 onward, the early SHMs are followed inside the embryo by the closely adjacent posterior late SHMs. One cluster of posterior late secondary head mesoderm (lSHMp) cells delaminates from the surface epithelium flanking the posterior lip of the stomodeum; a second lSHMp cluster appears at the same stage at a slightly more posterior level. The first cluster seems to contribute to the hemocyte population; the posterior cluster gives rise to the nephrocytes forming the subesophageal body (also called garland cells; labeled by CG32094). Garland cell precursors are initially arranged as a paired cluster latero-ventrally of the esophagus primordium; subsequently, the clusters fuse in the midline and form a crescent underneath the esophagus. Garland cells are distinguished from crystal cells by their size, location, and arrangement: crystal cells are large, round cells grouped in an oblong cloud dorso-anterior to the proventriculus. Garland cells are smaller, closely attached to each other, and lie ventral of the esophagus (de Velasco, 2005).

During stages 10 and 11, cells delaminate beside and anterior to the stomodeum, originating from the anlage of the esophagus and the epipharynx (labrum). These cells, called anterior late secondary head mesoderm cells (lSHMa), can be followed by their expression of tin. Two groups can be distinguished. The tin-positive cells delaminating from the esophageal anlage (es) give rise to the visceral musculature (vm) surrounding the esophagus. These cells lose tin expression soon after their segregation, but can be recognized by other visceral mesoderm markers such as anti-Connectin. More dorsally, in the anlage of the clypeolabrum (cl) delaminate, the dorsal subpopulation of the lSHMas, which rapidly migrates posteriorly on either side and slightly dorsal of the esophagus, can be found. These cells retain expression of tin into the late embryo. They assemble into two longitudinal rows stretching alongside the roof of the esophagus primordium. During late embryogenesis, they move posteriorly along with the esophagus towards a position behind the brain commissure. Many of the tin-positive SHMs apparently undergo apoptosis: initially counting approximately 25 on either side, they decrease to 12-15 at stage 14 to finally form a single, irregular row of about 15 cells total in the late embryo. These cells come into contact with the anterior tip of the dorsal vessel. This formation of previously undescribed cells, for which the term 'procephalic vascular cells', is proposed, is interpreted as a rudiment of the head aorta, which forms a prominent part of the dorsal vessel in many insect groups (de Velasco, 2005).

On the basis of additional molecular markers, the tin-positive procephalic vascular cells are further subdivided into two populations. The first subpopulation expresses the muscle and cardioblast-specific marker Dmef2; the second type is Dmef2-negative. In the dorsal vessel of the trunk, tin-positive cells also fall into a Dmef2-positive and a Dmef2-negative population. Dmef2-positive cells of the trunk represent the cardioblasts, myoendothelial cells lining the lumen of the dorsal vessel. Dmef2-negative/tin-positive cells form a somewhat irregular double row of cells attached to the ventral wall of the dorsal vessel. The ultimate fate of these cells has not been explored yet. However, preliminary data suggest that they develop into a muscle band that runs alongside the larval dorsal vessel. This would correspond to the situation in other insects in which such a ventral cardiac muscle band has been described (de Velasco, 2005).

The role of tinman in the formation of the procephalic vascular rudiment was investigated by assaying tin-mutant embryos for the expression of Dmef2. Similar to the cardioblasts of the trunk, the Dmef2-positive cells of the procephalic vascular rudiment are absent in tin mutants. It is quite likely that the (Dmef2-negative) remainder of the procephalic vascular rudiment is affected as well by loss of tin, but in the absence of appropriate markers (besides tin itself, which is not expressed in the mutant), it was not possible to substantiate this proposal (de Velasco, 2005).

At the time of appearance of the ventral furrow, segmental markers such as hh do not allow the distinction between distinct 'preoral' segments. Thus, hh is expressed in a wide procephalic stripe in front of the regularly sized mandibular stripe. During stage 7, the procephalic hh stripe splits into an anterior, antennal stripe and a posterior, short, intercalary stripe. The anterior lip of the ventral furrow (domain PHMA) coincides with the anterior boundary of the antenno-intercalary stripe. Thus, the primary head mesoderm and endoderm originating from within the anterior ventral furrow can be considered a derivative of the antennal and intercalary segments. This interpretation is supported by the expression of the homeobox gene labial (lab) found in the intercalary segment. The labial domain covers much of the anterior ventral furrow, including domains PHMB-C (de Velasco, 2005).

Morphogenetic movements in the ventral head, associated with the closure of the ventral furrow, the formation of the stomodeal placode, and the subsequent invagination of the stomodeum result in a shift of head segmental boundaries. The antennal segment tilts backward, as can be seen from the orientation of the antennal hh stripe that from stage 8 onward forms an almost horizontal line, connecting the cephalic furrow with the sides of the stomodeal invagination (which falls within the ventral realm of the antennal segment, in Drosophila as well as other insects). Since the expression of hh, like that of engrailed (en), coincides with the posterior boundary of a segment, the territory located ventral to the antennal hh stripe falls within the intercalary segment. This implies that most, if not all, of the posterior late SHM, is intercalary in origin. It is further plausible to consider that the anterior lSHM belongs to the intercalary and antennal segment. The vascular cells of the head, a conspicuous derivative of the anterior lSHM in Drosophila, are derived from the antennal mesoderm in other insects. The labrum, with which much of the anterior lSHM is associated, represents a structure that has always been difficult to integrate in the segmental organization of the head. Most likely the labrum represents part of the intercalary segment; this would help explain some of the unusual characteristics of the head mesoderm (de Velasco, 2005).

In conclusion, several fundamental similarities are found between the mesoderm of the head and that of the trunk regarding the tissues they give rise to, and possibly the signaling pathways deciding over these fates. After an initial phase of structural and molecular homogeneity, the trunk mesoderm becomes subdivided into a dorsal and a ventral domain by a Dpp-signaling event that emanates from the dorsal ectoderm. The dorsal domain, characterized by the Dpp-dependent continued expression of tinman, becomes the source of visceral and cardiogenic mesoderm, among other cell types. A role of Dpp/BMP signaling in cardiogenesis seems to be conserved among insects and vertebrates. Subsequent signaling steps, involving both Wingless and Notch/Delta, separate between these two fates and further subdivide the cardiogenic mesoderm into several distinct lineages, such as cardioblast, pericardial cells, and secondary hemocyte precursors (lymph gland). As a result of these signaling events, Tinman and several other fate-determining transcription factors become restricted to their respective lineages: tin to the cardioblasts, odd to pericardial cells and hemocyte precursors, zfh1 and srp to hemocyte precursors and fat body. Dmef2 and several other transcription factors become restricted to various combinations of muscle types (somatic, visceral, cardiac) (de Velasco, 2005).

In the head mesoderm, the above genes are associated with similar fates. Tin and Dmef2 appear widely in the procephalic ventral furrow and the anterior lSHM before getting restricted to the procephalic vascular rudiment and/or the pharyngeal musculature, respectively. In contrast with the initially ubiquitous expression of Tin and Dmef2 in the trunk mesoderm, those parts of the head mesoderm giving rise to hemocytes (PHMC, posterior lSHM) never express these mesodermal genes. Previous work has shown that the head gap gene buttonhead (btd) is responsible for the early repression of tin in the above mentioned domains of the head mesoderm. The early absence of Tin and Dmef2 in the head mesodermal hemocyte precursors is paralleled by the presence of Srp and Zfh1 in these cells. Interestingly, Srp/Zfh-positive cells of the head produce only hemocytes and no fat body, suggesting that an as-yet-uncharacterized signaling step prevents the formation of fat body in the head. It is tempting to speculate that there exists within the mesoderm a 'blood/fat body equivalence group'. Blood cells and fat body share not only the expression of fate-determining genes such as srp and zfh1, but also, later, functional properties that have to do with immunity. In the trunk, the blood/fat body equivalence group gives rise mostly to fat body, producing only a limited number of hemocyte precursors in the dorsal mesoderm of the thoracic segments. In the head, on the other hand, all cells of the equivalence group become hemocytes (de Velasco, 2005).

Attention is drawn to another mesodermal lineage that produces related, yet not identical, cell types in the trunk and the head: the nephrocytes. Nephrocytes are defined by their characteristic ultrastructure (membrane invaginations sealed off by junctions) that attests to their excretory function. In the trunk, nephrocytes are represented by the pericardial cells that settle beside the cardioblasts; a newly discovered nephrocyte population ('star cells') invading the Malpighian tubules is derived from the mesoderm of the tail segments. In the head, nephrocytes aggregate near the junction between esophagus and proventriculus as the subesophageal body, also called garland cells. The fact that from the early stages of development onward different transcription factors are expressed in garland cells and pericardial cells suggests that these cells perform similar, yet not fully overlapping, functions (de Velasco, 2005).

Six-4, complementing the dorsal mesodermal determinant tinman, plays a key role in ventral cell identity of Drosophila mesoderm

Patterning of the Drosophila embryonic mesoderm requires the regulation of cell type-specific factors in response to dorsoventral and anteroposterior axis information. For the dorsoventral axis, the homeodomain gene, tinman, is a key patterning mediator for dorsal mesodermal fates like the heart. However, equivalent mediators for more ventral fates are unknown. This study shows that Six4, which encodes a Six family transcription factor, is required for the appropriate development of most cell types deriving from the non-dorsal mesoderm: the fat body, somatic cells of the gonad, and a specific subset of somatic muscles. Misexpression analysis suggests that Six4 and its likely cofactor, Eyes absent, are sufficient to impose these fates on other mesodermal cells. At stage 10, the mesodermal expression patterns of Six4 and tin are complementary, being restricted to the dorsal and non-dorsal regions respectively. These data suggest that Six4 is a key mesodermal patterning mediator at this stage that regulates a variety of cell-type-specific factors and hence plays an equivalent role to tin. At stage 9, however, Six4 and tin are both expressed pan-mesodermally. At this stage, tin function is required for full Six4 expression. This may explain the known requirement for tin in some non-dorsal cell types (Clark, 2006).

A fundamental question in developmental biology concerns the means by which uncommitted cells become specified to form a diversity of tissues according to their spatial location. In general, it is clear that a relatively small number of signaling and transcription factors are expressed in response to positional information, and in turn, these act combinatorially to regulate the expression of more specialized cell type regulatory factors. There is much interest in understanding the combinatorial regulation of cell type factors, particularly through computational analysis of their cis-regulatory regions. This is hampered, however, by an incomplete understanding of the identity and function of the upstream regulatory mediators themselves (Clark, 2006).

The specification of the mesoderm in Drosophila provides a tractable model system in which to study how the expression of cell type regulators is patterned within a large group of cells that are initially identical. A diverse range of organs derives from the Drosophila mesoderm, including the heart, the somatic and visceral muscles, the fat body and the somatic component of the gonad. For parasegments 4-12, an approximate fate map can be constructed outlining the mesodermal regions that give rise to these organs. Transplantation experiments show that fate determination is dependent on cell position, and therefore, patterning the mesoderm requires positional information. This is provided in part by inductive signaling from the overlying ectoderm, which results in the establishment of specific expression patterns for mesodermal transcription factors (Clark, 2006).

Along the anteroposterior axis, the parasegmental mesoderm is divided into two domains that correspond to the action of the pair rule genes, even skipped (eve) and sloppy paired (slp). The eve domain includes the cells underlying ectodermal stripes of hedgehog (hh) and engrailed (en) expression, and these genes participate in the development of the tissues that derive from this region. The action of hh is antagonized by that of wingless (wg), which signals to cells of the slp domain leading to body wall muscle and heart development. In the slp domain, twist (twi) is expressed at a high level and contributes to the development of the somatic muscles, while Notch signaling modulates twi to low levels in the eve domain (Clark, 2006 and references therein).

In the dorsoventral axis, the homeodomain transcription factor, Tinman (Tin), plays a central role in establishing dorsal mesoderm fates. In the dorsal region, ectodermal Decapentaplegic (Dpp) signaling maintains the expression of tin, which is lost from the remainder of the mesoderm following gastrulation. Tin and Dpp combine with factors involved in anteroposterior patterning to establish the primordia of the various dorsal mesodermal organs. For example, in the dorsal slp domain, Tin cooperates with Wg to activate specific sets of target genes, leading to heart and dorsal muscle development. Conversely, the visceral mesoderm is formed in the dorsal eve domain through the activation of bagpipe by Tin and Dpp and its repression by wg/slp. Apart from its dorsal function, tin also has a poorly understood role in the development of more ventral mesodermal fates (Clark, 2006 and references therein).

Outside the dorsal domain, it has been suggested that the non-dorsal mesoderm is divided into ventral and dorsolateral domains. This was based on the response of fat body cells to Wg signaling, although it is not clear whether this distinction has a genetic basis. The dorsolateral domain contains cells with dual fat body/somatic gonadal precursors (SGP) competence, although normally only those cells in parasegments 10-12 take on an SGP fate. Apart from the role of Tin dorsally, the patterning of mesodermal fates in the dorsoventral axis is poorly understood. Nevertheless, recently it has been demonstrated that Pox meso exhibits an early function, partially redundant with the function of lethal of scute, in demarcating the 'Poxm competence domain', a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. A major unanswered question is whether there are factors in the non-dorsal mesoderm that perform functions complementary to those of Tin in the dorsal region. A candidate for such a factor is the Six family homeodomain protein, Six4. In mouse, Six1, Six4, and Six5 genes are coexpressed during myogenesis, while Six1 and Six4 at least are required during mesoderm development. In human, reduction of SIX5 expression may underlie some of the abnormalities associated with Type 1 Myotonic Dystrophy (DM1). In Drosophila, the sole Six4/Six5 homologue, Six4, is the only Six homeoprotein expressed in the early mesoderm, and its mutation disrupts gonad and muscle development (Clark, 2006).

Evidence that Six4 is a key mesodermal patterning factor and is necessary for the correct development of various cell types deriving from the non-dorsal mesoderm, including fat body, SGP, and somatic muscles. Correspondingly, at stages 10/11, Six4 is expressed in non-dorsal mesoderm in a complementary pattern to tin. Moreover, with its cofactor Eyes absent (Eya), Six4 is sufficient to drive the specification of certain non-dorsal fates. In addition, these results clarify the function of tin in ventral mesodermal cells: it is proposed that earlier in development (at stages 8/9), part of tin's function ventrally is to initiate expression of Six4 (Clark, 2006).

Using a GFP reporter gene construct (referred to as Six4-III-GFP), an enhancer was identified within the Six4 third intron that activates GFP in a pattern corresponding closely to the mesodermal expression of Six4 RNA. At stage 9, Six4-III-GFP is coexpressed with D-Mef2 in a broad mesodermal domain. Subsequently, by stage 10, GFP expression becomes largely restricted ventrally, although some perduring protein remains in the dorsal region. At this point, lateral/ventral Six4-III-GFP expression is complementary to the dorsal expression of Tin (although the two are coexpressed earlier). Once restricted, the dorsal limit of Six4-III-GFP expression coincides with that of serpent (srp) protein, which marks the dorsolateral fat body cells. The levels of Six4 mRNA and Six4-III-GFP expression are modulated in the anteroposterior axis of the segment, being stronger in the slp domain (between the dorsolateral Srp clusters of the eve domain). This anteroposterior modulation of Six4 expression resembles that of twi, raising the possibility that different levels of protein have different functional consequences (Clark, 2006).

At stage 10, inductive dpp signaling from the dorsal ectoderm acts to maintain tin expression, thereby driving the dorsal restriction of Tin. Conversely, the ventral restriction of Six4 may depend on an inhibitory effect of dpp signaling. Consistent with this, misexpression of dpp throughout the mesoderm reduces expression of Six4 RNA to a low level. Thus, it is suggested that dpp signaling acts to establish two, non-overlapping spatial domains of gene expression in the mesoderm: a dorsal domain expressing tin and a ventral and lateral domain in which Six4 is expressed. Six4 is therefore a candidate for the counterpart of tin in patterning more ventral mesodermal fates (Clark, 2006).

Six4 is a key factor for the development of a variety of tissues that originate from the non dorsal mesoderm. It is required for the SGPs, fat body precursors and specific lateral and ventral muscles and is likely to be a competence factor or patterning mediator, acting to regulate a variety of key tissue and cell identity genes, such as srp for the fat body and ladybird for the segment border muscle founder cells. Different target genes would be regulated in different locations by the combinatorial action of Six4 and other factors involved in dorsoventral and anteroposterior axis patterning. Six4 may play additional roles later in gonad development, since its expression is maintained in SGPs throughout embryogenesis, whereas it is expressed transiently in most of the mesoderm (Clark, 2006).

Defective Six4 function results partly in failure of cell fate maintenance and/or cell survival. This is a common mutant phenotype of members of the Six and Eya gene families. Strikingly, however, expression of Six4 with its cofactor, Eya, throughout the mesoderm causes the expansion of Six4-dependent cell types (fat body and SGPs) with the concomitant disruption of other mesodermal derivatives, including the cardioblasts and visceral mesoderm. This supports an active role for Six4 in initial patterning of cell fates. It is possible, therefore, that maintenance/survival phenotypes are a secondary effect of defects in the initial establishment of cell identity (Clark, 2006).

Specific aspects of muscle cell identity are affected in Six4 mutant embryos. The phenotype is variable, but the external lateral and some ventral muscles are consistently disrupted. When Six4 and Eya are misexpressed/overexpressed in the mesoderm, an aberrant but regular muscle pattern is formed, suggesting that they have a patterning role, as opposed to a function in differentiation or myoblast fusion. It is likely that Six4 participates in the activation of certain muscle identity genes in founder myoblasts. Expression of the SBM identity gene, ladybird, requires Six4, while misexpression of Six4 and eya specifically in founder cells (using a dumbfounded-Gal4 driver) results in a muscle phenotype indistinguishable from that of embryos misexpressing these genes throughout the mesoderm (Clark, 2006).

The relationship between Six4 and tin is complex, partly because it changes over time and also because tin has functions in the ventral and lateral mesoderm that have remained obscure. The best characterized functions of tin concern the dorsal mesoderm, reflected in its restricted dorsal expression at stage 10/11. At this time, Six4 expression is complementary to that of tin, and there are no discernable effects on dorsal mesoderm structures in Six4 mutants. It is proposed that these two genes play complementary roles in their respective domains, promoting the development of specific cell types in conjunction with additional patterning factors. Despite their complementary expression patterns at this stage, there is no evidence that tin and Six4 are mutually antagonistic: although Tin can act as a repressor as well as an activator, there is no significant expansion of Six4 expression along the dorsoventral axis in a tin mutant and vice versa (unpublished data). It is more likely that, like tin, Six4 is directly regulated by dpp signaling (Clark, 2006).

In addition to dorsal mesoderm defects, tin mutant embryos show SGP, fat body and specific lateral and ventral muscle defects that presumably depend on its early pan-mesodermal expression. At least one tin function appears to depend on its regulation of Six4 in the early mesoderm before their mutually exclusive refinement of expression. Like Six4, tin is required for correct SGP development: a reduced number of SGPs appear at stage 11, and the number diminishes further until stage 13 when germ cell migration defects become apparent. However, the ventral expression of tin is lost before the SGPs are apparent, suggesting that another factor mediates its function in SGP development. Six4 may be this factor, since initially the two genes are transiently coexpressed broadly in the mesoderm, and Six4 expression is partly dependent on tin function. At this stage, Tin could be a direct transcriptional activator of Six4, since there are a number of sequences in the third intron that match the core E-box of the canonical Tin binding site (ACAAGTGG) (Clark, 2006).

The pattern of lateral and ventral muscle defects in embryos lacking Tin is different from that of Six4 mutants. Muscles affected by tin include LL1, LO1, VL3, VL4, and VT1, which do not require Six4 or Eya. Conversely, muscles that are severely affected by Six4 mutation appear normal in tin mutants, including VA3, the SBM, and the external lateral muscles LT1, LT2, LT3, and LT4. Based on these findings, it is proposed that muscles fall into at least three categories. The visceral, cardiac, and dorsal somatic muscles all require tin function directly through persistent dorsal tin expression. A second group, comprising a subset of ventral and lateral muscles, requires tin function via its transient pan-mesodermal expression, either directly or perhaps through unknown patterning mediators. A third group, a different subset of lateral and ventral muscles, is dependent on Six4/eya function and is not affected in tin single mutants, presumably because the reduced Six4 expression in these embryos is sufficient for their patterning. Muscles in this last category resemble the fat body precursors in their functional requirements, being dependent on an early, partially redundant function of tin and zfh-1, which is necessary to initiate Six4 expression in most parasegments. Confirmation of this model awaits a comprehensive characterization of muscle identity gene expression in founder cells in tin and Six4 mutant embryos (Clark, 2006).

The role of Six4 in mesoderm patterning appears to be conserved in other organisms. Expression of human SIX5 is reduced in Type I Myotonic Dystrophy, which may suggest a role in myogenesis since the most severe forms of this condition display muscle developmental defects (Harper, 1989). The murine orthologues, Six4 and Six5, are both expressed during myogenesis, although their precise roles are not yet established as single gene knock-out models have no clear muscle defects, perhaps owing to compensatory interactions. Six4 mutation, however, strongly exacerbates the muscle loss of mice mutant for the more divergent homologue, Six1. It is striking in particular that hypaxial progenitors (which contribute to limb muscles) lose their identity in Six1 Six4 double mutant mice. These muscle progenitors require the function of an lb homologue, Lbx1, and there is evidence that Lbx1 may be a target of Six/Six4. Thus, it appears that the function of Six4/5 genes might be conserved to a high degree (Clark, 2006).

The requirement for Six4 in diverse cell types, linked by their location of origin during mesoderm patterning, may represent a primordial state. The C. elegans homologue (unc-39) is also required for a number of mesodermal cell types. Although knowledge of Six4 and Six5 function is incomplete, it is notable that Six1 is required for the development of diverse organs such as muscle, kidney, and otic vesicle. It is interesting to note that Lbx1 regulation may be achieved by the combinatorial action of Six1/4 and Hox genes, which would thus behave as patterning factors in a similar way to Six4. The current studies suggest that diverse roles of SIX genes in vertebrate organogenesis as apparent cell- or tissue-type regulators may have their evolutionary origins in a general primordial developmental patterning mechanism, part of which may be preserved more clearly in the role of Six4 in mesoderm development in Drosophila (Clark, 2006).

Defective decapentaplegic signaling results in heart overgrowth and reduced cardiac output in Drosophila; Dpp represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number

During germ-band extension, Dpp signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. A second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. The data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. This study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects (Johnson, 2007).

A previous study suggested that a second round of Dpp dorsal ectoderm-to-mesoderm signaling, stimulated by enhancers located in the dpp disk region, initiates during germ-band retraction (stage 12; Johnson, 2003). This is referred to as the second round of signaling because a distinct set of enhancers located in the dpp Haplo-insufficiency (Hin) region activates Dpp dorsal ectoderm-to-mesoderm signaling during germ-band extension (stage 8). Further, the data revealed that dpp dorsal ectoderm expression driven by the Hin region enhancers persists long after germ-band retraction. These studies showed that Hin-region-driven dpp expression is sufficient for Dpp ectodermal functions such as dorsal closure and dorsal branch migration (Johnson, 2007).

Given these data, it appears that the dppd6 inversion prevents the augmentation of dpp expression in the dorsal ectoderm during germ-band retraction that is normally provided by disk region enhancers. The presence of numerous mesodermal phenotypes in dppd6 mutants (Johnson, 2003) suggests that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals so that they can reach the underlying mesoderm. Perhaps there are barriers of distance or extracellular matrix density between these germ layers that must be overcome (Johnson, 2007).

The data are wholly consistent with the hypothesis that the dppd6 inversion prevents the augmentation of dpp expression provided by disk region enhancers during germ-band retraction. The data further suggest that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals such that they can reach the underlying mesoderm. Finally, this study shown that during germ-band retraction Dpp signals maintain the boundary between pericardial cells and dorsal muscle cells via two distinct mechanisms: the regulation of gene expression and the restriction of cell proliferation. To regulate gene expression, Dpp signals directly to pericardial cells and restricts Odd and Tin expression in a zfh1-dependent manner. Dpp also limits Tin expression, independently of zfh1, by repressing the expression of mid, a stimulator of proliferation (Johnson, 2007).

With respect to zfh1-dependent regulation, the data support the hypothesis that Dpp restricts Zfh1 expression to regulate the number of pericardial cells derived solely from symmetrically dividing lineages. Lineage analyses have identified both symmetric and asymmetric cell divisions of myogenic and pericardial precursor cells. Pericardial cells are derived from four separate lineages that arise from four distinct precursor cells. Asymmetric precursor cell divisions initiating between stages 8 and 10 give rise to the Odd-positive/Seven up (Svp)-positive pericardial cells and the Eve-positive/Tin-positive pericardial cells (EPCs). In contrast, symmetric division, initiating at the same stage, establishes the Odd-positive/Svp-negative pericardial cells (OPCs) and the Tin-positive/Eve-negative pericardial cells (TPCs). dpp mutations do not affect the number of EPCs or the number of Odd-positive/Svp-positive cells. However, embryos bearing dpp mutations show an increase in the number of OPCs and TPCs. Therefore, the ectopic pericardial cells seen in dpp mutants derive from symmetrically dividing lineages (Johnson, 2007).

Previous reports have shown that regulation of asymmetric cell division is a key mechanism in establishing boundaries among the various cell types in the dorsal mesoderm. For instance, in the absence of Numb, a Notch pathway antagonist, asymmetric progenitor cell division is abrogated and the number of Odd-positive/Svp-positive cells and EPCs increases at the expense of the Svp-expressing cardial cells and Eve-expressing dorsal muscle cells, respectively. This study extends these observations by showing that pericardial cell types derived from symmetrically dividing lineages are also under strict regulatory control (Johnson, 2007).

With respect to zfh1-dependent regulation of pericardial cell number, Dpp restricts cell proliferation and, in turn, Tin expression by limiting mid expression. In wild-type embryos, cell division in the dorsal mesoderm is largely complete by the early stages of germ-band retraction (stage 11), whereas in dppd6 embryos cell proliferation in the dorsal mesoderm continues through stage 13. Interestingly, the number of cells expressing Zfh1 increases from stage 12 to stage 13 in wild-type embryos in the absence of cell division, demonstrating that patterning events subsequent to cell division regulate cell fate choices in the dorsal mesoderm. This hypothesis is supported by the fact that tracing pericardial cell lineages requires inducing mitotic clones by stage 8. Therefore, the ectopically dividing mesoderm cells observed in dppd6 embryos are derived from cells with the potential to become Tin-expressing cells (Johnson, 2007).

During stage 12, tin expression is reactivated in a subset of cardiac cells in a mid-dependent fashion, suggesting that tin expression in precursor cells alone is not sufficient for specifying the ultimate fate of their daughter cells. Moreover, misexpression of mid results in both ectopic cell division and expanded tin expression. Lineage studies support the necessity of reactivating Tin by showing that a single precursor cell gives rise to two Tin-positive/Eve-negative pericardial cells and two siblings that do not express Tin. Thus tin is not reactivated in all subpopulations of pericardial cells. The data suggest that, during stage 12, Dpp prevents tin reactivation in cells occupying lateral regions of the dorsal mesoderm by limiting mid expression (Johnson, 2007).

Development of the dorsal musculature initiates when founder cells are specified in the mesoderm. These founder cells then fuse with neighboring cells to form syncitial myofibers. In the absence of Dpp, the pericardial cell domain expands into the dorsal muscle domain and reduces expression from the dorsal muscle genes Kr and Eve. Since the separation between pericardial and dorsal muscle cells is lost in dpp mutant embryos, it is concluded that Dpp maintains the pericardial-dorsal muscle cell boundary after it is established. Moreover, reducing pericardial cell number increases Kr expression after germ-band retraction, suggesting that cross-repressive interactions between pericardial and dorsal muscle cells contribute to patterning of the dorsal mesoderm. The presence of ectopic pericardial cells in the dorsal mesoderm reduces the number of myofibers comprising the dorsal muscles even though the dorsal muscle founder cells are, for the most part, correctly specified. pMad does not accumulate in Kr-expressing founder cells yet Kr expression is significantly reduced in dpp mutant embryos. Therefore, changes in Kr and Eve expression observed in embryos with altered dpp or zfh1 activity reflect alterations in the number of myoblast fusion events in the dorsal mesoderm (Johnson, 2007).

These data extend a previous study showing that misexpressing Zfh1 reduces dMef2 expression in somatic muscles. This study demonstrates that misexpression of Zfh1 induces ectopic pericardial cells and that the presence of pericardial cells in the dorsal muscle domain reduces myoblast fusion. Therefore, reduced dMef2 expression in embryos misexpressing Zfh1 is likely the result of reduced myoblast fusion and not of direct repression of dMef2 expression by Zfh1. Further, analysis of lmd mutants that have reduced numbers of myoblasts revealed that they also contain an excessive number of pericardial cells. Together, these results suggest that maintaining the pericardial-dorsal muscle cell boundary requires Dpp-mediated cross-repressive interactions between these cell types. Thus, in the absence of Dpp, the transformation of dorsal muscle cells into pericardial cells reduces the number of myoblasts available for fusion (Johnson, 2007).

Experiments in the larvae of Drosophila and other insects suggested that pericardial cells act as nephrocytes that filter the hemolymph. These studies also showed that pericardial cells secrete proteins into the hemolymph, suggesting that one pericardial cell function may be to provide short- or long-range signals. Consistent with this, reducing pericardial cell number reduces heart rate and increases the cardiac failure rate, suggesting that pericardial cells influence the development of cardiac cells (Johnson, 2007).

This study shows that pericardial cell hyperplasia reduces the luminal distance of the heart during systole as well as diastole, resulting in an overall decrease in average pulse distance of each contraction. However, pericardial overgrowth does not alter heart rate, indicating that cardiac cells develop appropriately in the presence of ectopic pericardial cells. Luminal measurements suggest a role for pericardial cells in the mechanics of heart function. One hypothesis for this is based on the fact that pericardial cell hyperplasia results in excess levels of extracellular matrix protein Pericardin (Prc) in the extracellular matrix (ECM) surrounding the heart. Prc is a collagen IV-like ECM protein secreted at high levels from pericardial cells. In dpp mutants, excess Prc is seen predominantly in the posterior of the heart where the pulse-distance reduction was observed. It is proposed that Prc secreted by pericardial cells limits the width of the dorsal vessel at diastole and thus modulates the pulse distance of each heart contraction. Pericardial cell overgrowth would increase Prc deposition, thereby reducing the size of the diastolic heart and the pulse distance. Consistent with this hypothesis, excessive expression of ECM proteins, including collagen IV, was correlated with heart failure in patients presenting with end-stage cardiomyopathy (Johnson, 2007).

It is well documented that many of the early events driving Drosophila embryonic heart development have been conserved in vertebrates. The data provide the first basis upon which to determine if Dpp regulation of Zfh1 or Tin late in heart development is also conserved (Johnson, 2007).

Two orthologs of zfh1, Sip1 and Kheper, have been identified in vertebrates. Zebrafish embryos injected with the Dpp homolog BMP4 show reduced Kheper expression while Xenopus embryos injected with the BMP antagonist Chordin display elevated Sip1 expression. These results suggest the possibility that Dpp repression of zfh1 expression may be conserved in vertebrates. In addition, mammalian Sip1 plays an essential role in heart development. In mice, Sip1 is expressed in neural crest cells (NCCs), paraxial mesoderm, and neuroectoderm. The subset of NCCs that express Sip1 give rise to the septum and large arteries of the heart. Sip1 knockout mice fail to form these NCCs and these mice die midway through gestation with numerous heart defects. Mice lacking the BMP receptors BMPRIA or ALK2 specifically in NCCs also display numerous cardiac phenotypes. In conditional knockout of ALK2 in NCCs, abnormalities are seen in the heart's outflow tract, and conditional knockout of BMPRIA in NCCs results in heart failure and early embryonic lethality similar to Sip1 knockout mice. Thus BMP signals are required for proper specification of NCCs, and loss of BMP signaling in NCCs phenocopies Sip1 knockout mice to an extent. It is tempting to speculate that, as in Drosophila, BMP signals regulate the Zfh1 ortholog Sip1 to correctly specify NCCs and, in turn, to properly pattern the mammalian heart (Johnson, 2007).

With regard to the conservation of late-stage Dpp regulation of Tin, a recent article describing a study of mice with a conditional knockout of Nkx2.5 where expression is missing only during late stages of heart development (post E14.5) is highly relevant. Utilizing rescue of Nkx2.5 mutant embryos with BMP-signaling-pathway components, the study identified a direct connection among BMP4 signaling, Nkx2.5 activity, and heart cell proliferation. Since Nkx2.5 is the Tin homolog, BMP4 is the Dpp homolog, and the mutant phenotype (heart cell hyperplasia) is the same in both species, this suggests that this aspect of Dpp signaling is conserved in mammals. Together with this study, these results suggest that defects in late-stage BMP signaling may play a role in congenital heart defects (Johnson, 2007).

The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation

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).


Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA+ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

Gene expression changes during metamorphosis also foreshadow both larval muscle breakdown and adult myogenesis. At approximately 2 hours APF, the anterior larval musculature begins to break down. This breakdown lasts until approximately 6 hours APF. Genes encoding both structural and regulatory components of muscle formation are down-regulated as early as 4 hours BPF (see Muscle-specific genes regulated during metamorphosis). In addition to the repression of genes encoding components of thin and thick filaments, genes encoding other muscle-specific molecules are also repressed, including factors that compose the mesh in which these filaments lie and regulatory factors involved in the specification of muscle tissue. The mRNAs of all these repressed genes decrease substantially many hours before histolysis of the anterior larval muscles and therefore predict the occurrence of this morphological event well before it begins. Twenty-four hours APF, adult myogenesis is well underway. The genes DMef-2, bagpipe, and tinman are all up-regulated at 12 hours APF from the baseline at PF, coincident with the prepupal pulse of ecdysone. It is suggested that induction of these regulatory factors initiates the development of the adult musculature, which will establish itself several hours later (White, 1999).

Effects of Mutation or Deletion

Embryos that are deficient for the chromosomal region 93C-F, a region that includes the tinman gene, show normal mesoderm invagination and dorsal spreading. However, later in development no visceral muscle and dorsal vessel differentiation can be detected, though some skeletal muscles do form, albeit abnormally. There is, however, no defect in major morphogenetic movements. Twist protein is found in mesodermal cells below the dorsal region of the embryo in tinman mutants (Bodmer, 1990).

In tinmutant embryos, bagpipe expression is not activated in the dorsal mesoderm. Probably as a consequence, neither visceral mesoderm nor midgut musculature are formed in these mutants, and the absence of visceral mesoderm results in strong disruptions of endoderm migration and midgut morphogenesis. In addition to visceral mesoderm development, tin is required for the formation of the heart from dorsal mesoderm and for the specification of founder cells for particular body wall muscles (Azpiazu, 1993).

The function of tinman is required for visceral muscle and heart development. Embryos that are mutant for the tinman gene lack the appearance of visceral mesoderm and of heart primordia, and fusion of anterior with posterior endoderm is impaired. Even though tinman mutant embryos do not have a heart or visceral muscles, many of the somatic body wall muscles appear to develop, although abnormally. When the tinman cDNA is ubiquitously expressed in tinman mutant embryos, via a heatshock promoter, formation of heart cells and visceral mesoderm is partially restored. tinman seems to be one of the earliest genes required for heart development and the first gene reported for which a crucial function in the early mesodermal subdivisions has been implicated (Bodmer, 1993). In tinman mutants, bagpipe is not activated in dorsal mesoderm. Consequently, neither visceral mesoderm nor midgut musculature are formed in tinman mutants (Azpiazu, 1993)

Fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene is involved in early directional migration of the Drosophila mesoderm. New data is provided that (1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function; (2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and (3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl (Michelson, 1998).

The involvement of Htl in mesodermal founder cell fate specification was tested by reducing its activity under conditions where earlier cell migration is not compromised. This was accomplished by ectopic expression of a dominant negative of the Htl Fgf receptor. Dominant negative Htl induces numerous defects in mesodermal structures at multiple positions along the dorsoventral axis and at different stages of development. In late stage embryos, somatic muscles are missing from ventral, lateral and dorsal groups, and gaps occur in the rows of cardial and pericardial cells. These defects can be traced to an earlier stage where the corresponding precursor cells are found to be lacking. For example, dominant negative Htl prevents the formation of progenitors of the Eve-expressing pericardial and somatic muscle cells. Small gaps in the normally continuous rows of visceral mesodermal precursors are also observed (Michelson, 1998).

Ectopic mesodermal expression of a constitutively active form of Ras1 is capable of partially rescuing a strong hypomorphic htl mutant. Partial rescue of a null htl mutation by activated Ras1 also is manifest in the expression of Eve in the dorsal mesoderm. No Eve-positive cells are found in the complete absence of htl function, whereas a hypomorphic mutant contains a markedly reduced number of Eve-expressing segments. Although the Epidermal growth factor, a second receptor tyrosine kinase, is involved in development of Eve muscle founders, all of the Eve-positive cells generated by activated Ras1 in htl mutant embryos are confined to the dorsal mesoderm in their usual segmental pattern, consistent with the involvement of Ras1 in both the migration and cell fate specification functions of Htl (Michelson, 1998).

Interestingly, loss-of-function mutations in tinman and htl have identical affects on the development of Eve pericardial and somatic muscle cells. Similarities are also seen between the cardial and dorsal somatic muscle phenotypes of these two genes. However, tinman differs significantly from htl in the mechanism of action since mesoderm migration is completely normal in tinman mutants. This implies that tinman is involved in only one of the processes affected by htl, namely the determination of dorsal mesodermal cell fates. Since Ras1 functions in the Htl signaling pathway and activation of this signal transduction has the opposite effect on Eve progenitor development as tin loss-of-function, an epistasis experiment could be performed. Expression of activated Ras1 in a tinman mutant background results in an Eve expression phenotype corresponding to that of tinman. That is, tinman loss-of-function completely blocks the ability of activated Ras1 to promote the formation of Eve pericardial cell and somatic muscle progenitors (Michelson, 1998).

The following model is proposed for the role of Tinman and Htl in the formation of Dorsal mesoderm. When mesodermal cells reach the dorsal-most region of the ectoderm, they are induced by Dpp to express Tinman, thereby acquiring the competence to differentiate into visceral, cardiac, or dorsal somatic muscle derivatives. Superimposed on this process is the activation of the Ras1 pathway in a small subset of dorsal mesodermal cells. Ras1 activation is mediated by Htl in those cells destined to form the Eve pericardial progenitiors, whereas both Htl and Egf receptor function together to generate a Ras1 signal specific to Eve-positive somatic muscle fate. In this sense, Htl/Egfr/Ras1 signaling serves to distinguish a fate characterized by Eve expression from additional dorsal mesodermal fates that are also dependent on tinman. It should be noted that Tin and Ras1 regulation are not required to function in any particular order; one may precede the other or they may act simultaneously. The essential point is that both are absolutely required for the specification of Eve cardiac and somatic muscle fates in the dorsal mesoderm (Michelson, 1998).

In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most superficial cell from the promuscular cluster, thus suggesting a role for the overlying ectoderm during its segregation. . Since epidermal Wg and Hedgehog (Hh) signaling has been shown to influence muscle formation, the SBM-associated lb expression was examined in embryos carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM progenitors. The initial influence of these signals is no longer observed later in development. In addition to signals from the epidermis, the activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of the majority of SBM fibers. During promuscular cluster formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an unknown factor. The lack of neurogenic gene function, known to be involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).

A number of aspects of midgut development depend on the mesoderm: migration of anterior midgut (AMG) primordium and the posterior midgut (PMG) primordium, and transition to an epithelium. The extension of the midgut primordia is achieved by cell migration along the visceral mesoderm that forms a continuous layer of cells within the germ band. In mutant embryos lacking the entire mesoderm or failing to differentiate the visceral mesoderm, AMG and PMG are formed but do not migrate properly. In addition, they fail to form an epithelium and instead either remain as compact cell masses anterior and posterior to the yolk (in twist and snail mutant embryos) or only occasionally wrap around the yolk before embryogenesis is completed (in tinman-deficient embryos). Thus the visceral mesoderm serves as a substratum for the migrating endodermal cells; contact between visceral mesoderm and endoderm is required for the latter to become an epithelium (Reuter, 1993).

Each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior somatic gonadal precursor (SGP) fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. clift has been identified as a regulator of Drosophila gonadogenesis. When cloned, clift turned out to be identical to eyes absent. Mutations in clift abolish gonad formation and produce female sterility. Just as with abdominal A, clift is expressed within SGP as these cells first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in gonadogenesis. Using clift as a marker, it has been shown that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with a gene regulatory hierarchy involving abd A within the mesoderm. While loss of wg abolishes gonadal precursors, ectopic expression expands the population such that most cells within lateral mesoderm adopt gonadal precursor fates. Initial dorsoventral positioning of somatic gonadal precursors relies on a regulatory cascade that establishes dorsal fates within the mesoderm. tinman appears to mediate the role of ectodermally expressed decapentaplegic; in tinman mutants few or no SGP cells are detected. clift expression is subsequently refined through negative regulation by bagpipe, a gene that specifies nearby visceral mesoderm. Thus, these studies identify essential regulators of gonadal precursor specification and differentiation and reveal novel aspects of the general mechanism whereby somatic gonadal cell fate is allocated within the mesoderm (Boyle, 1997).

Analysis of a tinman;zfh-1 double mutant shows that Zn finger homeodomain 1 acts in conjunction with tinman, another homeodomain protein, in the specification of lateral mesodermal derivatives, including the gonadal mesoderm. It is unlikely, however that tin and zfh-1 fit neatly into a linear hierarchy controlling gonadal mesoderm determination. The early broad expression of tin is required for SGP development. However, zfh-1 is not required for this expression, suggesting that zfh-1 is not upstream of tin. Furthermore, since germ cell association with SGPs is blocked in zfh-1 mutants but not in tin mutants, it seems unlikely that tin acts upstream of zfh-1 in SGP development. These observations suggest that tin and zfh-1 function in parallel in gonadal mesoderm development (Broihier, 1998).

In many animal groups, an interaction between germ and somatic lines is required for germ-line development. In Drosophila, the germ-line precursors (pole cells), which form at the posterior tip of the embryo migrate toward the mesodermal layer where they adhere to the dorsolateral mesoderm, which ensheaths the pole cells to form the embryonic gonads. These mesodermal cells may control the expression of genes that function in the development of germ cells from pole cells. However, such downstream genes have not been isolated. In this study, a novel transcript, indora(idr), is identified that is expressed only in pole cells within the gonads. The nucleotide sequence of the 1.5 kb cDNA predicts a protein of 131 amino acids. The amino acid sequence shows no significant homology to any known proteins. The putative Idr protein is highly basic (calculated isoelectric pH is 10.1). During normal development, the expression of idr transcripts become discernible in pole cells at the embryonic stage 14, when pole cells are incorporated into the gonads. Expression persisted in pole cells until the completion of embryonic development. idr expression is undetectable in the adult germ line. However, the possibility that a trace amount of IDR mRNAs is expressed in somatic cells as well as in the germ line throughout most of the life cycle cannot be excluded, because Northern blot analysis reveals that idr transcripts are detectable from late embryogenesis to adulthood (Mukai, 1998).

Reduction of idr transcripts by an antisense idr expression causes the failure of pole cells to produce functional germ cells in females. Furthermore, idr expression depends on the presence of the dorsolateral mesoderm, but it does not necessarily require its specification as the gonadal mesoderm. In order to determine the source of the mesodermal cue, idr expression was analyzed in the absence of the mesodermal cells that make up the gonads. The origin and development of the somatic components of the gonads are described. The somatic gonad precursors (SGPs) are specified from the dorsolateral mesoderm within PS 10-12 at stage 11. In tin;zfh-1 double-mutants, no dorsolateral mesoderm is formed, which results in loss of SGPs. In these embryos, pole cells pass through the midgut epithelium, but subsequently they are dispersed around the midgut. idr expression is drastically reduced in tin;zfh-1 double-mutants. This result shows the requirement of the dorsolateral mesoderm for idr expression in pole cells. It was next asked whether the specification of the dorsolateral mesoderm as SGPs is needed to induce idr expression in pole cells. To examine this, abd-A and iab-4 mutations were used. abd-A function is required in the mesodermal cells for the specification of SGPs. In abd-A mutant embryos, pole cells pass through the midgut wall and are normally associated with the dorsolateral mesoderm. However, they do not coalesce with the pole cells to form the gonads due to their failure to be specified as SGPs. Consequently, pole cells are released from the mesoderm and scattered throughout the embryo. In these embryos, the dispersed pole cells express idr during stages 14-16. Furthermore, a regulatory mutation in the abd-A locus, iab-4, also has no deleterious effect on idr expression. Thus, the specification of the dorsolateral mesoderm as SGPs is dispensable for idr expression. These findings suggest that the induction of idr in pole cells by the mesodermal cells is required for germ-line development (Mukai, 1998).

During the development of peripheral nerves, pioneer axons often navigate over mesodermal tissues. During wildtype embryonic development, the transverse nerve efferents that innervate abdominal segments associate with glial cells located on the dorsal aspect of the CNS midline (transverse nerve exit glia). These glial cells have cytoplasmic extensions that prefigure the transverse nerve pathway from the CNS to the body wall musculature prior to transverse nerve formation. Transverse nerve efferents extend to the periphery along this scaffold where they fasciculate with projections from a peripheral neuron--the LBD. In tinman mutants, the transverse nerve exit glia appear to be missing, and efferent fibers remain stalled at the CNS midline, without forming transverse nerves. In addition, fibers of the LBD neurons are often truncated. These results suggest that the lack of exit glia prevents normal transverse nerve pathfinding (Gorczyca, 1994).

Recent evidence suggests that cardiogenesis in organisms as diverse as insects and vertebrates is controlled by an ancient and evolutionarily conserved transcriptional pathway. In Drosophila, the NK-2 class homeobox gene tinman (tin) is expressed in cardiac and visceral mesodermal progenitors and is essential for their specification. In vertebrates, the tin homolog Nkx2-5/Csx and related genes are expressed in early cardiac and visceral mesodermal progenitors. To test for an early cardiogenic function for Nkx2-5 and to examine whether cardiogenic mechanisms are conserved, the mouse Nkx2-5 gene and various mutant and chimeric derivatives were introduced into the Drosophila germline, and tested for their ability to rescue the tin mutant phenotype. While tin itself strongly rescues both heart and visceral mesoderm, Nkx2-5 rescues only visceral mesoderm. Other vertebrate ‘non-cardiac’ NK-2 genes (Nk2-1, Nk2-2 and Nk2-4) rescue neither heart nor visceral mesoderm. The potent transactivation domain of Tin is shown not to be required for cardiogenic function (Ranganayakulu, 1998).

Previous studies have demonstrated that sequences within the C terminus of Nkx2-5 strongly inhibit transcriptional activity in transient transfection assays. Deletion of the conserved NK2SD leads to a modest increase in transcriptional activity, further enhanced by removal of all sequences C-terminal to the homeodomain. Whether the NK2SD masks the activity of a transactivation domain or influences DNA-binding affinity is not yet known. To determine whether the C terminus of Nkx2-5 masks a cardiogenic function in Drosophila, two C-terminal deletion mutants similar to those examined in vitro were tested for their ability to rescue heart and visceral mesoderm in tin mutants. A mutant lacking only the NK2SD (Nkxdelta NK) is stable and produces three new effects when compared to wild-type Nkx2-5: (1) it rescues visceral mesoderm to a greater extent, as judged by Fasciclin III staining; (2) it activates the DMef-2 enhancer-lacZ reporter in visceral mesoderm and (3) it rescues 2 or 3 clusters of Eve-positive cells in a few (~10%) mutant embryos. Thus, deletion of the NK2SD improves both the extent and integrity of the rescued visceral lineage. A negative regulatory function for this domain can therefore be demonstrated both in vitro and in vivo. In rescuing a few Eve-positive cells, the deletion mutant may also unmask a weak cardiogenic activity for Nkx2-5 (Ranganayakulu, 1998).

The cardiogenic domain of Tin was mapped to a unique region at its N terminus. When transferred to Nkx2-5, this region confers a strong ability to rescue heart. To assess whether the cardiogenic function of Tin is wholly confined to the N terminus, the mutant Tin deltaC, lacking most amino acids C-terminal to the homeodomain, was examined. This mutant is able to rescue Eve-positive pericardial cells strongly, but Zfh-1 and DMef-2-positive pericardial and cardial cells only weakly. Thus, while the N terminus of Tin carries the strong cardiogenic domain, its efficacy for certain downstream cardiogenic functions appears to be modulated by the C terminus. Substitution of the C terminus of Nkx2-5 with that of Tin does not confer on Nkx2-5 the ability to rescue heart formation. The cardiogenic domain of Tin was further mapped by making a series of N-terminal deletion mutants. Constructs containing N-terminal amino acids 1-220 or 1-134 of Tin fuse to the homeodomain. C terminus of Nkx2-5, both are able to strongly rescue heart formation in tin mutant embryos. These results suggest that the region of Tin spanning amino acids 1-134 contains the cardiogenic activity. Since deletion of amino acids 43-123 within this region has no deleterious effect, it appears that the essential region is contained either within amino acids 1-42 or 124-134. N-terminal amino acids 1-52 of Tin were fused to amino acids 53-319 of Nkx2-5 and tested for rescue. This N-terminal Tin domain confers full cardiogenic activity on Nkx2-5. Thus, the cardiac and visceral mesodermal functions of NK-2 homeogenes are separable in the Drosophila assay. The results suggest that, while tin and Nkx2-5 show close functional kinship, their mode of deployment in cardiogenesis has diverged possibly because of differences in their interactions with accessory factors. The distinct cardiogenic programs in vertebrates and flies may be built on a common and perhaps more ancient program for specification of visceral muscle (Ranganayakulu, 1998).

Phylogenetic comparisons suggest that the hearts of vertebrates and invertebrates evolved as independent adaptations of pulsatory muscular vessels, themselves derived from visceral musculature. The results of the rescue experiments also suggest a link between visceral mesoderm and heart, and allow the formulation of a more precise evolutionary hypothesis: that the genetic circuitry underlying cardiac development in mammals and insects has been built on a common and more ancient program for specification of visceral muscle, one which utilizes NK-2 homeogenes. The ability of zebrafish nkx2-5 to rescue the function of pharyngeal muscles in nematodes lacking the NK-2 gene ceh-22 may also reflect the common ancestral role for NK-2 genes in visceral development. In this context, it is interesting that ceh-22 clearly lies within the ‘non-cardiac’ clade by phylogenetic analysis, the group that appears to lack rescue function in the fly assay. Thus, the nematode rescue assay reveals a myogenic function for both cardiac and non-cardiac genes (Ranganayakulu, 1998).

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. An analysis has been carried out of how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. Cells in different branches are found exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. Alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and a model is proposed for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints (Franch-Marro, 2000).

Tracheal cells are first specified as clusters of ectodermal cells at the embryonic surface. Since tracheal cells invaginate and form the tracheal pits they occupy the grooves between the muscle precursors of adjacent metameres. The formation of this groove is independent of tracheal invagination because it also forms between metameres that do not have tracheal placodes and it also develops in trh mutant embryos, which do not undergo tracheal invagination. A subset of the tracheal cells moves anteriorly, whereas another subset moves posteriorly until they reach the cells from the adjacent placodes. These cells will form the dorsal trunk, the most prominent tracheal branch that spans the embryo longitudinally. Those cells migrate across the adjacent precursors of somatic muscles and separate the precursors of the most dorsal muscles from the precursors of more ventral muscles. Other cells, those from the dorsal side of the tracheal pit, move dorsally along the longitudinal groove to form the dorsal branches that will end up fusing with the dorsal branches coming from the contralateral hemisegments. In the ventral side, the tracheal cells follow two different paths along the two clusters of lateral muscle precursors at each side of the groove. Anterior ventral cells will form the anterior lateral trunk while the posterior ventral cells will form the posterior lateral trunk. Finally, another group of cells from a midposition in the tracheal pit will migrate inward and will form the visceral branch (Franch-Marro, 2000).

The migrating cells of the dorsal trunk do not recognize any preexisting gap between the muscle precursor cells. Thus, the role of the lateral mesoderm in the migration of tracheal cells was studied. The lateral mesoderm, which comprises the fat body and the somatic gonadal precursors, is determined by the early functions of tinman (tin) and zinc-finger homeodomain protein-1 (zfh1). In embryos mutant for either zfh-1 or tin, the number of somatic gonadal and fat body precursors is reduced. Consistent with a role for the lateral mesoderm in the migration of the dorsal trunk cells, it was observed that the development of the dorsal trunk is impaired in these mutants. This is a nonautonomous effect since tin and zfh-1 are not expressed in the tracheal cells. The effect is quite mild, however. Since tin and zfh-1 have overlapping and partially redundant functions, mutant embryos for both genes were examined. In those embryos, the somatic gonadal mesoderm and the fat body precursors are virtually absent and the tracheal dorsal trunk is almost completely absent, but formation of the dorsal and ventral branches is not impaired. This defect is not due to a failure of bnl expression since it appears at the right position between the tracheal pits. (Franch-Marro, 2000).

In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example, holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. Whether a similar mechanism of brain development occurs in the protostome Drosophila has been examined; the foregut and mesoderm have been found to act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and deuterostomes had a prototype of the brains present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ layers, are actually homologous (Page, 2002).

As the Drosophila foregut invaginates, it normally becomes ensheathed by visceral mesoderm. Thus, when the foregut is ablated, visceral mesoderm is displaced from its normal position adjacent to the brain. How much does the loss of mesoderm contribute to the brain phenotype seen in foregut ablated animals? Embryos lacking function of the NK-2 class transcription factor Tinman have defects in forming mesoderm around the foregut, as revealed using mesodermal markers as Fasciclin III expression, but do form foregut ectoderm. In 65% of Tinman loss-of-function embryos there were excess cells at the dorsal midline of b1; the area occupied by neuronal nuclei was increased when compared with wild type in this region of the brain, and the preoral brain commissure was abnormally thin (Page, 2002).

Why are there excess cells at the dorsal midline in foregut- and mesoderm-ablated embryos? During normal brain development, more neurons are born than will be present in the adult brain and apoptosis eliminates the excess cells. Defects in apoptosis could contribute to the observed defects in brain patterning by failing to remove excess cells. To see if apoptosis was perturbed when the foregut and mesoderm were ablated, Acridine Orange staining, which labels apoptotic cells, was carried out. In forkhead loss-of-function embryos, the pattern of apoptosis in the brain at the level of the preoral brain commissure was clearly different from wild type at late ES13. In the wild-type b1 neuromere, there were groups of apoptotic cells at the dorsomedial edges of the hemispheres. This correlates with previous observations regarding the expression of the apoptosis regulatory protein Reaper. In forkhead loss-of function embryos, which are defective in foregut development, there was a clear reduction in the number of these cells. Examination of tinman loss-of-function embryos, which are defective in mesodermal development, showed that removal of mesoderm results in a similar reduction in the number of apoptotic cells at the dorsal midline, thus suggesting that the mesoderm and possibly the foregut have an influence on the normal pattern of apoptosis in brain development (Page, 2002).

The results of foregut and mesoderm ablation experiments strongly suggest that the brain is patterned by induction from these tissues. Did ablation of these tissues remove inductive signals required for normal brain development? What molecular signals could be mediating this effect? In vertebrates, Hedgehog signaling originating from the prechordal plate functions in forebrain patterning. Thus, the Hedgehog pathway in Drosophila seemed a good place to begin to look for inductive signals involved in brain development. Null mutations in Drosophila Hedgehog result in a phenotype in 70% of embryos that strongly resembles the one seen in the foregut ablation experiments. In brain segment 1 (b1), the right and left hemispheres of the brain are joined at the midline or separated by an abnormally small space because of excess cells in this region, and the preoral brain commissure shows abnormal defasciculation. In addition, in b1 the frontal commissure is missing, and there is a significant decrease in the area occupied by neuronal nuclei and the number of glia. In b2-S3 (S3 is ventrolateral to the foregut), the longitudinal connectives are disrupted, and the area occupied by neuronal nuclei and the number of glia is significantly reduced, and the number of Fasciclin II-expressing neurons is reduced (Page, 2002).

The final overall shape of the salivary gland and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. This study focuses on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. The salivary gland turns and migrates along the visceral mesoderm (VM) to become properly oriented with respect to the overall embryo. Salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. A role for integrin function in salivary gland migration is demonstrated. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells (Bradley, 2003).

These studies suggest that the VM is required for salivary gland migration and predicts that other mutations affecting VM formation would cause similar salivary gland defects. During development, mesodermal precursor cells that migrate dorsally are exposed to the dorsally localized Decapentaplegic (DPP) signaling molecule, which is required to specify dorsal mesoderm derivatives, including cardiac and VM precursors. In embryos lacking thickveins (tkv), which encodes a DPP receptor, breaks of variable width and position in the VM are observed, similar to the breaks observed in the htl VM. Correspondingly, salivary gland migration defects were observed in tkv mutants similar to those seen in htl mutants. Such migration defects were not observed, however, in embryos expressing a negative regulator of the DPP pathway, Daughters against dpp. Together, these results suggest that DPP signaling, like FGFR1, is not required in the salivary gland cells for their normal migration, but rather that DPP signaling is required for VM formation, which in turn is required for the directed migration of the salivary gland (Bradley, 2003).

In htl, tkv, and tinman, the residual fragments of VM express Fas3, have a VM-like structure, and are able to direct salivary gland migration if present along its migratory path. Thus, the residual structures appeared to be differentiated VM with wild-type properties. To determine whether salivary gland migration requires a differentiated VM, embryos with mutations in the VM-specific gene biniou (bin) were examined. In bin mutant embryos, VM precursors segregate from dorsal mesoderm and move internally where they coalesce into the typical VM band; however, all tested VM-specific genes, including Fas3, fail to be expressed in bin mutants. Thus, an intact structure formed from VM precursors is present in bin mutants, but the VM precursor cells fail to express markers indicative of differentiation from a general mesodermal cell into a VM-specific cell. The salivary glands in bin mutants had no defects in turning or posterior migration, suggesting that guidance of salivary gland posterior migration by the VM requires neither the terminal differentiation of the precursors nor the function of any VM gene whose expression is bin-dependent (Bradley, 2003).

The VM forms a contiguous structure that may physically block salivary cells from further dorsal movement, thereby causing the cells to move posteriorly, in the path of least resistance. Alternatively or additionally, there may be a bin-independent factor (or factors) that guides salivary gland migration in a more instructive way, perhaps via a secreted signal or a transmembrane guidance molecule. If the mesodermal cue were informational, a signaling pathway functioning within salivary gland cells would have to be involved. A screen of several candidate pathways revealed that mutations disrupting the FGFR1-, FGFR2-, EGF-, DPP-, JNK-, or Wg-signaling pathway did not have phenotypes consistent with a role in the salivary cells for their migration. Thus, focus was placed on molecules known to have a more direct role in migration, specifically the integrin family of cell adhesion molecules, which are heterodimers of two transmembrane proteins, an alpha and a ß subunit. In Drosophila, each of the five identified alpha subunits (alphaPS1-5) is thought to dimerize with the ßPS subunit encoded by the myospheroid (mys) gene. The alpha subunit of alphaPS2ßPS (PS2) integrin is expressed in all mesodermal cells beginning at a very early stage, suggesting that PS2 integrin is likely to be present in the VM precursor cells prior to bin-dependent differentiation. Indeed, alphaPS2 RNA expression was observed in the mesoderm of binR22 homozygotes. In embryos mutant for inflated (if), the gene encoding the alphaPS2 subunit, migration of two tissues along the VM is affected, the endoderm and the tracheal visceral branch. Thus, the PS2 integrin is required to make the VM a suitable substrate for the migration of at least two distinct cell populations (Bradley, 2003).

Whether PS2 integrin is required for salivary gland migration was examined by staining if mutant embryos for several salivary gland proteins. In if homozygotes, salivary cells appear to invaginate normally. The first group of salivary cells to be internalized reaches the approximate level of the wild-type turning point but fails to migrate. During subsequent stages, the remaining if salivary cells continue to internalize, but the distal tip remains at the approximate VM turning point, and the tube is often slightly bent. By late stages, if salivary tubes are frequently folded in half with the distal tips oriented anteriorly. The apparent lack of salivary gland migration in if mutants is distinct from the mismigration phenotypes in htl, hbr, tkv, and tin mutants (Bradley, 2003).

The tinman and bagpipe genes are members of the NK homeobox gene family of Drosophila, so that tin occupies a higher position than bap in the regulatory hierarchy. Little is known about the level and pattern of genetic polymorphism in homeobox genes. Nucleotide polymorphism was analyzed in 27 strains of Drosophila melanogaster and one each of D. simulans and D. sechellia, within two closely linked regions encompassing a partial sequence of tin and the complete sequence of bap. The two genes exhibit different levels and patterns of nucleotide diversity. Two sets of sharply divergent sequence types are detected for tin. The haplotype structure of bap is more complex: about half of the sequences are identical (or virtually so), while the rest are fairly heterogeneous. The level of silent nucleotide variability is 0.0063 for tin but significantly higher, 0.0141, for bap, a level of polymorphism comparable to the most polymorphic structural genes of D. melanogaster. Recombination rate and gene conversion are also higher for bap than for tin. There is strong linkage disequilibrium, with the highest values in the introns of both genes and exon II of bap. The patterns of polymorphism in tin and bap are not compatible with an equilibrium model of selective neutrality. It is suggested that negative selection and demographic history are the major factors shaping the pattern of nucleotide polymorphism in the tin and bap genes; moreover, there are clear indications of positive selection in the bap gene (Balakireva, 2004).

The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. This study analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate (Mann, 2009).

The specification of a subset of mesodermal cells towards a cardiac fate requires well-orchestrated interactions of a plethora of factors. Drosophila is the model system of choice to decipher the complex transcriptional network that initiates and sustains a cardiac lineage. The data place tup as an essential component in the early transcriptional network that specifies cardiac mesoderm (Mann, 2009).

After the initially broad expression domain of Tin has become restricted to the dorsal mesodermal margin, Tup expression is first seen in the cardiac mesoderm in ~10 small clusters, which co-express Eve. Slightly later, Tup is present throughout the Tin-positive cardiac mesoderm and gene expression analyses in tupisl-1, tin346, pnrVX6 and Df(3L)DocA embryos demonstrate that all four factors are required to maintain each other's expression. Additionally, analyses of cardiac gene expression in embryos that are transheterozygotic for tup and tin, pnr or Doc, showed that these factors interact genetically to specify heart cells (Mann, 2009).

Although it might be expected that Tup expression is lost in tin mutants since these embryos are devoid of heart cells, it is interesting that Tup expression in the early cell clusters is still initiated. This finding is somewhat reminiscent of the observation that the initiation of Doc expression is also independent of tin. According to the temporal appearance of Tup in the cardiac mesoderm with respect to Tin and Doc, tup is required for their maintenance rather than their initiation. By contrast, the onset of mesodermal Pnr and Tup expression appears to coincide. It was not resolved whether Tup is induced by Pnr or directly by Dpp. A direct regulation by Dpp was implicated by the reduced expression of Tup after mesodermal overexpression of UAS-brinker, which is known to bind to dpp-response elements of dpp target genes. Conversely, it was shown that dpp expression depends on tup and the present data suggest that this regulation requires pnr (Mann, 2009).

Germ layer-specific inhibition of Tup using a construct that lacks the homeodomain, but contains the two LIM domains, revealed that Tup can regulate cardiogenesis in the mesoderm as well as from the ectoderm. Since the 69B-Gal4 driver has been reported not to be strictly ectodermal, it is possible that mesodermal Tup function was also interfered with. However, the mesodermal expression of 69B-Gal4 seems to be negligible. The effect of ectodermal Tup inhibition on cardiogenesis in the mesoderm can only be explained if the function of a secreted growth factor is impaired. dpp expression was analyzed, and a slight downregulation of its transcripts was observed in embryos expressing UAS-tupδHD in the ectoderm. Since this effect might not be sufficient to account for the strong Tin phenotype, further experiments will be required to determine whether additional growth factors are affected (Mann, 2009).

To better determine the germ layer-specific contribution of Tup in cardiogenesis, attempts were made to rescue the Tin phenotype by co-expressing the full-length tup cDNA. Somewhat unexpectedly, a better rescue was obtained when both constructs were expressed in the ectoderm rather than in the mesoderm. Since the LIM domains present in tupδHD can sequester LIM-domain-binding proteins, a simple explanation for this finding is that Tup interacts with proteins that are present in the mesoderm but not in the ectoderm. It is reasonable to hypothesize that in the mesoderm the LIM domains of tupδHD not only act as a dominant-negative for Tup, but additionally for another, perhaps as yet unidentified, LIM-domain containing protein. Since it has been shown that Pnr can bind Tup through the LIM domains, it is likely that Pnr function was interfered with by overexpressing UAS-tupδHD. The requirement of the LIM domains for proper cardiac specification is shown by the reduction of Tin-expressing cells after mesodermal expression of the UAS-tupδLIM construct. Further experiments are under way to better resolve the molecular function of Tup in the different tissues (Mann, 2009).

Since the mesodermal expression of UAS-tupδHD resulted in a strong reduction of Tin-expressing cells at early stages of cardiac mesoderm formation, it was surprising to observe a rather low reduction of Dmef2-positive myocardial cells at later stages (15/16). To exclude the possibility that the twi-Gal4 driver does not sufficiently express UAS-tupδHD throughout embryogenesis, this experiment was repeated using the combined mesodermal driver twi-Gal4; 24B-Gal4. However, the phenotypes were not enhanced. A time course for Tin expression in these crosses revealed that Tin appears to recover over time. A similar phenomenon can be seen in tupisl-1 mutants, although it might not be as obvious because the mutants also lack ectodermal tup expression. In any case, the data is suggestive of a different temporal requirement for tup with respect to tin expression. It is known that tin expression depends on different transcriptional activation events. Consistent with the onset of Tup expression in the cardiac mesoderm at mid-stage 11, the earlier phases of Tin expression are unlikely to depend on Tup. Hence, the initial Tin expression at stages 8-10 is sufficient to generate a considerable number of Dmef2-positive myocardial cells at later stages (Mann, 2009).

These analyses further implicate that Tup might act as a transcriptional activator or repressor depending on the cellular context and on the factors with which it is co-expressed. This is most strikingly observed with respect to the Odd-expressing pericardial and lymph gland cells. In tup mutants, Odd-positive cells are missing in both organs. A similar phenotype is seen when Tup is overexpressed in the mesoderm using the twi-Gal4 driver. The loss of Odd-expressing cells in lymph glands is reminiscent of the phenotype observed in tup mutants, although it is less severe. This differential occurrence of the phenotype indicates that tup can differentially regulate factors involved in cardiogenesis versus lymph gland development. This is substantiated by the finding that mesodermal overexpression of tup results in an increase in Hand expression in the lymph glands, while Hand expression throughout the dorsal vessel is only mildly affected. Despite the loss of Odd-positive cells after early mesodermal tup overexpression, Tup is required in the pericardial and lymph gland cells at later stages to maintain Odd expression. Moreover, overexpressing tup in the pericardial cell lineage yields additional Odd-expressing pericardial cells and rescues Odd expression in the lymph glands (Mann, 2009).

To obtain more insight into possible functional interactions with other cardiac transcription factors, tup was overexpressed in combination with pnrD4. The latter is a highly active variant of wild-type pnr that contains an amino acid substitution in the N-terminal zinc finger, which abolishes binding of Ush to Pnr. Mesodermal overexpression of pnrD4 results in robust ectopic activation of Tin and embryos co-overexpressing tup and pnrD4 exhibit the same phenotype. Most likely, a possible influence of Tup on Pnr activity, regardless of whether it is positive or negative, is concealed by the strong gain-of-function pnr allele. However, analysis of Eve expression does provide insight into possible regulatory interactions between Tup and Pnr. Mesodermal overexpression of each factor alone yields opposing phenotypes, and when both factors are co-overexpressed PnrD4 can efficiently counteract Tup activity and prevent the overspecification of Eve cells. Vice versa, Tup can, although only moderately, counteract the effect of PnrD4. It has been shown that during patterning of the thorax, Tup can antagonize the proneural activity of Pnr by forming a heterodimer, and that the physical interaction between Pnr and Tup is mediated by the two zinc fingers of Pnr. Hence, the somewhat weak, but possibly antagonistic, function of Tup towards PnrD4 in Eve-positive cell specification could be due to the amino acid substitution encoded in the pnrD4 allele, which might weaken the interaction between the two factors, as compared with wild-type Pnr. Overexpression of a Tup construct that lacks both LIM domains did not result in expanded Eve-positive clusters, which strongly suggests that the effect of Pnr on Tup activity, as seen when both factors are co-expressed, requires the presence of the LIM domains (Mann, 2009).

In summary, these data demonstrate the crucial role of tup in the proper specification of cardiac mesoderm in an invertebrate organism. Therefore, tup/Isl1 should be added to the core set of ancestral cardiac transcription factors. Consequently, this implicates that the evolution of the vertebrate four-chambered heart does not necessarily require the acquisition of a novel network of cardiac transcription factors. At least, it is unlikely that tup/Isl1 is part of a regulatory network separate from that of tin/Nkx2.5, pnr/Gata4 and Doc/Tbx5/6 because it is an essential factor for the formation of the simple linear heart tube in the fly (Mann, 2009).

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, this study demonstrates that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, it was determined that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. It is concluded that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis (Xu, 2012).

Proper dorsal vessel morphogenesis is critically dependent upon intercellular signaling and the regulation of gene expression. Although great progress has been made in the study of heart development, it is not known exactly how many genes and pathways are involved in this cardiogenic process or how many of these factors cooperate together. Previous genetic screens have identified genes that play roles in the specification, morphogenesis, and differentiation of the heart, including mastermind and tup. The current sensitized screen has also proved to be an efficient method to find additional factors in this process, suggesting that much remains to be learned about the molecular components involved in correct dorsal vessel formation (Xu, 2012).

Spir is required for the proper timing of cytoplasmic streaming in Drosophila, and loss of spir leads to premature microtubule-dependent fast cytoplasmic streaming during oogenesis, the loss of oocyte polarity, and female sterility. Even though it is known that spir is an important actin filament nucleation factor, the findings are the first report to describe a function of spir for cell division. Through phenotypic analysis of the spir mutant phenotype, it was found that many cardioblast nuclei are partially or completely divided. However, the cytoplasm is not divided in the absence of spir, which is consistent with the function of spir in cytoplasmic movement. Thirteen rapid nuclear division cycles without cell division initiate Drosophila embryo development, followed by three waves of cell division. The first wave of cell division occurs in the mesoderm at cell cycle 14. After this initial division, cells migrate, spread dorsally and undergo a second round of cell division at cell cycle 15. The third wave of cell division in the mesoderm occurs at the end of germband extension during cell cycle 16. There are two different types of cardioblast precursor cells: one type divides into two identical Tin-positive cardioblasts (TC), and the other type divides into one Svp-positive cardioblast (SC) and one Svp-positive pericardial cell (SPC). Based on the comparison of CycA and spir mutant phenotypes, a tentative cell division model is proposed to demonstrate spir function in determining cardiac cell fate (see A cell division model of spir function during heart development). In a wild-type background, one Svp-positive super progenitor (SSP) divides into two Svp-positive progenitors (SP), then each of these cells divides into one SPC and one SC. For Tin-positive super progenitors (TSP), after each divides into two Tin-positive progenitors (TP), each TP further divides into two identical TCs. In the current model, division from the super progenitor to progenitors takes place at cell cycle 15, and division from progenitors to full differentiated heart cells occurs at cell cycle 16. In CycA mutants, mitosis 16 is blocked such that both SPs and TPs stop cell division. This results in the two SPs assuming a myocardial fate. Thus the number of SCs remains normal, but half of the TCs are missing in the CycA mutants. The data suggest that in spir mutant embryos, most of the SPs fail to undergo full cell division at cycle 15 resulting in a SPC fate with paired nuclei. A subset of these cells are able to undergo the 15th cell division but are arrested at cycle 16 as double-nucleated cells which exhibit both Svp and Mef2 staining, characteristic of the SCs seen in the CycA mutants. Similarly, for TPs, cycle 16 was also blocked such that it resulted in two double-nucleated cells. In summary, Spir affects mitosis 16 for Tin-positive cell division and both mitosis 15 and 16 for Svp-positive cell division (Xu, 2012).

Antibody staining suggests that Spir is expressed ubiquitously before stages 12-13 and is located in both nuclei and cytoplasm. After cell cycle 16 cell division stops, occurring during stage 10-11. The expression of Spir in the cytoplasm then decreases gradually. At stage 15, the staining pattern shows mostly nucleus expression with some cytoplasmic expression and by stage 16 the nuclei become distinct indicating nucleus staining only. It is hypothesized that expression of Spir decreases in the cytoplasm but remains constant in the nuclei when cell division halts (Xu, 2012).

The genetic analysis of spir, Doc, pnr and tin suggests that these factors may regulate each other during dorsal vessel formation, and especially significant is the interaction between spir and pnr. Pnr is a GATA class transcription factor expressed in both the dorsal ectoderm and dorsal mesoderm, where it is required for cardiac cell specification. Proper dorsal vessel formation is inhibited in pnr loss-of-function embryos due to failure in the specification of the cardiac progenitors. In spir mutants, the expression pattern of Pnr remains normal. However, Spir is over-expressed in the cardiac mesoderm in pnr mutants, suggesting that Pnr may repress the expression of the spir (Xu, 2012).

In conclusion, Spir is a newly-identified factor functioning in cell division during dorsal vessel formation. Tin-, Eve- and Svp-positive heart cells are all affected in the absence of spir. Also, spir expression depends on the transcription factors Doc, tin and pnr. Genetic interaction data also show that spir cooperates with CycA in heart cell division (Xu, 2012).

tinman: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.