Myocyte enhancer factor 2

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of Mef2 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Mef2 gene expression is first detected during Drosophila embryogenesis within mesodermal precursor cells during gastrulation [Images], prior to specification of the somatic and visceral muscle lineages (Lilly, 1994). MEF2 protein is expressed throughout the mesoderm following gastrulation. Later in embryogenesis, its expression is maintained in precursors and differentiated cells of the somatic and visceral musculature, as well as the heart. Like Nautilus, Mef2 seems to be confined to single precursor cells.

The three major types of musculature in the Drosophila embryo requireMEF2 for later aspects of their differentiation, including body wall muscles, gut musculature, and the heart . tinman is restricted to the visceral mesoderm, the tissue that gives rise to cardiac muscle cells, and nautilus is restricted to somatic mesoderm, the tissue that gives rise to skeletal cells, however MEF2 is found in both tissues. (Bour, 1995).

In Drosophila, much has been learned about the specification of neuronal cell fates but little is known about the lineage of mesodermal cells with different developmental fates. During development, individual mesodermal precursor cells are initially singled out to become the founder cells for specific muscles. The selection of muscle founder cells is thought to employ a Notch-mediated process of lateral inhibition, similar to what is observed for the specification of neural precursors. These muscle founder cells then seem to fuse with the surrounding, uncommitted myocytes, inducing the formation of muscle fiber syncytia. In contrast, the differentiated progeny of neural precursor cells are usually the result of a fixed pattern of asymmetric cell divisions that are directed, in part, by interactions among Numb (a localized intracellular-receptor protein); Sanpodo (Spdo, a potential tropomodulin homolog), and Notch (a transmembrane receptor protein). The roles of these neural lineage genes have been examined in the cell fate specification of muscle and heart precursors. numb and spdo mutations have opposite effects in the specification of muscle founder cells. In all numb mutant embryos examined, the number of Kruppel and S59/NK1 expressing muscle cells is dramatically reduced or absent in stage 12/13 embryos. In spdo mutant embryos the number of S59 and Kr expressing muscle founders is increased (Park, 1998).

A progenitor cell that generates both a pericardial heart cell and a somatic muscle founder cell was the focus of investigation. The two sibling cells studied were a single dorsal muscle, DA1, and a non-muscle pericardial cell (termed EPC), which is associated with the heart. Both cell types express Eve, however, they can be distinguished from one another morphologically. The precursor for both the EPCs and the putative founders of DA1 muscle emerges from a small cluster of mesodermal Eve-expressing cells in each hemisegment at mid-stage 11. At first, these mesodermal Eve cells are indistinguishable from one another, and they co-express Mef2, which is expressed in the entire early mesoderm and later in all (contractile) muscle types. Subsequently Mef2 expression ceases in the future EPCs as they begin to differentiate as non-muscle, pericardial cells. The putative DA1 founder seems to maintain Mef2 expression and begins fusing with surrounding myocytes. In Mef2 mutants, no fusion occurs but the putative muscle founders maintain expression of their muscle precursor markers, such as Eve. The asymmetric segregation of Numb into one of these daughter cells antagonizes the function of Notch and Spdo by preventing the presumptive muscle founder from assuming the same fate as its cardiac sibling. In numb mutants, most DA1 muscles are physically absent and the remaining ones lack Eve (and Kr) expression; in addition, the number of EPCs is doubled. These data suggest that in numb mutants, the putative DA1 founders are transformed into EPCs, because the Eve progenitor cells that normally give rise to DA1 founders and EPCs now only produce EPCs. Overexpression of numb or loss-of-spdo-function result in a failure to generate EPCs but allow for the formation of DA1 muscles. Similarly, expression of constitutively active Notch leads to a failure of DA1 muscle formation and an increase in the number of EPCs. Studies of double mutants indicate that spdo is epistatic to numb (in numb;spdo double mutant embryos, the spdo phenotype is apparent), suggesting that it acts downstream of numb. These results suggest that asymmetric cell divisions, in addition to the previously-documented inductive mechanisms, play a major role in cardiac and somatic muscle patterning. In addition, the cytoskeleton may have a role in the asymmetrical localization of cell fate determinants (Park, 1998).

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:

• Domain A (PHMA): the anterior lip of the T-bar, defined by the expression of sine oculis (so), giant (gt), and a reduced level of heartless (htl).
• Domain B (PHMB): the posterior lip of the T-bar, defined by high levels of htl and tinman (tin) expression and absence of serpent (srp).
• Domain C (PHMC): the anterior part of the procephalic ventral furrow proper, defined by the expression of srp and other endoderm markers and by low levels of htl or tin.
• Domain D (PHMD): the posterior part of the procephalic ventral furrow (anterior gnathal mesoderm), which initially resembles the trunk mesoderm in expressing high levels of htl and tin. Secondarily, much of the gnathal mesoderm switches to srp expression as it adopts the fate of blood cell precursors (de Velasco, 2005).

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.

• The early secondary head mesoderm (eSHM) ingresses and delaminates during stages 7-8 from the ventral epithelium flanking the endoderm.
• The posterior late secondary head mesoderm (lSHMp) ingresses during stages 9-11 from a similar position as the eSHM. Its cells give rise to blood cells and nephrocytes (the subesophageal body or garland cells).
• The anterior late secondary head mesoderm cells (lSHMa) delaminate lateral and anterior to the stomodeum. Coming from within the anlagen of the esophagus and epipharynx, these cells produce esophageal visceral muscle cells, as well as a novel tin-positive cell type that is interpreted as a rudimentary cephalic vascular structure (de Velasco, 2005).

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).

Larval

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).

Mef-2 in cultured cells

Schneider SL2 cells activate the myogenic program in response to the ectopic expression of daughterless alone, as indicated by exit from the cell cycle, syncytia formation, and the presence of muscle myosin fibrils. Myogenic conversion can be potentiated by the coexpression of Drosophila Mef2 and nautilus with daughterless. In RT-PCR assays Schneider cells express two mesodermal markers, Nautilus and Mef2 mRNAs, as well as very low levels of Daughterless mRNA but no Twist. Full-length RT-PCR products for Nautilus and Mef2 encode immunoprecipitable proteins. RNA-i was used to demonstrate that both endogenous nautilus expression and Mef2 expression are required for the myogenic conversion of Schneider cells by daughterless. Coexpression of twist blocks conversion by daughterless but twist dsRNA has no effect. These results indicate that Schneider cells are of mesodermal origin and that myogenic conversion with ectopic expression of daughterless occurs by raising the levels of Daughterless protein sufficiently to allow the formation of Nautilus/Daughterless heterodimers. The effectiveness of RNA-i is dependent upon protein half-life. Genes encoding proteins with relatively short half-lives (10 h), such as Nautilus or Hsf, are efficiently silenced, whereas more stable proteins, such as cytoplasmic actin or beta-galactosidase, are less amenable to the application of RNA-i. These results support the conclusion that Nautilus is a myogenic factor in Drosophila tissue culture cells with a functional role similar to that of vertebrate MyoD. This is discussed with regard to the in vivo functions of Nautilus (Wei, 2000).

Effects of Mutation or Deletion

Complete loss of MEF2 protein in homozygous mutant embryos results in a dramatic absence of myosin heavy chain (MHC)-expressing myoblasts and lack of differentiated muscle fibers. Examination of earlier events involved in muscle development indicates that the specification and early differentiation of somatic muscle precursors are not affected because even-skipped-, nautilus-, and beta 3-tubulin-expressing myoblasts are present. However, these partially differentiated cells are unable to undergo further differentiation to form muscle fibers in the absence of Mef2.

The later aspects of differentiation of the visceral mesoderm and the heart are also disrupted in Mef2 mutant embryos, although the specification and early development of these tissues appear unaffected. Midgut morphogenesis is disrupted in the mutant embryos, presumably as a consequence of abnormal development of the visceral mesoderm. In the heart, the formation of cardial cells is not affected by the absence of Mef2, but differentiation here is deficient. even-skipped expressing paracardial cells are present in Mef2 mutants.

Mef2 deficiency is reminiscent of myogenin phenotype in mice. Myogenin is needed for the terminal differentiation of myoblasts, but not for their determination. Roles in myoblast determination have been ascribed to MyoD and Myf-5 (Bour, 1995).

Embryos deficient for MEF2 protein, due to a deletion of upstream transcriptional control sequences, fail to form muscle, suggesting that the gene is required for muscle cell differentation. In the somatic muscle lineage, Mef2 is required for the formation and patterning of body wall muscle. In the absence of somatic myogenesis, there is extensive apoptosis among the myoblast cell population. In contrast, in the cardiac muscle lineage, morphogenesis of the dorsal vessel occurs normally, but the three myosin subunit genes are not expressed. MeF2 is also expressed in adepithelial cells. Rare Mef2 transheterozygous mutant adults fail to fly, consistent with defects observed in the indirect flight muscles (Ranganayakulu, 1995).

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle 'pioneer' or 'founder' cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. Toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) have been used to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA (Misquitta, 1999).

The results from the injection of nautilus dsRNA point to a more general approach for the analysis of gene function during Drosophila development and suggest that the RNA interference method essentially would mimic a gene knock-out in the injected generation of Drosophila embryos. To test this idea a variety of cDNA clones were obtained representing a maternal gene expressed in the embryo (daughterless); additional genes involved in myogenesis (S59, DMEF2); homeobox genes (engrailed and S59); a gene important for gastrulation (twist), and a gene expressed in the adult eye (white). This panel of genes covers most stages of Drosophila development. twist was initiatially tested because the mutant has a clear phenotype that is easy to score when compared with wild-type larva. The injection of twist dsRNA (the complete coding region) into embryos produces a twisted larval phenotype that is indistinguishable from the original twist mutation. Similarly, injection of the first 1,200 bp of engrailed dsRNA produces the compressed dentical belt pattern characteristic of an engrailed null mutant. Daughterless mRNA is both maternally loaded and expressed zygotically, and the mutant phenotype produces very characteristic disruptions in the central nervous system (CNS) and peripheral nervous system (PNS). It has been shown previously that mex3, a maternally loaded RNA in C. elegans, can be ablated by dsRNA injection into the gonads. daughterless dsRNA (complete coding region) was injected and the characteristic neuronal phenotypes were sought by using the mAb MAB 22C10. The CNS as well as the PNS were disrupted to varying degrees in the injected embryos. The severity of the phenotype consistently shows a CNS disruption with a variable PNS pattern, possibly reflecting the fact that the CNS is formed before the PNS. This result suggests that maternally loaded as well as zygotically expressed RNA can be affected by RNA-i in Drosophila. The homeobox gene S59 marks a subset of muscle founder cells for 5 of 29 muscles in each hemisegment of the embryo corresponding to muscles 5, 18, 25, 26, and 27. Embryos with an S59 lacZ transgene marking muscles 18 and 25 were injected with S59 dsRNA (complete coding region). In this case, the S59-specific lacZ antibody-staining pattern is abolished. The total muscle pattern for embryos injected with S59 dsRNA, although disrupted, still shows the presence of poorly organized muscle groups in each hemisegment. This is unlike the almost complete absence of muscle observed with the injection of nautilus dsRNA. DMEF2, a member of the MADS domain transcription factor family, is essential for muscle formation in Drosophila. The DMEF2 / embryo has no muscle and is missing the characteristic gut constrictions found in the uninjected embryo. Injection of DMEF2 dsRNA (complete coding region) results in embryos that lack any detectable muscle and an absence of gut morphology (Misquitta, 1999).

mef2 genes encode alternatively spliced transcription factor isoforms that function in muscle differentiation in both Drosophila and vertebrates. Drosophila mef2 (Dmef2) has been shown to be required for the differentiation of a variety of distinct muscle types. However, many possible aspects of its function in muscle remain unexplored. There has also been no analysis in vivo of the activity of different Mef2 isoforms in any species. This investigation centers on the role of different levels of DMef2 in regulating diverse events of muscle differentiation in the Drosophila embryo and on the functional significance of Dmef2 alternative splicing. The GAL4/UAS system was used to both misexpress and overexpress individual DMef2 isoforms and to rescue the different aspects of the Dmef2 mutant phenotype. Ectopic ectodermal expression of DMef2 activates muscle gene expression and inhibits epidermal differentiation. Overexpression of DMef2 in the mesoderm disrupts differentiation of the somatic and visceral muscle and the heart. The use of different DMef2 levels in rescue experiments reveals an activity range compatible with differentiation of the different muscle types: the consequence of too little or too much DMef2 activity is disrupted differentiation. These rescue experiments also reveal that distinct DMEF2 thresholds are required for different properties within a cell and also for different cells within a muscle type and for different muscle types. For example, relatively low levels of DMef2 rescue myosin expression in the visceral muscle, together with, in almost all embryos, the midgut constrictions. Higher levels are required for complete rescue of the alary muscles, while the pharyngeal muscle and somatic musculature are only fully rescued with the higher levels produced at 29 degrees C. Complete rescue of myosin expression in the heart is only achieved in about 35% of the embryos at 29 degrees C., indicating that this tissue is the most difficult to rescue. Finally, each isoform functions equivalently in these experiments, including in the stringent test of rescue of the Dmef2 mutant phenotype (Gunthorpe, 1999).

The Him gene inhibits the development of Drosophila flight muscles during metamorphosis

During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. This study shows that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibers. Consistent with this, it was found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fiber degeneration. Then a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him, was uncovered. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibers. Lastly, evidence is provided for a link between Notch function and Him and mef2 in this balance (Solera, 2009).

This paper analysed events in the development of the DLMs, components of the indirect flight musculature, during Drosophila adult myogenesis. Him was found to be expressed in myoblasts, but not in developing adult muscle fibers, and consistent with this pattern of expression it was found that Him could inhibit DLM development. Template splitting was inhibited and subsequently remaining muscle fibers degenerated. This phenotype resembles that found when the myoblasts are removed by a genetic manipulation. It is inferred from this phenotypic similarity that over-expression of Him makes these myoblasts functionally defective. It was also found that this phenotype due to Him is suppressed by mef2. This genetic interaction, together with previous observations that mef2 hypomorph mutants also have impaired LOM splitting, is consistent with Him antagonising Mef2 activity in these myoblasts. It is noted, however, that in contrast to mef2 hypomorphic mutants, Him can induce degeneration of the DLMs. This could be because 1151-Gal4 driving UAS-Him results in a lower level of Mef2 activity than in the hypomorphic mef2 mutants, and/or because Him induces DLM degeneration by another route. The rescue of the degeneration phenotype, as well as of template splitting, by mef2 suggests that the effect of Him is at least substantially through the mef2 pathway (Solera, 2009).

It is apparent that there must be a mechanism to inhibit Mef2 function in the committed, but undifferentiated adult myoblasts associated with the wing discs, but it was not known how this is achieved. The current findings suggest that Him could be such an inhibitor. Him could antagonise the mef2-induced expression of myosin, a classic marker of muscle differentiation, in disc-associated myoblasts. Moreover, in normal development, Him is already expressed in the myoblasts when Mef2 is first expressed in the third larval instar. Him and Mef2 also remain co-expressed in the myoblasts during the early phase of pupal life, but later, as the muscle fibers differentiate, Mef2 is expressed in the fibers whereas Him is not, although it continues in surrounding myoblasts. This arrangement where Mef2 is expressed together with an inhibitor suggests a capacitor-like situation in which there could be a burst of Mef2 activity, a discharge, when the inhibition is relieved. This could result in a co-ordinated onset of a phase of the muscle differentiation programme (Solera, 2009).

This study has revealed a balance of Him and mef2 activities in the control of adult myoblast differentiation. Thus, additional Him expression, like reduced mef2 function, leads to disrupted DLM development, and the effect of Him can be suppressed by mef2. Moreover, although mef2 has a positive role in adult myogenesis, it is required for proper DLM development, it was found that over-expression of mef2 affected DLM formation and the total number of DLM fibers was somewhat reduced compared to wild type. It was also found that reduced expression of the Mef2 inhibitor Him had a similar effect. This indicates that too much of a positive signal, or too little of a negative, can affect muscle development. Taken together with other results, this suggests that the balance of antagonistic activities, which includes mef2 and Him, has to be precisely controlled to ensure correct implementation of the differentiation programme (Solera, 2009).

One other known input into the control of adult myogenesis is Notch. It has been shown to inhibit adult muscle differentiation, and it has been suggested that it may do this through twist, although much remains unclear about the molecular pathway(s) involved. The results link Notch with two other regulators. It was found that mef2 over-expression could partially suppress the inhibitory effect of N-intra on DLM development, and that N-intra resulted in Him expression in muscle fibers. The latter might be direct regulation of gene expression because a region upstream of the Him gene contains several binding sites for Su(H), a nuclear effector of Notch signalling, and either deletion of this region or a Su(H) genetic background results in down-regulation of Him expression in wing discs. Together these results indicate that Notch can upregulate Him expression, which in turn can inhibit mef2 function, and this will result in inhibition of DLM development (Solera, 2009).

These findings further understanding of the factors that influence the programme of differentiation during adult Drosophila myogenesis. They also pave the way for future studies into the mechanisms that control this intriguing system of muscle development, which includes tissue remodelling and is effectively a type of regeneration, and which also has certain parallels with mammalian muscle post-natal growth and repair. This work also illustrates the significance of a balance of activities in how adult progenitor cells might be maintained in a committed, but undifferentiated state and then be triggered to enter the differentiation pathway. This is of broad significance both in development and in potential applications of stem cell biology (Solera, 2009).

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date revised: 28 December 2011

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