FGF receptor 1


DEVELOPMENTAL BIOLOGY

Embryonic

In early embryos expression is specific to the mesodermal primordium and invaginated mesodermal cells, including cephalic mesoderm. At later stages, putative muscle precursor cells and cells in the central nervous system (CNS) express DFR1. Cells surrounding the hindgut and foregut are FR1 positive at stages 12 and 13 (Shishido, 1993).

A protein-null mutant of heartless and heartless expression were examined using anti-Heartless antibody. Mutant phenotypes are completely rescued by a genomic fragment from the heartless locus. After invagination, mesodermal cells expressing heartless undergo proliferation and spread out dorsally to form a monolayer beneath the ectoderm. In mutant embryos, however, the mesoderm is not capable of extending to the normal dorsal limit and consequently mesodermal cells fail to receive ectodermal signals and are thus rendered incapable of differentiating into primordia for the heart, visceral and somatic muscles. Thus there appears to be a specific defect in cell migration in heartless mutants. In heartless mutants the intensity of tinman expression decreases inappropriately and is no longer expressed in presumptive heart cells, although visceral mesodermal expression in maintained. Dorsal tin expression itself is likely to be independent of htl, but reduction in the number of mesodermal cells capable of receiving Dpp signals from the dorsal-most ectoderm causes changes in the dorsoventral subdivision of mesoderm, in particular, loss of the Tin domain for heart progenitors in htl mutant embryos. Fasciclin III is a marker for progenitors of midgut visceral mesoderm and Connectin is a cell surface protein which marks a subset of visceral midgut muscle precursors. The presence of Fas III and Con positive cells in htl mutants indicates that specification of visceral muscle precursors does take place, at least to some extent, in heartless mutant embryos, although Fas III and Con postive cells are greatly reduced in number. Dorsal oblique muscles decrease to 26% of those of the wild-type; ventral longitudinal and oblique muscles are reduced to 54% of wild-type. Pleural and dorsal transverse muscles in the lateral body wall show a moderate decrease. In a large fraction of presumptive somatic muscle precursors, Htl expression is wingless dependent. wingless is expressed in the mesoderm for only a short period after gastrulation, suggesting that the second phase of Htl expression may be controlled by Wingless signals from ectodermal cells (Shishido, 1997).

heartless is also required for normal development of the central nervous system. Most, if not all of the Heartless postitive CNS cells occupy positions corresponding to future longitudinal tracts, and they express Repo, an indication that these cells are interface glia: they consist of longitudinal glia and their immediate glial neighbors. Htl is expressed in the extended cytoplasmic sheets growing out of the glial cell bodies, which encircle the neuropil from both lateral and medial sides. The absence of heartless results in the failure of longitudinal glia to enwrap longitudinal axon tracts. heartless mutant phenotypes were partially mimicked by the targeted expression of activated Yan, thus demonstrating the involvement of the MAP kinase pathway in the differentiation of mesoderm. Yan is a downstream repressor of the MAP-kinase pathway; ectopic expression of activated Yan causes a reduction in Eve-expressing cells. A much less extensive effect of Fas II is also detected. Normal heart and somatic muscle formation does not occur, though midgut visceral muscle formation appears to proceed normally. (Shishido, 1997).

In contrast to later mesodermal expression of heartless, early expression in presumptive mesodermal cells prior to gastrulation is localized to the apical cell surface. During stage 9, the area expressing heartless divides into two domains: cephalic and postcephalic mesoderms. The pattern of heartless expression in the latter tissue begins to show signs of a metameric fluctuation along the anteroposterior axis. A similar segmental expression pattern has been reported for Twist -- high expression coincides with future parasegmental borders. During stage 10, heartless expression declines steadily with residual but strong expression remaining in a fraction of somatic muscle precursor cells. Little or no Htl protein is detected in heart precursors at stage 11. It is presumed that the second phase expression of Htl in most somatic muscle precursors starts during stage 11. Expression in heart precursors begins with stage 12. The heartless expressing heart precursors are Even-skipped negative. A second round of htl expression in visceral muscle precursors for midgut and hindgut is discernible at stage 14, when dorsal pharyngeal muscles and foregut visceral muscles, derivatives of the cephalic mesoderm, are Htl positive. From stage 12 onward, Htl is evident in a fraction of CNS cells (Shishido, 1997).

Neurons provide critical signals that regulate both the number and differentiation of glia. In addition, glia are attracted to and enwrap neuronal axonal processes. FGF-like signaling is thought to be one of the many potential axon-derived morphogenetic signals, however, the multiple roles of FGFs have made experimental tests of these signals difficult in vivo. In the Drosophila FGF receptor mutant heartless, glia migrate to axons, but fail to elongate around them. This study shows that in the similar but larger grasshopper CNS, FGF signaling is likely to mediate one step in the close interaction between glia and axons. To examine the expression of htl in the grasshopper embryo, antiserum against the extracellular portion of the fly protein was used to stain gel-resolved protein extracts of grasshopper embryos as well as intact ganglia. A single major protein species stains with the antiserum; in addition, the amount of phosphotyrosine is increased upon treatment of ganglia with FGF2. Staining for Htl in the embryo is seen in distinct punctate clusters at the axonal/glial interface in axon bundles, muscle precursors and a pair of neurons. Two large bundles of axons connect each ventral nerve cord ganglion and contain a central core of axons surrounded by glial cells. The glial nuclei are on the outer surface and their processes wrap around the outside of the axonal bundle. Staining for Htl is seen mostly at the neuronal glial interface. This striped Htl staining pattern at the glial/axonal interface is generally unilateral and discontinuous. FGF2-coated beads attract glia in the CNS and compete with axons for their resident, enwrapped glia. In addition, bath applied FGF2 causes mature axonal glia, which normally enwrap axon tracts, to round up. FGF2 activates the product of the grasshopper heartless FGF receptor gene and probably interferes with the normal function of an endogenous axon-associated FGF-like molecule. It is proposed that insect axons provide a critical spatially restricted FGF-like signal that induces glia to enwrap the axons (Condron, 1999).

Drosophila mesoderm migration behaviour during gastrulation

Mesoderm migration is a pivotal event in the early embryonic development of animals. One of the best-studied examples occurs during Drosophila gastrulation. Here, mesodermal cells invaginate, undergo an epithelial-to-mesenchymal transition (EMT), and spread out dorsally over the inner surface of the ectoderm. Although several genes required for spreading have been identified, the inability to visualise mesodermal cells in living embryos has hampered gathering of information about the cell rearrangements involved. Several mechanisms, such as chemotaxis towards a dorsally expressed attractant, differential affinity between mesodermal cells and the ectoderm, and convergent extension, have been proposed. This study resolved the behaviour of Drosophila mesodermal cells in live embryos using photoactivatable-GFP fused to alpha-Tubulin (PAGFP-Tub). By photoactivating presumptive mesodermal cells before gastrulation, it was possible to observe their migration over non-fluorescent ectodermal cells. The outermost (outer) cells, which are in contact with the ectoderm, migrate dorsolaterally as a group but can be overtaken by more internal (inner) cells. Using laser-photoactivation of individual cells, it was then shown that inner cells adjacent to the center of the furrow migrate dorsolaterally away from the midline to reach dorsal positions, while cells at the center of the furrow disperse randomly across the mesoderm, before intercalating with outer cells. These movements are dependent on the FGF receptor Heartless. The results indicate that chemotactic movement and differential affinity are the primary drivers of mesodermal cell spreading. These characterisations pave the way for a more detailed analysis of gene function during early mesoderm development (Murray, 2007).

Using a combination of whole mesoderm and single-cell photoactivation this study has observed the combination of cell behaviours employed by Drosophila mesodermal cells to form a monolayer, providing insights into the mechanisms responsible for this important part of gastrulation. The first observation was that outer cells moved dorsolaterally over the ectoderm. Although this is not unexpected, it nevertheless confirms a central prediction of the chemoattraction model: that cells migrate in a dorsolateral direction. Remarkably, it was then observed that inner cells are able to overtake outer cells to achieve a more dorsal position. Single-cell labelling then showed that these inner cells were likely to have originated from a position adjacent to the centre of the ventral furrow. Significantly, inner lateral (IL) cell progeny invariably move away from the midline, suggesting that they receive a directional guidance cue from the dorsal region of the ectoderm, again consistent with a chemoattraction model (Murray, 2007).

A complication in the simple chemoattraction model is that the two likely chemoattractants, Pyr and Ths, are initially expressed in quite broad lateral domains. During mesoderm migration, however, pyr expression does become restricted to the more dorsal parts of the ectoderm, whereas ths is expressed in a complementary fashion in the ventral regions of the neurogenic ectoderm. It has been suggested that the two ligands may have different binding affinities, and that the refinement of Pyr expression to more dorsal positions could guide mesodermal cells dorsally. An alternative is that those regions of the ectoderm that are not yet covered with mesodermal cells, such as the dorsal ectoderm, are highly attractive to mesodermal cells simply because the FGF ligands that they are producing are not being bound and internalised by outer cells already in contact with the ectoderm (Murray, 2007).

An alternative to chemoattraction that has been suggested is that FGFR activation is permissive rather than instructive and simply imparts a degree of motility to cells, allowing them to disperse until they are able to contact the ectoderm. This motility, combined with a steric hindrance effect, in which cells tended to move into unoccupied territory, could theoretically achieve a monolayer in the absence of directional cues. It would be expected, however, that if IL cell progeny were simply made motile and moved randomly, that cells adjacent to the midline would sometimes cross the midline to contact the ectoderm on the opposing side. This was never observed (Murray, 2007).

The movement of inner cells past the lateralmost outer cells is also consistent with the differential affinity model, according to which mesodermal cells form strong adhesions with the ectoderm. Cells not already in contact with the ectoderm would either intercalate between existing outer cells, or, as seen here, move past them. The fact that intercalation was not seen suggests either that outer cells adhere strongly to the ectoderm and do not easily move apart, or, again, that outer cells are masking FGF produced in the ectoderm. If a differential affinity model is active, the most likely candidate adhesion molecules would be integrins, which are expressed at the interface of the mesoderm and ectoderm, although there is, as yet, no published evidence for a functional role for integrins in this process (Murray, 2007).

During the initial migration of outer cells over the ectoderm it was found that cells maintained their position relative to their immediate neighbours. This result supports the argument against the convergent extension model. If convergent extension was a primary driving force behind lateral spreading, one would expect to see widespread intercalation throughout the mesoderm as inner cells pushed in between existing outer cells. This was not observed, although the possibility cannot be ruled out that some degree of intercalation does occur during this migration phase. Intercalation does, however, appear to play a part during the later stages of the formation of the monolayer, where inner medial (IM) cell progeny are seen appearing at the ectoderm. The timing of this event, at around the time of the second mitosis, suggests that the sudden lateral spreading that accompanies the second mitotic wave (50 minutes of development) may be due to the intercalation of a pool of inner cells. One possibility is that the adhesion between the mesodermal cells and the surrounding cells, both mesodermal and ectodermal, is decreased as they go through mitosis, permitting the inner cells access to their preferred position in association with the ectoderm. Thus, although a general convergent extension is not in evidence, intercalation does appear to contribute to mesoderm spreading (Murray, 2007).

On the basis of these observations, the following model of mesoderm cell behaviour following ventral furrow formation is presented. Following the breakdown of the epithelium, the first division results in a rapid spreading down onto the ectoderm, presumably due to decreased adhesion between mesodermal cells. Cells that are thereby placed in contact with the ectoderm start to polarise and proceed to migrate dorsolaterally as a group. Outer cells form a strong adhesive contact with the ectoderm, which prevents inner cells from intercalating between them and instead forces inner cells either to take up positions that outer cells vacate near the midline or move past them to more dorsal positions. Inner lateral cells receive a directional cue from the dorsal ectoderm guiding them laterally, over the outer cells. In this manner, by the time of the second mitosis the ectoderm is largely covered by mesodermal cells. Inner medial cell progeny that have failed to contact the ectoderm during the initial spreading are prevented from doing so by cells already strongly adhered to the ectoderm until the time of the second division. The second division then allows the remaining inner cells to contact the ectoderm. This intercalation produces a rapid lateral extension followed by a general retraction as the cells exit mitosis and re-establish adhesive contacts, with the ectoderm finally forming the monolayer (Murray, 2007).

The combination of behaviours observed may represent the most efficient way to rapidly spread one tissue over another. The tendency for cells to migrate dorsolaterally helps to constantly make space for those cells placed nearer the midline. If cells that contacted the ectoderm never moved away, it would mean that internal cells would have to travel further and further dorsally to find space on the ectoderm. In a similar manner, if chemotaxis towards a dorsally placed attractant was the only mechanism operating, one might expect that cells would continue moving dorsally, even if this resulted in an excess of cells in dorsal positions and a deficit closer to the midline. The tendency of mesodermal cells to develop and maintain a strong adhesive contact with the ectoderm would help ensure that all parts of the ectoderm remain covered. Finally, having a period of intercalation serves to give any remaining inner cells a chance to finally contact the ectoderm (Murray, 2007).

The resolution of mesodermal cell behaviour described in this study will make it possible analysis in greater detail of the migration defects in mutants such as htl and pebble. It will also make it possible to test whether cell rearrangements are normal in those situations in which directional information is lost, but in which spreading still occurs (e.g. rescue with activated Htl, or widespread, non-localised expression of FGF ligands). Finally, it will be of interest to determine whether the behaviors observed are typical of mesoderm migration in other systems. In mouse embryos, mesodermal cells emanating from the primitive streak migrate out over the basal surface of the primitive ectoderm to eventually form the mesodermal layer of cells. The cell rearrangements that occur during this process are not known. Photoactivatable GFP, which has provided such a versatile analysis tool here, could be applied to cultured mouse embryos to resolve these events (Murray, 2007).

Embryonic development of the Drosophila corpus cardiacum

The development of the Drosophila neuroendocrine gland, the corpus cardiacum (CC) was investigated, along with the role of regulatory genes and signaling pathways in CC morphogenesis. CC progenitors segregate from the blastoderm as part of the anterior lip of the ventral furrow. Among the early genetic determinants expressed and required in this domain are the genes giant (gt) and sine oculis (so). During the extended germ band stage, CC progenitor cells form a paired cluster of 6–8 cells sandwiched in between the inner surface of the protocerebrum and the foregut. While flanking the protocerebrum, CC progenitors are in direct contact with the neural precursors that give rise to the pars intercerebralis, the part of the brain whose neurons later innervate the CC. At this stage, the CC progenitors turn on the homeobox gene glass (gl), which is essential for the differentiation of the CC. During germ band retraction, CC progenitors increase in number and migrate posteriorly, passing underneath the brain commissure and attaching themselves to the primordia of the corpora allata (CA). During dorsal closure, the CC and CA move around the anterior aorta to become the ring gland (see Image). Signaling pathways that shape the determination and morphogenesis of the CC are decapentaplegic (dpp) and its antagonist short gastrulation (sog), as well as hedgehog (hh) and heartless (htl; a Drosophila FGFR homolog). Sog is expressed in the midventral domain from where CC progenitors originate, and these cells are completely absent in sog mutants. Dpp and hh are expressed in the anterior visceral head mesoderm and the foregut, respectively; both of these tissues flank the CC. Loss of hh and dpp results in defects in CC proliferation and migration. Htl appears in the somatic mesoderm of the head and trunk. Although mutations of htl do not cause direct effects on the early development of the CC, the later formation of the ring gland is highly abnormal due to the absence of the aorta in these mutants. Defects in the CC are also caused by mutations that severely reduce the protocerebrum, including tailless (tll), suggesting that additional signaling events exist between brain and CC progenitors. The parallels between neuroendocrine development in Drosophila and vertebrates are discussed (De Velasco, 2004).

In the larva, the ring gland forms a large and conspicuous structure located anterior to the brain and connected to the brain by a pair of tracheal branches and the paired nerve of the corpus cardiacum (NCC). Three different glands, the corpus allatum (CA; dorsally), prothoracic gland (laterally), and corpus cardiacum (CC; ventrally) form part of the ring gland. By far, most of its volume is taken up by the prothoracic gland whose cells, the source of ecdysone, grow in size and number as larval development progresses, whereas the cells of the CC remain small and do not appear to proliferate. Both the CC and CA, as well as axons innervating the ring gland, are FasII positive from the late embryonic stage onward. Labeling of the CC is stronger and starts earlier (stage 11) than that of the CA (stage 15), which makes it easy to distinguish between the two structures in the embryo. Another convenient marker of the CC is adipokinetic hormone (AKH), which is expressed exclusively in the CC from late embryonic stages onward (De Velasco, 2004).

The ring gland of the mature embryo is situated posterior to the brain hemispheres. The CC and CA occupy their positions ventral and dorsal to the aorta, respectively. The prothoracic gland cannot yet be recognized as a separate entity, possibly due to the fact that its precursors are small and few in number. Cells of the CC number around eight on each side and are arranged in a U-shape around the floor of the aorta. All cells are spindle shaped and send short processes ventromedially where they meet and form a bundle attached to the ventral wall of the aorta (subaortic processes) (De Velasco, 2004).

Several signaling pathways, notably Shh, BMP, and BMP antagonists, Wnt and FGF, specify the fate map of the head in vertebrates and also control later morphogenetic events shaping head structures. The same signaling pathways are active at multiple stages in Drosophila head development, and the pattern of activity and requirement of these pathways in regard to CC development was therefore investigated (De Velasco, 2004). .

The first signal acting zygotically in the Drosophila head is the BMP homolog Dpp, which forms a dorsoventral gradient across the blastoderm. The homolog of the BMP antagonist Chordin, short gastrulation (Sog), is expressed in the ventral blastoderm, overlapping with the ventral furrow. Loss of sog results in the absence of the CC, while the SNS is still present, which reflects ventral origin of the CC. Sog seems to be the only signal, of those tested, required for CC determination, since mutation of all other pathways does not eliminate the CC but merely effects its size, shape, or location (De Velasco, 2004).

Following its early widespread dorsal expression, Dpp becomes more confined during gastrulation to a narrow mid-dorsal stripe and an anterior cap that corresponds to parts of the anlagen of the esophagus and epipharynx. From this domain segregates the most anterior population of head mesoderm cells that give rise to the visceral muscle of the esophagus and which maintain Dpp expression. The visceral mesoderm of the esophagus flanks both CC and SNS. Loss of Dpp causes absence of the SNS; the CC is still present and expresses AKH but does not migrate posteriorly (De Velasco, 2004).

Activity of the MAPK signaling pathway is widespread in the Drosophila head from gastrulation onward. Beside a wide anterior and posterior domain traversing the lateral and dorsal domain of the head ectoderm, the primordia of the foregut, including the SNS, and head mesoderm show a dynamic MAPK activity. At least two RTKs, EGFR and FGFR/heartless, drive the MAPK pathway in the embryonic head. EGFR is responsible for activation in the ectoderm and foregut. Loss of EGFR causes widespread cell death in the head and the absence of the SNS. The CC is still present, although reduced in size. Activation of MAPK by Heartless (Htl) occurs in a narrow anterior domain of head mesoderm that gives rise to the dorsal pharyngeal muscles. The foregut, SNS, and CC develop rather normally in htl mutants. However, the CC shows variable defects in shape and location, which are most likely due to the absence of the aorta and CA, both of which are derivatives of the dorsal mesoderm, which is defective in htl loss of function and to which the CC is normally attached (De Velasco, 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).

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the mesoderm

Blood progenitors arise from a pool of pluripotential cells ('hemangioblasts') within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. This paper shows that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ('cardiogenic clusters') that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the MAPK pathway by EGFR and FGFR is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts (Grigorian, 2011).

In this study pursued two goals: to elucidate the precise location and cellular composition of the cardiogenic mesoderm, and to analyze the mechanism by which Notch becomes activated in the restricted subset of these cells that become blood progenitors. The findings show that the cardiogenic mesoderm is comprised of segmentally reiterated pairs of clusters (cardiogenic clusters) defined by high expression levels of Dl, L'sc and activated MAPK. The MAPK pathway, activated through both EGFR and FGFR signaling, is required for the specification (EGFR) and maintenance (EGFR and FGFR) of all cardiogenic lineages. As shown previously, the default fate of all cardiogenic cells is cardioblasts. Notch activity triggered by Dl is required for the specification of blood progenitors (thoracic cardiogenic clusters) and nephrocytes (abdominal cardiogenic clusters), respectively. One of the downstream effects of MAPK signaling is to maintain high levels of Dl in the cardiogenic clusters, and to help localize Dl expression toward a dorsal subset of cells within these clusters, which will become cardioblasts. Dl stimulates Notch activity in the surrounding cells, which triggers the blood progenitor/nephrocyte fate in these cells (Grigorian, 2011).

The cardiogenic clusters form part of a larger population of mesodermal cells defined by high expression levels of l'sc. Based on l'sc in situ hybridization, these authors mapped 19 clusters of l'sc expressing cells within the somatic mesoderm. Many of these clusters (called 'myogenic clusters' in the following) give rise to one or two cells that transiently maintain high levels of l'sc, whereas the remaining cells within the cluster lose expression of l'sc. The l'sc-positive cells segregate from the mesoderm to a more superficial position, closer to the ectoderm, undergo one final mitotic division, and differentiate as muscle founder cells. Dl/Notch mediated lateral inhibition was shown to act during the singling-out of muscle founders from the myogenic clusters. Loss of this signaling pathway caused high levels of L'sc to persist in all cells of the myogenic clusters, with the result that all cells developed as muscle founders. Interestingly, loss of l'sc had only a mild effect, consisting of a slight reduction in muscle founders. This is similar to what was find in this paper in l'sc-deficient embryos, which show only a mild reduction in cardioblasts and other cardiogenic lineages (Grigorian, 2011).

The developmental fate of most of the L'sc-positive clusters within the dorsal somatic mesoderm is different from that of the ventral and lateral myogenic clusters discussed above, even though several parallels concerning the morphogenesis, proliferation, and dependence on Dl/Notch signaling are evident. The somatic (anterior, Wg-positive) mesoderm is divided into a dorsal and ventral domain based on the expression of Tin. Initially expressed at high levels in the entire mesoderm, this gene is maintained only in the dorsal mesoderm, as a result of Dpp signaling from the dorsal ectoderm. The dorsal somatic mesoderm, which is called 'early cardiogenic mesoderm', includes four L'sc-positive clusters, C2 and C14-C16. The development of C2 has been described in detail. C2 gives rise to a progenitor that divides twice; two of the progeny become the Eve-positive pericardial cells. Meanwhile, C15 which appears later at the same position as C2, seems to behave like a 'normal' myogenic cluster. It produces a progenitor that divides once and forms the founders of the dorsal muscle DA1. As shown in this paper, the two remaining dorsal clusters, C14 and C16, give rise to the cardioblasts. It is noted that the Eve-positive progenitors, as well as the cardioblasts, resemble the muscle founders derived from the typical myogenic clusters in three aspects. First, they segregate toward a superficial position, close to the ectoderm, relative to the remainder of the cells within the clusters. Secondly, they undergo one (in case of C2: two) rounds of division right after segregation. And third, they are restricted in number by Dl/Notch signaling: in all cases, they are increased in number following Dl or Notch loss of function (Grigorian, 2011).

Cardiogenic clusters, like myogenic clusters, also depend on the MAPK signaling pathway. Past studies have shown that in the Eve-positive C2 and C15 clusters, Ras, is capable of inducing the formation of additional Eve-positive progenitors. Ras is a downstream activator of both the EGFR and FGFR tyrosine kinase pathways, both of which have been seen to be important for the formation of the Eve-positive progenitors. With a loss of the FGFR pathway, Eve-positive progenitors of both the C2 and C15 cluster are lost; by contrast, the EGFR pathway affects only C15. The balanced activity of MAPK and Notch, which in part depends on reciprocal interactions between these pathways, determines the correct number of C2/C15 derived progenitors. Ras-induced MAPK activation upregulates the expression of other MAPK signaling pathway members (autoregulatory feed-back loop), but also stimulates the antagonist Argos, as well as Dl. Dl-activated Notch, in turn, inhibits MAPK signaling (Grigorian, 2011).

Both Dl/Notch and MAPK signaling are active in the C14 and C16 clusters, which constitute the definitive cardiogenic mesoderm. MAPK activity is required for the maintenance of all lineages derived from these clusters, as shown most clearly in the EGFR LOF phenotype that entails a lack of cardioblasts, blood progenitors, and pericardial nephrocytes. Overexpression of Ras results in an increased number of all three cell types, which indicates that the C14/C16 clusters attain a larger size, possibly by an additional round of mitosis. The phenotype seen in embryos suffering from loss- or overexpression of Dl/Notch pathway members can be interpreted in the framework of a classical lateral-inhibition mechanism: Dl is upregulated in the C14/C16 derived cardioblast progenitors (analogous to the Eve-progenitors of C2/C15), from where it activates Notch in the remainder of the C14/C16 cells; these cells are thereby inhibited from forming cardioblasts, and instead become nephrocytes/blood progenitors. The level of Notch activity affects the expression of tin (low Notch) and the GATA homolog srp (high Notch), which triggers the fate of cardioblasts and blood progenitors/nephrocytes, respectively (Grigorian, 2011).

MAPK is required for the initial activation of Dl in the cardiogenic clusters (just as in the myogenic clusters). Input from the pathway is most likely also instrumental in the subsequent restriction of Dl to the cardioblast progenitors. The positive interaction between MAPK and Notch signaling could occur at several levels. A mechanism shown for the ommatidial precursors of the eye disc involves Ebi and Strawberry notch (Sno), which are thought to act downstream of EGFR signaling and lead to an upregulation of Dl through the Su(H) and SMRTER complex (Grigorian, 2011).

Generally, when one progenitor cell is seen to give rise to two different cell types it is accomplished in one of two ways. One: there is an asymmetric division, where a factor expressed by the progenitor is segregated into only one daughter cell; two: a non-uniformly expressed extrinsic signal effects one cell, but not its neighboring sibling. In the posterior (abdominal) segments of the Drosophila cardiogenic mesoderm, inhibition of Notch by Numb accounts for the asymmetric activity of Notch in a small set of cardiogenic mesoderm cells, the Svp-positive cells. If Numb function is removed, these cells, which normally produce two cardioblasts and two pericardial nephrocytes, instead give rise to four cardioblasts. However, multiple nephrocytes per segment remain in numb loss-of-function mutations; furthermore, loss of numb does not cause any defect in the blood progenitors, where asymmetrically dividing Svp-positive cells are absent. This suggests that in addition to the numb-mediated mechanism, directional activation of Notch by one of its ligands is required for the majority of nephrocytes and all of the blood progenitors. It is proposed in this study that the spatially restricted upregulation/maintenance of Dl in nascent cardioblasts acts to activate Notch in the remainder of the cells within each cardiogenic cluster, which promotes their fate as blood progenitors and nephrocytes (Grigorian, 2011).

The Notch signaling pathway is typically associated with members of two different types of bHLH transcription factors. One type act as activators, while the other act as repressors. In the context of lateral inhibition, best studied in Drosophila neurogenesis, activating bHLH transcriptions factors, including genes of the AS-C like l'sc, are expressed at an early stage in clusters of ectodermal or mesodermal cells, where they activate genetic programs that promote differentiative pathways such as neurogenesis, or myogenesis/cardiogenesis. Subsequently, Notch ligands initiate the Notch pathway in these clusters; cells with high Notch activity turn on members of the Hairy/E(spl) (HES) family of bHLH genes which act as repressors and abrogate the transcriptional programs that had been set in motion by the activating bHLH factors. This paper shows that the gene cassette consisting of the Notch signaling pathway, as well as activating and repressing bHLH factors, operates in the cardiogenic mesoderm to determine the balance between cardioblasts and blood progenitors/nephrocytes. As discussed in the following, the same cassette also appears to be centrally involved in the specification of vascular endothelial cells and hematopoietic stem cells in vertebrates, which adds to the list of profound similarities between Drosophila and vertebrate blood/vascular development (Grigorian, 2011).

Even prior to the appearance of hemangioblasts, the lateral mesoderm of vertebrates is prepatterned by sequentially activated signaling pathways and transcriptional regulators similar to those that act in flies. The Wnt/Wg pathway, for example, separates subdomains of the mesoderm in vertebrates and Drosophila, as well as more ancestral ecdysozoans. Notch signaling plays an essential role in generating boundaries between segmental, as well as intra-segmental, subdomains within the ectoderm and mesoderm. The FGF signaling pathway predates the appearance of Bilaterians and plays a highly conserved role in early mesoderm patterning. Likewise, specific sets of transcriptional regulators are the targets of these signaling pathways (e.g., twist, zfh and myostatin) and play a role during the establishments of cell fate in the mesoderm. It appears, therefore, that the bilaterian ancestor featured a mesodermal subdomain, the 'cardiogenic/lateral mesoderm, in which signals of the Wg, BMP, Notch, and FGF pathways and conserved sets of transcriptional regulators established boundaries and cell fate in the mesoderm (Grigorian, 2011).

The vertebrate gene encoding an activating bHLH factor with sequence similarity to the Drosophila AS-C genes is SCL. SCL expression in the lateral mesoderm marks the first appearance of hemangioblasts; note that SCL is also expressed widely in the developing vertebrate CNS. In Zebrafish, from their site of origin in the lateral mesoderm, SCL-positive hemangioblasts migrate dorso-medially and form the intermediate cell mass (ICM). The ICM is the site of primitive endothelial blood vessel and hematopoietic cell specification. Gain of function studies carried out in zebrafish embryos have shown SCL to be one of the genes important in specifying the hemangioblast from the posterior lateral plate mesoderm. The specification of hemangioblast here comes at the expense of other mesodermal cell fates, namely the somitic paraxial mesoderm. In mice, lack of SCL affects blood and vascular development as SCL mutants are bloodless and show angiogenesis defects in the yolk sac (Grigorian, 2011).

Vertebrate homologs of the repressive Drosophila Hairy/E(spl) family of bHLH genes are the Hes and Hey (hairy/Enhancer-of-split related with YRPW motif) genes. A well studied member of the Hey family in zebrafish is gridlock, which is required for the specification of hematopoietic progenitors from the ICM. Hey 2 mutations in mice lead to severe congenital heart defects. In addition, the Hes protein plays a role in hematopoiesis as it is a positive regulator of Hematopoietic Stem Cell (HSC) expansion (Grigorian, 2011).

Genetic studies of the Notch receptors and their ligands in vertebrates support the idea that this pathway does indeed play a crucial role in the initial determination of hematopoietic stem cells. The yolk sac and the para-aortic splanchnopleura (P-Sp)/AGM (aorta gonad mesonephros) of Notch null mouse embryos lack HSCs. A similar phenotype is observed in mutants of Jagged 1, one of the Notch ligands. Notch is thought to be the deciding factor between hematopoietic and endothelial cell fates when the two originate from a common precursor or hemangioblast. In murine mutants exhibiting lower Notch1 mRNA levels, a lack of hematopoietic precursors is seen and is accompanied by an increase in the number of cells expressing endothelial cell markers. Likewise, in Drosophila, loss of Notch is associated with an increase in cardioblast number and a loss of blood precursor cells (Grigorian, 2011).

Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. This study examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. Two transcriptional regulators were identified that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, the Drosophila heparan sulfate proteoglycan, Trol, was identified as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches (Dragojlovic-Munther, 2013).

Larval

DFR1 mRNA is observed in several imaginal discs. FR1 expression in the wing and leg discs takes place in probable myoblasts in a pattern similar to that of twist, a mesodermal gene. The mRNA was also detected in the morphogenetic furrow and the posterior region of the eye disc and around the proliferation center of the brain. These results suggest that DFR1 is involved in the development of mesodermal and neuronal cells constituting the adult body (Emori, 1993).

Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli

The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. This study shows that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation (Wu, 2017)

The use of discrete neuropil compartments for organizing and signaling information is widespread in invertebrate and vertebrate nervous systems. In both the fly antennal lobe and vertebrate olfactory bulb, axons from different ORN classes are segregated into distinct glomeruli. The rodent barrel cortex also uses discrete compartments, the barrels, to represent individual whiskers. This study shows that FGF signaling between neurons and glia mediates neural compartment formation in the Drosophila antennal lobe (Wu, 2017)

Members of the FGF family have diverse functions in a variety of tissues in both vertebrates and invertebrates. Vertebrate FGFs regulate not only neural proliferation, differentiation, axon guidance, and synaptogenesis but also gliogenesis, glial migration, and morphogenesis. Many of these roles are conserved in invertebrates. For example, Ths and Pyr induce glial wrapping of axonal tracts, much like the role other FGF members play in regulating myelin sheaths in mammals. Ths and Pyr also control Drosophila astrocyte migration and morphogenesis; likewise, FGF signaling promotes the morphogenesis of mammalian astrocytes. Therefore, studying the signaling pathways in Drosophila will extend understanding of the principles of neural development (Wu, 2017)

In ensheathing glia, whose developmental time course and mechanisms have not been well documented before this study, a glial response was observed to FGF signaling reminiscent of the paradigm shown previously; however, the exquisite compartmental structure of the Drosophila antennal lobe and genetic access allowed this study to scrutinize further the changes of neuropil structure and projection patterns that occurred alongside morphological phenotypes in ensheathing glia. The requirement for Ths in LNs was demonstrated, although it is possible that ORNs and PNs also contribute. The function was tested of the other ligand, Pyr, in antennal lobe development. No change was detected in ensheathing glia morphology with pyr RNAi, and double RNAi against ths and pyr did not enhance the phenotype compared with ths knockdown alone (Wu, 2017)

FGF signaling in glomerular wrapping appears to be highly local. In overexpression experiments, the hyperwrapping effect was restricted to the glomerulus where the ligand is excessively produced and did not spread to nearby nonadjacent glomeruli. These experiments suggest that Ths communicates locally to instruct glial ensheathment of the glomeruli rather than diffusing across several microns to affect nearby glomeruli. Because heparan sulfate proteoglycans are known to act as FGF coreceptors by modulating the activity and spatial distribution of the ligands, it is speculated that Ths in the antennal lobe may be subject to such regulation to limit its diffusion and long-range effect (Wu, 2017)

The data showed that deficient ensheathment of antennal lobe glomeruli is accompanied by imprecise ORN axon targeting. However, it was not possible to determine whether these targeting defects reflect initial axon-targeting errors or a failure to stabilize or maintain the discrete targeting pattern. Previous models for the establishment of antennal lobe wiring specificity suggested that the glomerular map is discernible by the time glia processes start to infiltrate the antennal lobe. Because of a lack of class-specific ORN markers for early developmental stages, the relative timing between when neighboring ORN classes refine their axonal targeting to discrete compartments and when ensheathing glia barriers are set up still remains unclear. Nevertheless, this discovery that FGF signaling functions in the formation of discrete neuronal compartments in the antennal lobe highlights an essential role for glia in the precise assembly of neural circuits (Wu, 2017)

Drosophila Heartless acts with Heartbroken/Dof in muscle founder differentiation

The formation of a multi-nucleate myofibre is directed, in Drosophila, by a founder cell. In the embryo, founders are selected by Notch-mediated lateral inhibition, while during adult myogenesis this mechanism of selection does not appear to operate. It is here shown, in the muscles of the adult abdomen, that the Fibroblast growth factor pathway mediates founder cell choice in a novel manner. It is suggested that the developmental patterns of Heartbroken/Dof and Sprouty result in defining the domain and timing of activation of the Fibroblast growth factor receptor Heartless in specific myoblasts, thereby converting them into founder cells. These results point to a way in which muscle differentiation could be initiated and define a critical developmental function for Heartbroken/Dof in myogenesis (Dutta, 2005).

During myogenesis in the Drosophila embryo a single precursor cell is chosen by Notch-mediated lateral inhibition. The daughters of the precursor cell form two embryonic muscle founder cells -- each with a characteristic pattern of expression of markers that specify its identity -- or they form an embryonic muscle founder cell and an adult myoblast progenitor. This latter cell type proliferates during larval life and its progeny, the adult myoblasts, are associated with imaginal discs and larval nerves. While embryonic founder cells shut down the expression of Twi, a marker of myoblast identity, the adult myoblasts retain Twi expression during their proliferative phase during larval life. At the onset of metamorphosis, Twi levels decline in a group of cells, the adult founders, that express duf-lacZ at high levels and are located at the sites of myofibre formation. Twi expression is also shut off in other myoblasts as they fuse with the founder to form multi-nucleate cells (Dutta, 2005).

Interestingly, adult myoblasts, like the embryonic founders from whose siblings they are derived, express duf-lacZ (albeit at low levels) throughout larval life. As adult muscle differentiation begins, this low-level expression changes dramatically to a pattern in which one founder cell -- expressing duf-lacZ at high levels -- is chosen to seed each muscle fibre. How is this founder cell chosen? Removal of Notch signalling in adult myoblasts does not result in an increase in the number of founders. This suggests that lateral inhibition mediated by Notch, the process that operates in the embryo, is not the mechanism by which adult founders are chosen. Indeed, the requirements are quite different; adult myoblasts all express duf-lacZ at low levels, suggesting (consistent with their origins as siblings of embryonic founders) that they all already have some properties similar to founder cells. In choosing adult founder cells, therefore, duf-lacZ is to be up-regulated in cells that will become founders and down-regulated in others that will become fusion-competent cells. The results of this study show that the Htl pathway plays a key role in choosing adult founders. It is suggested that Htl does this using an unusual mechanism in which an intracellular positive regulator plays an important role (Dutta, 2005).

Adult myoblasts in the third larval instar express Twi, Hbr/Dof, Htl, and sty-lacZ. At the onset of adult abdominal myogenesis, Twi expression declines. With this, the expression of Hbr and Sty declines in myoblasts. It is suggested that, in the third instar larva, the presence of Sty prevents the activation of the Htl receptor, even if the ligand and Hbr/Dof are available. However, since both Hbr/Dof and sty-lacZ expression decline with Twi, at the onset of myogenesis, the Htl receptor will still be unable to function, because Hbr/Dof is necessary for the function of the Htl receptor. It is suggested that, as Sty and Hbr/Dof expression decline (as Twi expression shuts down at the onset of myogenesis), the Htl receptor is active in some myoblasts. Htl signalling maintains Hbr/Dof expression in these cells by a positive feedback mechanism. Maintenance of Hbr/Dof expression reinforces the Htl signal, which in turn up-regulates the expression of founder-specific genes such as duf in these cells, thereby imparting them with founder properties. Consistent with this hypothesis, activating the Htl receptor results in the maintenance of Hbr/Dof in adult myoblasts. This prolonged activation of Hbr/Dof, and therefore of duf, could be the cause of morphological changes associated with the excess founder cells (Dutta, 2005).

How could this localised activation of the receptor occur? One way is via the localised availability of the Htl ligand. Proximity of some of the cells to the source of the ligand could cause higher levels of Htl signalling in those cells than others, thus biasing their fate towards that of a founder. Examining the expression pattern of the recently identified ligands of Htl should be able to resolve whether this indeed is the case. A second, and more likely, mechanism for localised activation of receptor is via a process that does not involve the localised presence of the ligand. This possibility is suggested because the continued mis-expression of Hbr/Dof in all adult myoblasts results in an increased number of founders and muscle fibres. Since Hbr/Dof function is dependent on ligand activation of the receptor, the ligand must be available to Htl on all myoblasts. Local activation of the receptor could occur by Hbr/Dof being maintained briefly in a founder cell pattern in some myoblasts even as all of the others down-regulate Sty and Hbr/Dof at the onset of myogenesis (with the decline of Twi expression). This continued expression of Hbr/Dof in some myoblasts, and the absence of Sty, could allow local activation of the receptor and the consequent maintenance of Hbr/Dof in a founder pattern (Dutta, 2005).

The problem then shifts to deciphering the mechanism by which the (hypothetical) localised activation of Hbr/Dof takes place. Since abdominal myoblasts are associated with nerves, one possibility is that the signal could come from the nerves. This 'solution' has two problems, however. (1) It is not clear how a precise periodicity of signal, expressed along the nerve and seen by associated myoblasts, would be generated to organise the correct spacing of founder cells. More pertinent perhaps is the observation that (2) surgical removal of the nerve does not affect the number of muscle fibres. Thus, nerves are unlikely to be the source for the signal that organises myoblasts in a founder pattern. Another possible source for a signal that maintains and elevates Hbr/Dof expression in a founder pattern could be the epidermis. The abdominal epidermis develops from ectodermal cells, the histoblasts. As the epidermis differentiates during metamorphosis, muscle tendon precursor cells (specified by and expressing the stripe locus) can be identified. The tendon precursor cells, given that they are in proximity to the differentiating myoblasts, could possibly be a source of organising signal that modulates Hbr/Dof expression to a founder pattern. Thus, the precise segmental and regional patterning of the epidermis could organise the pattern of founder cells in the developing abdominal musculature. In favour of this hypothesis is the finding that reduction of stripe-expressing cells in the dorsal thoracic disc results in the reduction of duf-lacZ expression in the larval templates that give rise to the thoracic dorsal longitudinal muscles, and increasing stripe expression in the ectoderm results in the increase of duf-lacZ expression in the developing dorsal longitudinal muscles. It is not known yet if these results apply to the abdomen (Dutta, 2005).

A third possible mechanism of localised activation of Htl, not exclusive of either of the ones mentioned earlier, is that a dynamic interaction between ligands, other activators, and repressors results in the activation of Htl in a specific pattern. Such a process has been described in the embryo, e.g., in the anterior patterning of follicle cells in the Drosophila egg (Dutta, 2005).

In conclusion, while many mechanistic details still remain elusive, the implication of the FGF pathway as a key player in adult founder cell choice provides the molecular tools to identify missing elements in the pathway. Integrated within the broad question of founder cell specification are more specific questions pertinent to the different muscle groups. Activation of Htl signalling produces a less prominent effect on the dorsal muscles than on the lateral muscles. Also, the extra founders of the dorsal muscles are located in a characteristic fashion (altered in orientation) that is different from that observed for the excess lateral founders. These observations raise questions about whether the dorsal and lateral groups of founders have different levels of sensitivity to the FGF pathway and whether they employ the pathway in different ways (Dutta, 2005).

The results allow the testing of whether this pathway operates in a similar manner during myogenesis in other contexts in Drosophila and in other animals, in particular the higher vertebrates. Vertebrate muscles are composed of multiple fibres, which make them similar to Drosophila adult muscles. Vertebrate myogenesis shares several features with Drosophila myogenesis, at the level of genetic and molecular regulatory mechanisms. The FGF pathway in vertebrates, mediated by multiple isoforms of the receptor and the ligand, has been found to play an instructive role in induction and commitment of myogenic cells. In Xenopus, for instance, an FGF-mediated pathway controls specification and differentiation of myotomal progenitors. Also, signalling via FGFR4 positively regulates myogenic differentiation during avian limb muscle development. The present study, showing the role of Htl in muscle differentiation, highlights yet another similarity. This study also provides directions for probing how the number and location of fibres are regulated in vertebrates, questions that remain to be resolved in the field of vertebrate myogenesis (Dutta, 2005).

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early fetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis this study found that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis it was found that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, this study has identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and has shown that the molecular circuitry required is lineage specific (Avet-Rochex, 2012).

The correct control of gliogenesis is crucial to CNS development and the Drosophila post-embryonic nervous system is a powerful model for elucidating the molecular players that control this process. This study has identified two separate glial populations that proliferate extensively and have defined the key molecular players that control their genesis and proliferation. Perineural and cortex glia both use insulin and FGF signalling in a concerted manner, but the requirements for these pathways are different in each glial type. The data suggest a model that describes the molecular requirements for post-embryonic gliogenesis in each of these glial types in the brain (Avet-Rochex, 2012).

The results show that Pyramus is expressed by perineural glia to activate FGF signalling in adjacent glia and acts in parallel to InR/TOR signalling (activated by the expression of Dilp6). These two pathways act synergistically to generate the correct complement of perineural glia. The results also show that cortex glia proliferation is controlled by FGF signalling through FGFR (Htl) and the Ras/MAPK pathway. Pyr expression is required from both glia and neurons and acts non-cell-autonomously. Neuronal Pyr expression activates the FGFR on adjacent cortex glia, thereby coordinating neurogenesis and glial proliferation. InR is also partially required in cortex glia and is likely to signal through the Ras/MAPK pathway (Avet-Rochex, 2012).

Using both pan-glial inhibition and LOF clonal analysis this study has shown that the InR/TOR pathway is required for perineural glia proliferation. InR/TOR signalling has widespread roles in nervous system development and a role has been demonstrated for this pathway in the temporal control of neurogenesis (Bateman, 2004; McNeill, 2008). InR can be activated by any one of seven DILPs encoded by the Drosophila genome, which can act redundantly by compensating for each other. dilp6 is expressed in most glia during larval development, including perineural and cortex glia, and that dilp6 mutants have reduced gliogenesis. The dilp6 phenotype is weaker than that associated with the inhibition of downstream components of the InR/TOR pathway, suggesting that other DILPs might be able to compensate for the absence of dilp6 expression in glia (Gronke, 2010). Pan-glial inhibition and clonal analysis also demonstrated that the FGF pathway is required for normal levels of perineural glia proliferation. FGF signalling is activated in perineural glia by paracrine expression of Pyr. Inhibition of either the InR/TOR or FGF pathway reduced perineural glia proliferation by about half, so tests were performed to see whether these two pathways act together. The data demonstrate that inhibition of both pathways simultaneously has a synergistic effect, suggesting that these two pathways act in parallel, rather than sequentially, and that their combined activities generate the large numbers of perineural glia found in the adult brain (Avet-Rochex, 2012).

Cortex glia employ a molecular mechanism distinct from that of perineural glia to regulate their proliferation. Cortex glia have a clear requirement for InR, as InR mutant cortex clones are significantly reduced in size. The early events in post-embryonic gliogenesis are poorly understood, but FGF signalling is likely to be required during this stage as LOF clones for components of this pathway almost completely block cortex gliogenesis. These data suggest that InR acts in parallel to FGF signalling in these cells, as loss of InR combined with activation of FGF signalling only partially rescues the InR phenotype. Interestingly, the PI3K/TOR pathway is not required in cortex glia, suggesting that InR signals through the Ras/MAPK pathway to control cortex glia proliferation (Avet-Rochex, 2012).

The FGF pathway in cortex glia responds to paracrine Pyr expression from both glia and neurons. Expression from both glia and neurons is required to activate the pathway and stimulate cortex gliogenesis. Neuronal regulation of glial FGF signalling enables cortical neurogenesis to modulate the rate of gliogenesis, so that the requisite number of glia are generated to correctly enwrap and support developing cortical neurons. Recent studies have also identified a mechanism by which DILP secretion by glia controls neuroblast cell-cycle re-entry in the Drosophila early post-embryonic CNS. Thus, neurons and glia mutually regulate each other's proliferation to coordinate correct brain development (Avet-Rochex, 2012).

This study has shown that two major glial populations in the larval brain, perineural and cortex glia, are generated by glial proliferation rather than differentiation from neuroglioblast or glioblast precursors. Differentiation of most embryonic glia from neuroglioblasts in the VNC requires the transcription factor glial cells missing (gcm), which is both necessary and sufficient for glial cell fate. In the larval brain the role of gcm is much more restricted and it is not expressed in, nor required for, generation of perineural glia. Thus, the developmental constraints on gliogenesis in the embryonic and larval CNS are distinct. The larval brain undergoes a dramatic increase in size during the third instar, which might favour a proliferative mode, rather than continuous differentiation from a progenitor cell type (Avet-Rochex, 2012).

Glial dysfunction is a major contributor to human disease. The release of toxic factors from astrocytes has been suggested to be a contributory factor in amyotrophic lateral sclerosis and astrocytes might also play a role in the clearance of toxic Aβ in Alzheimer's disease. Rett syndrome is an autism spectrum disorder caused by LOF of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Astrocytes from MeCP2-deficient mice proliferate slowly and have been suggested to cause aberrant neuronal development. This hypothesis was recently confirmed by astrocyte-specific re-expression of Mecp2 in MeCP2-deficient mice, which improved the neuronal morphology, lifespan and behavioural phenotypes associated with Rett syndrome. Characterisation of the molecular control of gliogenesis during development might lead to a better understanding of such diseases (Avet-Rochex, 2012).

Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation

During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).

During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).

Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).

Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).

Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several RasGTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of RasGTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).

The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active PointedP1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).

This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).

In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of RasGTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).

Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes

Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. This study characterized a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. The Fibroblast growth factor (FGF) receptor Heartless was demonstrated to autonomously control astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. It was further shown that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. The data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo (Stork, 2014).

Astrocytes are among the most abundant cell types in the mammalian CNS and fulfill diverse functions in brain development and physiology. In the mature brain, astrocytes buffer ions and pH, metabolically support neurons, and clear neurotransmitters. Astrocytes can sense neuronal activity, react with transient increases of intracellular calcium ion concentration, and in turn modulate neuronal activity. The diverse homeostatic and modulatory roles for astrocytes are essential for neuronal function, and evidence is mounting that this tight physiological relationship between astrocytes and neurons is highly regulated and provides astrocytes with the capacity to exert powerful and dynamic control over neuronal circuits (Stork, 2014).

Astrocytic functions are critically dependent on the intimate spatial relationship between astrocytes and neurons, and accordingly astrocytes exhibit a highly ramified morphology. Primary cellular extensions radiate from the soma of gray matter astrocytes, which then branch into hundreds of increasingly finer cellular processes, ultimately forming a dense meshwork in the brain that associates closely with synapses, neuronal cell bodies, and the brain vasculature. Intriguingly, individual mature mammalian astrocytes occupy unique spatial domains within the brain, apparently 'tiling' through a mechanism akin to dendritic tiling, such that the processes of neighboring astrocytes exhibit very limited overlap. Whether these unique spatial domains are functionally important remains a point of speculation (Stork, 2014).

Despite recent advances in understanding the molecular basis of astrocyte fate specification, control of synapse formation, and neuronal signaling, pathways regulating astrocyte morphogenesis in vivo remain poorly understood. While there appears to be a spatial restriction of astrocyte subtypes to particular regions of the vertebrate CNS, it is not clear whether astrocytes selectively associate with predetermined subsets of neurons. The morphology of individual mammalian astrocytes is quite variable, suggesting that sculpting of their morphology may be stochastic and shaped by cell-cell interactions (Stork, 2014).

This study characterizes a glial cell type in Drosophila remarkably similar to mammalian protoplasmic astrocytes. Drosophila astrocytes dynamically and progressively invade the synaptic neuropil late in embryonic development, associate closely with synapses throughout the CNS, and tile with one another to establish unique spatial domains. The Heartless FGF receptor signaling pathway was identified as a key mediator of astrocyte outgrowth into synaptic regions and the size of individual astrocytes. Through ablation studies, it was demonstrated that individual astrocytes have a remarkable potential for growth, and the establishment of astrocyte spatial domains is mediated by astrocyte-astrocyte inhibitory and/or competitive interactions. This work provides insights into cell-cell interactions governing astrocyte growth in vivo and demonstrates that the requirement for astrocytes is an ancient feature of the nervous system of complex metazoans (Stork, 2014).

Drosophila astrocytes form a highly ramified and dense meshwork of processes that infiltrate the entire neuropil and associate closely with synapses. This close spatial relationship is reminiscent of the mammalian 'tripartite synapse,' thought to be critical for neurotransmitter clearance and the modulation of synaptic activity during complex behaviors. In the L3 VNC, the majority of synapses were in close proximity to astroglial processes, although not directly ensheathed. Nevertheless, using the iGluSnFR reporter, it was demonstrated that local increases in extracellular glutamate readily reached astrocyte membranes, indicating that they are within the functional range of synapses (Stork, 2014).

Functional roles of Drosophila astrocytes also appear well conserved when compared to mammals. The glutamate transporter EAAT1 is expressed in Drosophila astrocytes and is essential for coordinated locomotor activity in larvae and prevention of excitotoxicity in the adult. This study demonstrates astrocyte-specific expression the GABA transporter Gat and partial loss of Gat impeded larval locomotion. GABA transporter inhibitors also impair larval coordinated locomotion, and Manduca and Trichoplusia Gat homologs are high-affinity GABA transporters, supporting the notion that gat-depleted animals experience disruption of GABA neurotransmitter clearance. Despite apparently normal CNS morphology, gat null animals die as late embryos. Astrocytic Gat is therefore essential for viability, and it is proposed that Gat plays a central role for astrocyte-mediated GABA clearance even before animal hatching (Stork, 2014).

Ca2+ microdomain signaling in mammalian astrocytes is emerging as a key mechanism by which astrocytes respond to and regulate neuronal activity. Drosophila cortex glia, cells closely associated with neuronal cell bodies, also exhibit microdomain Ca2+ oscillations, and glial Ca2+ signaling events can modulate fly circadian behavior and seizure activity. Interestingly, this study found Drosophila astrocytes exhibit spontaneous, local Ca2+ transients in vivo and seem to be coupled with respect to Ca2+ signaling: laser-induced injury of a single astrocyte in the larva induced an increase in intracellular calcium in the injured cell, which subsequently spread into neighboring astrocytes (Stork, 2014).

These data taken together argue strongly that Drosophila astrocytes will prove an excellent in vivo system in which to study many fundamental aspects of astrocyte biology and astrocyte-neuron interactions (Stork, 2014).

This study has shown that Drosophila astrocytes are critically important for animal survival. Partial ablation of mouse astrocytes during development also led to death at birth. Interestingly, astrocyte depletion by ~30% in selected spinal cord domains led to atrophy and loss of neuropil and synapses. In Drosophila larvae lacking the majority of astrocytes, gross CNS morphology was surprisingly normal. Therefore, fly astrocytes may not be strictly required for neuronal survival, although earlier ablation or ablations in the adult could yield different results. Alternatively, other subtypes of CNS glia (e.g., ensheathing or cortex glia) might functionally substitute for astrocytes and promote neuronal survival (Stork, 2014).

Depletion of astrocytes from large regions of the mammalian CNS did not lead to a repopulation of these zones by astrocytes from neighboring domains, suggesting that astrocytes possess a high regional specificity and low invasive behavior (Tsai, 2012). However, while dramatic movement of populations of astrocytes was not observed, it is less clear whether astrocytes at the border of astrocyte-depleted regions react more locally with increased growth. Regional astrocyte ablation studies in mammals followed by the use of markers that highlight single-cell astrocyte morphology will be essential to definitively resolve these question (Stork, 2014).

It has been proposed that astrocyte domain organization and association with specific subsets of neurons has an important role in the proper function of neuronal networks. While Drosophila astrocytes are quite stereotyped in cell number and cell body position, the domains of the neuropil covered by astrocyte processes show variability in size and shape. It therefore seems unlikely that individual astrocytes are genetically programmed to associate with particular regions of the brain or specific synapses (Stork, 2014).

Astrocytes appear to harbor a massive growth potential but exert a strong growth-inhibiting effect on one another. First, when adjacent cells are ablated, astrocytes expand their territories while tiling where they are in contact with other astrocytes. Second, when htl or dof mutant clones that failed to infiltrate the neuropil, the space adjacent to these clones was efficiently infiltrated by other astrocytes. Finally, while enhancing Htl signaling increased domain size, neighboring cells still 'tiled' and the overlap of astrocytic domains did not increase noticeably. How tiling of astrocytes occurs remains unclear but could be accomplished through contact-dependent growth inhibition or competition for neuropil growth factors. Nevertheless, based on the multiple lines of evidence presented in this study, it is proposed that astrocyte morphology is shaped dynamically during development by neuron-astrocyte and astrocyte-astrocyte interactions (Stork, 2014).

Finally, while the relative overlap of neighboring astrocytes appears to be higher in Drosophila compared to mammalian astrocytes, it is important to note from a mechanistic perspective that the size of a Drosophila astrocyte is smaller compared to mouse and that the absolute overlap of astrocyte processes in mouse and fly seem comparable. This discovery of tiling behavior in Drosophila suggests that fly and mammalian astrocytes may share common molecular mechanisms by which neighboring cells define their territories (Stork, 2014).

Loss of the FGF receptor Htl, its ligands Pyr and Ths, or the downstream signaling molecule Dof/Stumps blocked the infiltration of astrocyte processes into the neuropil, demonstrating that the Htl signaling pathway is critical for effective astrocytic growth into the synapse-rich neuropil. The level of Htl signaling is also critically involved in the regulation cell and domain size of astrocytes, with increased Htl signaling leading to increased astrocyte volume. Expression data, clonal analysis, and astrocyte-specific rescue experiments all indicate that Htl and Dof function autonomously in glia. Precisely where the FGF ligands Pyr and Ths are generated during development was more difficult to determine. However, based on its expression pattern and the ability to rescue astrocyte outgrowth when expressed in neurons, it is proposed that at least Ths is primarily derived from neurons (Stork, 2014).

Ectopic expression of Pyr or Ths away from the neuropil or astrocytic expression of a constitutively active form of Htl is able to partially restore astrocyte infiltration. These observations suggest a permissive role for the Htl signaling pathway in astroglial growth. However, expression of Pyr or Ths is also able to promote the outgrowth of ectopic astroglial branches outside of the neuropil, indicating that these ligands can provide directional cues for astrocyte outgrowth. Pyr and Ths appear different in their signaling abilities: single neuron expression revealed Pyr was unable to promote extension of astrocyte processes, while Ths drove robust astrocytic process outgrowth, suggesting that the promotion of outgrowth by Ths can act at a short range (Stork, 2014).

How can Pyr and Ths direct astrocyte process growth into the neuropil even when ectopically expressed? FGF signaling is critically dependent on heparan sulfate proteoglycans (HSPGs) in vivo. Two of the four HSPGs in Drosophila, Dally-like and Syndecan, have been reported to be prominently enriched in the embryonic neuropil, where they have been shown to act in Slit-dependent axon guidance. Expression of Sdc in the neuropil was confirmed, ectopic Sdc expression was found to be sufficient to redirect astrocyte membranes outside of the neuropil, and loss of Sdc led to a defect in the ventral migration of astrocyte cell bodies and, to a lesser extent, problems in early neuropil infiltration. Based on these observations it is speculated that Sdc plays a modulatory role in the development of astrocytes by concentrating the FGFs Pyr and Ths in the neuropil to drive directional infiltration even when the ligands are provided ectopically. Finally, Pyr and Ths might act redundantly with additional unidentified neuropil-restricted factors that can provide directional information for astrocytic process outgrowth (Stork, 2014).

ths null mutants showed a slight decrease in the number of astrocytes in late embryos and L3 larvae, while embryonic htlAB42 mutants did not show a reduction in cell counts. sdc mutants also showed a similar slight reduction in total cell number in L3 larvae. These data suggest that astrocytes are generated in the embryo at normal numbers in FGF-pathway mutants but that individual cells might be outcompeted by neighbors during process outgrowth, resulting in death of individual cells. Since it was not possible to uniquely identify the presumptive ventral cell among the dorsally located cells, it is not clear whether the nonmigrating presumptive ventral cells preferentially die or whether cell death is stochastic among all astrocytes. While the mechanism of such adjustment of cell numbers through cellular competition remains poorly understood, it might be based on competition for trophic factors or a more active form of cell killing by 'winning' neighbors. The data deepen understanding of the diverse roles FGF signaling plays in insect glial development, where FGFs have been shown to also regulate glial proliferation, survival, migration and ensheathment of axons, and glial wrapping of FGF2-coated beads in grasshopper (Stork, 2014).

FGF signaling has also been implicated in mammalian astrocyte development. Mammalian FGFs can act as mitogens for glial precursors and potentiate the ability of secreted factors CNTF and LIF to promote astroglial fate in neural progenitors. In addition, FGF application can induce maturation of astroglia in cell culture by controlling morphological stellation in two dimensions and the expression of GFAP and glutamine synthetase. FGF receptors 1-3 are expressed in astrocytes and their precursors. In particular, FGFR3 is highly enriched in the radial precursor cells in the ventricular zone and immature and mature astrocytes and in FGFR3 and other FGF pathway mutants, GFAP expression in astrocytes is perturbed in vivo. Furthermore FGFR1/2 mutants show a reduction in GFAP-positive astrocytes in the cortex and impaired Bergmann glia morphology in the cerebellum. The exact roles of mammalian FGFRs and their ligands in astrocyte ramification, association with neurons and synapses, and establishment of astrocytic domain size, however, remain to be tested. Observations of an essential requirement for FGF signaling in astrocyte development in vivo in Drosophila suggests that a detailed analysis of FGF signaling pathways in mammalian astrocyte development should prove fruitful. FGF signaling is known to be perturbed in glioma, and this study's observations of the key role for FGFs in astrocyte process outgrowth may ultimately provide insight into the highly invasive nature of glioma cells in the brain (Stork, 2014).


FGF receptor 1 continued: Biological Overview | Evolutionary Homologs | Regulation | Effects of Mutation | References

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