The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998).

Epidermal growth factor receptor signals through the conserved RAS pathway. One of the final effectors of the Ras pathway is MAP kinase. MAP kinase is activated by the dual phosphorylation of threonine and tyrosine residues, carried out by MEK. The recent production of a monoclonal antibody that specifically recognizes the active, dual phosphorylated form of MAP kinase allows one to follow in situ the activation pattern of receptor tyrosine kinase pathways such as Egfr. To identify when and in which cells Egfr signaling is required to promote early CNS development, the presence of active MAP kinase was assayed in wild-type, Egfr, vein, spitz and rhomboid;vein mutant embryos. During early embryogenesis, active MAP kinase is present in two temporally and spatially distinct patterns within the neuroectoderm in wild-type embryos. Prior to and during gastrulation active MAP kinase is first found in two broad bilaterally paired longitudinal bands of cells that run down the length of the neuroectoderm. At stage 10, active MAP kinase is again expressed in the neuroectoderm in the most medial neuroectodermal cells that flank the midline. In Egfr and in rhomboid vein mutant embryos active MAP kinase is not present in either pattern. In embryos singly mutant for either vein or spitz, the first wave of active MAP kinase appears in its normal pattern although at reduced levels. However, the second wave of active MAP kinase is absent in spitz, but normal in vein, mutant embryos. Thus, early CNS defects correlate with the absence of the first but not the second wave of active MAP kinase as clear CNS defects occur in Egfr and rhomboid;vein but not in spitz mutant embryos. MAP kinase is found to be present in the medial and intermediate but excluded from the lateral neuroectodermal column. It is concluded that Egfr is required for the activation of proneural genes and functions to subdivide the neuroectoderm (Skeath, 1998).

Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells. Individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of L'sc preclusters. One precluster, preC1, is found in the ventral mesoderm, and the other, preC2, is localized to the dorsal mesoderm. PreC2 encompasses the territory in which dorsal L'sc clusters C2 and C14-C17, the subject of this paper, subsequently develop. All mesodermal cells within preC2 precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), these RTKs are seen to be activated in small clusters of cells within the original competence domain (precluster). Each cluster represents an equivalence group because all members initially resemble progenitors in their expression of both L'sc and mesodermal identity genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes. The singling out of progenitors from mesodermal equivalence groups depends on lateral inhibition mediated by the neurogenic genes. Moreover, Egfr and Htl are differentially involved in the specification of particular progenitors (Carmena, 1998).

Transduction of RTK signals occurs, at least in part, via the Ras/MAPK cascade. By use of an antibody that is specific for the diphosphorylated or activated form of mitogen-associated protein kinase (MAPK) (diphospho-MAPK), it is possible to identify localized sites of RTK signaling in the Drosophila embryo. This reagent was used to monitor the spatial and temporal involvement of Egfr and Htl in the formation of mesodermal L'sc clusters and the specification of the corresponding progenitors. The earliest activation of MAPK in the fully migrated mesoderm occurs in C2, coincident with the restriction of L'sc and prior to the appearance of Eve in this cluster. These findings are consistent with the known requirement of Htl for C2 development. Diphospho-MAPK persists in C2 after the onset of Eve expression , but fades from most C2 cells during progenitor selection. Activated MAPK remains transiently in P2 (the paracardial precursor singled out in the C2 cluster) and then disappears. Simultaneously, C15 begins to express both diphospho-MAPK and Eve. The activation of MAPK in C15 is expected from the involvement of Htl and Egfr in C15 formation. P2 then divides to yield sibling founder cells, neither of which initially contains activated MAPK. However, by the time P15 is singled out, MAPK is reactivated in one of the sibling F2s. As is the case with P2, diphospho-MAPK remains at high levels in P15. Moreover, the persistence of MAPK activation correlates with the maintenance of Eve expression in both progenitors. Additional diphospho-MAPK expression is observed in cells derived from C14, C16, and C17, in agreement with the involvement of Htl in the formation of these clusters. Thus, both the temporal and spatial patterns of diphospho-MAPK expression are consistent with a requirement for RTK signaling in the formation of mesodermal L'sc clusters, the induction of mesodermal Eve expression, and the singling out of muscle and cardiac progenitors (Carmena, 1998).

The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobe primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwig’s organ cells that are called Bolwig’s organ founders. Phenotypic analysis of tll loss- and gain-of-function mutant embryos using specific markers for Bolwig’s organ and the optic lobe, reveals that tll functions to drive cells to an optic lobe fate, as opposed to a Bolwig’s organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwig’s organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwig’s organ pioneers. The data further suggest that the Bolwig’s organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwig’s organ cells where it activates the Epidermal growth factor receptor signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive, no evidence for regulation by the 'early eye genes' so, eya and ey, or by Egfr signaling is found (Daniel, 1999).

Epidermal growth factor receptor is activated in midline regions of the head neurectoderm, in particular in the anlage of the visual system. Moreover, increased and/or ectopic activation of Egfr results in a 'cyclops' phenotype very similar to what has been described for ectopic tll expression. Egfr signaling has been shown to be required in both chordotonal organs and compound eye for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated: (1) is Egfr signaling required for tll expression in the optic lobe and (2) is Egfr signaling involved in the recruitment of the secondary (non-atonal-expressing) Bolwig’s organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for the presence of Egfr-relevant mRNAs or proteins: Rhomboid mRNA, which would be expected to be present only in the signaling cells, and phosphorylated MAPK, Pointed and Argos mRNAs, which would be expected to be expressed in all cells receiving an Egfr-mediated signal. In stage 12 embryos, rho is expressed only in the small group of Bolwig’s organ founder cells (the same cells expressing ato). In contrast, activated (phosphorylated) MAPK is present in a larger cluster of cells including the entire Bolwig’s organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both known to be switched on in cells receiving the Spi signal, are expressed at the same stage throughout the entire Bolwig’s organ primordium. These gene expression and MAPK activation patterns are consistent with the idea that the Spi signal is activated by rho in the Bolwig’s organ founders and passed to the neighboring secondary Bolwig’s organ cells where it activates the Egfr signaling cascade. Supporting this view, only 3-4 photoreceptor neurons are found in the Bolwig’s organ of embryos lacking rho or spi; furthermore, the size of the posterior lip of the optic lobe is reduced in such embryos. The fact that absence of secondary Bolwig’s organ cells in rho or spi mutant embryos can be rescued by blocking cell death in the background of a deficiency that takes out the reaper complex of genes indicates that the Spi signal is not necessary for the specification (recruitment) of secondary Bolwig’s organ cells, but rather, for their maintenance (Daniel, 1999).

Fgf and Egf signaling require the Mapk pathway for tracheal development

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. The development of the air sacs of the dorsal thorax of Drosophila was used to study cellular events and their regulation via cell-cell signaling. Two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable (Cabernard, 2005).

The air sac of the dorsal thorax grows from a bud that arises during the third larval instar from a wing disc-associated tracheal branch. To illustrate the development of the air sac, a GFP trap line was used that rather ubiquitously expressed membrane bound GFP; tracheal cells were counterstained with an mRFP1-moesin construct under the direct control of the trachea-specific breathless (btl) enhancer. From the early to late third instar stage, a bud-like structure grows out of the transverse connective and spreads on the wing imaginal disc epithelium; this outgrowth corresponds to the primordium of the air sac of the dorsal thorax (Cabernard, 2005).

It has been proposed that the air sac of the dorsal thorax forms de novo from a small group of wing imaginal disc cells, and that the resulting sac subsequently generates a tracheal lumen by an unknown process (Sato, 2002). Since, in the early Drosophila embryo, the lumen arises from an epithelial invagination via cell migration, it was asked whether the cells in the growing air sac are epithelial in nature with a clear apical/basal polarity. For this purpose, a Dα-Catenin-GFP (Dα-Cat-GFP) fusion construct was expressed in the developing air sac and the distribution of GFP from early to late third instar larvae was analyzed. Dα-Cat-GFP labels the adherens junctions (AJs) of epithelial cells. Clearly, the growth of the air sac was accompanied from the early stages onward by an out-bulging of an AJ network, suggesting that most or all of the cells in the growing bud were epithelial in nature, and that a luminal space was generated at the apical side of the epithelial tracheal cells during outgrowth. To confirm this interpretation, use was made of the recent identification of a protein, Piopio (Pio), which is apically secreted into the tracheal lumen in the embryo (Jazwinska, 2003). Indeed, the prospective luminal space in the outgrowing air sac is filled with Pio protein, demonstrating that the air sacs consist of a sac-like epithelial sheet, generating a luminal space as they grow (Cabernard, 2005).

To test whether all cells maintained an apical-basal polarity during air sac budding, single tracheal cells were labelled by using a recently developed assay system that allows for the visualization (and manipulation) of individual tracheal cells in vivo (Ribeiro, 2004). When this scenario was used in the presence of a UAS-Dα-Catenin-GFP chromosome, it was found that, in virtually all cases, such individually labeled air sac cells contacted the lumen and formed AJs with neighboring cells, even when these cells were located at the tip of the outgrowing air sac. The same conclusion was reached when the expression of GFP-moesin was analyzed in single air sac cells; cells at the tip made clear contact with the lumen. Therefore, it is concluded that the air sac is sculpted from an epithelial cell layer, which expands and at the same time generates an apical luminal space filled with secreted proteins (Cabernard, 2005).

It was of interest to better understand how Ras can be used in the same tissue at the same time for different cellular processes. egfr mutant cells can contribute to the tip of the growing air sac, although the clones are relatively small. In the stalk of the air sac, cells lacking Egfr often appeared fragmented, a sign of cell death. Indeed, when egfr mutant cells were sustained with anti-Drice, a marker for apoptotic cells, a strong accumulation of this protein was found. When p35, a viral antiapoptotic protein, was expressed in egfr mutant cell clones, these clones grew to larger sizes and were able to populate the air sac tip at a significantly higher frequency than in the absence of p35. These experiments establish that Egfr is dispensable for migration, and that migration is exclusively triggered by one of the two RTKs, Btl/Fgfr. The experiments also demonstrate that, during the growth phase, Btl/Fgfr signaling is dispensable for cell division; clones can grow to large sizes, although they fail to populate the tip. This same result was obtained with two other components, which are exclusively used by the Fgfr signaling pathway in the air sac (and not the Egfr pathway), namely, Dof and Pointed. Thus, migration and cell division are controlled by two different RTKs, but both RTKs signal via the activation of Ras and the Map kinase pathway to regulate these different cellular outcomes (Cabernard, 2005).

How does Ras control cell migration in the tip and cell division in the remaining air sac? To start to address these questions, whether high levels of constitutive active Ras were compatible with directional cell migration was tested and RasV12 was expressed in wild-type tissue in small cell clones. Interestingly, such clones expanded considerably and grew to large sizes in the center of the air sac or in the stalk, resulting in bulgy outgrowths; however, clones expressing RasV12 never contributed to the tip of the air sac. This finding suggests that unrestricted levels of Ras in a cell perturb its capacity to read out the migratory cues (presumably the Bnl/Fgf ligand); wild-type cells were apparently much better in taking up the leading position. In line with this interpretation, expression of an activated version of Btl (Torso-Btl/Fgfr) also resulted in bulky outgrowths. In addition, cells expressing the chimeric Btl receptor never populated the tip. Quite in contrast, activated Egfr (Egfr fused to a lambda dimerization site) was not able to perturb air sac guidance, but it also triggered higher division rates in clones, generating bulgy outgrowths (Cabernard, 2005).

To test whether single cells expressing activated receptor constructs changed their behavior with regard to cytoskeletal dynamics, the expression of either the activated version of Fgfr or Egfr was induced in early third instar stages and the behavior of such cells was analyzed with live imaging of cultured discs. Cells in the stalk of the air sac expressing activated Fgfr showed extremely dynamic cytoskeletal activity and formed large lamelipodia extending away from the air sac, similar to cells at the tip. Quite in contrast, cells expressing activated Egfr did not show increased lamelipodia formation, and their basal side remained relatively inactive (Cabernard, 2005).

Since the expression of constitutive active versions of the two different RTKs during air sac growth had different effects, whether the endogenous receptors activated the Ras/Mapk pathway to different levels in wild-type air sacs was investigated. In order to monitor the strength of Mapk signaling, an antibody recognizing the double-phosphorylated form of Erk, dpErk were used. Indeed, high levels of dpErk was detected in the nucleus of tip cells; lower dpErk levels were found in the cells in the center of the air sac, and dpErk was mostly cytoplasmic (Cabernard, 2005).

From all of the above-mentioned data, it is concluded that air sac development makes use of two distinct RTKs to control directed organ extension via cell migration (Fgfr) and organ growth via cell division (Egfr). This study carefully analyzed air sac outgrowth from early to late stages, by using a number of different markers labeling either membranes or AJs of individual air sac cells, or the apical luminal compartment. It was found that the thoracic air sac is modeled out of the existing tracheal epithelium, and that a luminal space is generated by the migration of a few cells away from the cuticle of the existing tracheal branch; the luminal space is then expanded by increasing the cell number in the sac-like epithelial structure via cell division. During this process, all cells remain within the epithelium and only round up when they divide. Even those cells that send out filopodia and lamelipodia and migrate in the direction of Bnl/Fgf remain embedded within the epithelium, contact the lumen, and form AJs with their neighbors. Thus, the directed outgrowth of the thoracic air sac during larval development is very similar to the budding of tracheal branches in the early embryo, in that epithelial cells form extensions from the basal side, ultimately resulting in cell movement toward the Fgf ligand. In contrast, during tubule formation of MDCK cells in culture, cells initially depolarize and migrate to form chain-like structures before they repolarize and form the luminal cavity; tubulogenesis is thus accompanied with partial epithelial-to-mesenchymal as well as mesenchymal-to-epithelial transitions. The tube-forming process has been subdivided into different stages such as cyst, extension, chain, cord, and tubule. In the case of the MDCK model system, growth factors have been proposed to trigger branching by inducing a dedifferentiation that allows the monolayer to be remodeled via cell extension and chain formation. Similar to the MDCK system, it was found in Drosophila that growth factor signaling induces the formation of cellular extensions, the first sign of outgrowth. Also, in both systems, cell division is an integral part of the process, but it occurs randomly throughout the structure and not locally at the point of outgrowth. However, two different RTKs are used in the air sac to control extension (migration) and cell division, and chain and chord stages are not observed. It thus appears that both similarities and differences exist between these different cellular systems (Cabernard, 2005).

It has already been reported that cells divide during air sac formation. The cell division rates have been semiquantified and it was found that the elongating structure does not grow preferentially at the tip. The genetic analysis demonstrates that the Egfr is essential for cells to divide and survive efficiently in the air sac. Egfr signals via Ras and the Mapk pathway, but it does not require the Pnt transcription factor to regulate cell division. It is not yet known which ligand activates Egfr, and whether expression of this ligand is induced at early stages of development by Fgf signaling. As shown before (Sato, 2002), the complete lack of Fgfr signaling results in the absence of air sacs; Fgf signaling might thus be used at the onset of the budding process to initiate or trigger cell division, but it is clearly dispensable in later stages. Since cells in the tracheal branch, which gives rise to the air sac primordium, also divide in the absence of Fgf signaling, it is possible that the role of Fgf signaling consists in generating an outgrowth via directed cell movement, triggering cell division indirectly (Cabernard, 2005).

Interestingly, a recent study addressing the role of GDNF/Ret signaling in kidney branching morphogenesis in vivo has shown that ret mutant cells (which are unable to respond to GDNF) can contribute to the primary outgrowth of the ureteric bud, but are excluded from the ampulla that forms at its tip. Apparently, a Ret-dependent proliferation of tip cells under the influence of GDNF controls branch outgrowth. This study found that in Drosophila, in the developing air sac, cells lacking Fgfr are also excluded from the tip. However, evidence is provided that Fgf signaling is translated into directed migration in the leading structure and not into a local increase in cell proliferation. The isolation and cultivation of wing imaginal discs allows for using 4D imaging to document cell behavior during air sac growth. It was found that numerous tip cells extend long filopodia and lamelipodia, similar to the findings reported earlier (Sato, 2002). Tip cells not only produce extensions, but they indeed change their respective position with time, and move forward over the substrate in the direction of the filopodia/lamelipodia. Thus, tip cells are clearly motile and migrate in the direction of Bnl/Fgf. Cell clones incapable of responding to different families of ligands were produced and marked and were examined with regard to their capacity to populate the air sac tip. Among the receptors analyzed, only Btl/Fgfr was strictly required for cells to populate the leading tip of the air sac. Considering the observation that cells in the tip actively migrate, that Btl/Fgfr signaling is required for tracheal cell migration in the embryo, that tracheal cells migrate to ectopic sources of Bnl/Fgf in the embryo and the larva (Sato, 2002), and that cells form numerous filopodia and lamelipodia upon constitutive activation of the Fgf signaling pathway, it is concluded that Fgf steers cell migration in the tip of the air sac and leads to its directional outgrowth on the surface of the wing imaginal disc. The demonstration that the MARCM system can be used to analyze gene function with regard to cell migration in the developing air sac prompted an investigation of the role of Ras and the Mapk pathway in Fgf-directed cell movement (Cabernard, 2005).

Using the MARCM system, it was found that Ras activation is essential for cells to migrate at the tip of the air sac. The requirement for Cnk and Ksr strongly suggests that one important branch downstream of Ras in the control of cell migration is the Mapk pathway. This interpretation is supported by the somewhat surprising finding that the transcription factor Pnt is also strictly required for cell migration. In the Drosophila embryo, genes regulated by Fgf signaling at the transcriptional level and essential for migration have not been identified so far; although both pnt itself and blistered/DSrf are targets of Fgf signaling with important functions in tracheal morphogenesis, they are not required for migration. One possible target of Fgf signaling in the dorsal air sac cells might be the btl/fgfr gene itself. Attempts were made to rescue the pointed defects in air sac development by supplementing a btl transgene under the control of UAS sequences. It was found that even when Btl/Fgfr is provided by the transgenes, pnt mutant clones do not reach the tip. A second gene that might have been a transcriptional target of Pointed is dof; however, it was found that Dof protein is still present in pnt mutant clones (Cabernard, 2005).

The results demonstrate that the outgrowth of the dorsal air sac along the underlying wing imaginal disc is controlled by Btl/Fgfr and Egfr. Fgf signaling is required for directional outgrowth via cell migration, and Egf signaling is required for organ size increase sustaining cell division/cell survival. Both signals use the Ras/Mapk pathway to elicit their cellular responses. To what extent these two pathways regulate different downstream targets is not known at present. However, this study shows that Pointed is only required downstream of Fgf signaling in the control of directed cell migration, and not downstream of Egf signaling in the control of cell division/survival. Since the activation of the Map kinase pathway is much stronger in the cells at the tip as compared to the cells in the central portion or in the stalk of the air sac (according to the levels of dpErk), it is thought that the local availability of the Bnl/Fgf ligand results in a local signaling peak. Egf signaling in more central and proximal cells does not result in a strong activation of the Map kinase pathway, yet this activation is apparently sufficient to control cell division and survival. The independent regulation of cell migration and cell division by two different RTKs might be even more important in later stages of dorsal air sac development, when the growing tip is yet farther away from the main body of the air sac. It will be interesting to find whether other growing branched tissues use similar mechanisms to uncouple directional expansion and size increase (Cabernard, 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).

The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity

Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. This study shows that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network (Caviglia, 2013).

This work has investigated how the choice between the two types of specialized tip cells in the tracheal system is controlled. The transcriptional repressor Aop plays a key role in linking tracheal tip cell fate selection with branch identity. First, a novel tube morphogenesis phenotype is described in aop mutants, which is due to the massive mis-specification of regular epithelial cells into specialized tracheal tip cells. aop is specifically required for controlling tracheal cell fate, whereas aop, like pnt, is dispensable for primary tracheal branching, thus uncoupling roles of RTK signaling in cell fate specification and cell motility. The finding that tracheal branching morphogenesis proceeds normally in the presence of excess tip cell-like cells suggests that collective cell migration is surprisingly robust and that mis-specified cells apparently do not impede the guided migration of the tracheal primordium. Second, it was demonstrated that in the absence of inhibitors of MAPK signaling (aop and sty), all tracheal cells are competent to assume either TC or FC fate. The transcriptional repressor Aop globally blocks both TC and FC differentiation, but high-levels of MAPK signaling in tip cells relieve Aop-mediated inhibition, thus permitting differentiation. Third, the results suggest that in the DT region Aop limits FC induction through a distinct mechanism by antagonizing Wg signaling in addition to MAPK signaling. Conversely, in the other branches, Aop limits TC differentiation by blocking MAPK-dependent activation of Pnt. Fourth, it was shown that the region-specific choice between the two cell fates in the DT is determined by Wg signaling and by the selector gene salm. Based on these results, a model is proposed in which a single repressor, Aop, integrates MAPK and Wg signals to couple tip cell fate selection with branch identity. High levels of Bnl signaling trigger Pnt activation and Aop degradation in tracheal tip cells. It is proposed that in the DT, unlike in other tracheal cells, MAPK-induced degradation of Aop releases inhibition of Wg signaling. This is consistent with recent work showing an inhibitory effect of Aop on Wg signaling, possibly through direct interaction of Aop and Arm, or through Aop-mediated transcriptional repression of Wg pathway component. The current work extends the evidence for this unexpected intersection between two major conserved signaling pathways, suggesting that this function of Aop is likely to be more widespread than previously appreciated. The findings also provide an explanation for the puzzling observation that, in pnt mutants, TCs are lost, while FCs become ectopically specified. As pnt is required for expression of the feedback inhibitor sty, loss of pnt is expected to lead to MAPK pathway activation and consequently to increased Aop degradation. This would release Aop-mediated repression of Wg signaling, resulting in extra FCs, whereas TCs are absent because of the lack of pnt-dependent induction. This suggests that excessive FC specification in the DT of aop and sty mutants is mainly due to deregulated Wg signaling, rather than to de-repression of pnt-dependent MAPK target genes. Consistent with this notion, it was shown that pnt is not required for Delta and Dys expression in tracheal cells, although constitutively active AopACT represses their expression (Caviglia, 2013).

The results further show that salm function constrains the fate that is chosen by cells when released from the Aop inhibitory block. MAPK signaling triggers Aop degradation in all tip cells, but only in the absence of salm does this signal lead to TC induction. In salm-expressing cells, degradation of Aop releases Wg signaling, resulting in FC specification. Thus, salm biases the choice between two morphologically different types of seamless tubes. This is reminiscent of the role of salm in switching between different cell types in the peripheral nervous system and in muscles. salm expression is sufficient to repress TC formation. The genetic results, consistent with biochemical data showing that Salm acts as a transcriptional repressor, suggest that salm promotes FC fate by repressing genes involved in TC development. However, salm is not sufficient to overcome the requirement for Wg signaling in FC induction, indicating that Wg does not act solely via salm to induce FC fate. Indeed, FC induction requires genes whose expression is independent of salm (esg, dys). In addition, it is proposed that a feedback loop between Wg signaling and salm expression maintains levels of Wg signaling in the DT sufficiently high to induce FC fate. Taken together, these results suggest that the default specialized tip cell fate, and possibly an ancestral tracheal cell state, is TC fate. Although FCs and TCs differ in their morphology, they share a unique topology as seamless unicellular tubes. FCs and TCs might therefore represent variations of a prototypical seamless tube cell type. Salm might modify cellular morphology by repressing TC genes, including DSRF, which mediates cell elongation and shape change. Intriguingly, Wg-dependent salm expression in the DT of dipterans correlates with a shift towards FC as the specialized fate adopted by the tip cells of this branch. This study has shown that salm expression inhibits TC fate, while promoting the formation of a multicellular main tracheal tube by inhibiting cell intercalation. It is therefore tempting to speculate that the salm-dependent switch from a branching towards an anastomosing tip cell type in the DT may have evolved with the acquisition in higher insects of a main tube that connects separate tracheal primordia to generate a tubular network. It will be of great interest to identify the relevant target genes that mediate the effect of Salm on tube morphology and tip cell fate (Caviglia, 2013).

The mechanisms of tip cell selection during angiogenesis in vertebrates are beginning to be understood at the molecular level. However, the signals that control the formation of vascular anastomoses by a particular set of tip cells are not known. Intriguingly, the development of secondary lumina in aop mutants is reminiscent of transluminal pillar formation during intussusceptive angiogenesis, which is thought to subdivide an existing vessel without sprouting. Although the cellular basis for this process is not understood, it is conceivable that specialized endothelial cells are involved in transluminal pillar formation. This work provides a paradigm for deciphering how two major signaling pathways crosstalk and are integrated to control cell fate in a developing tubular organ. It will be interesting to see whether similar principles govern tip cell fate choice during tube morphogenesis in vertebrates and invertebrates (Caviglia, 2013).


A new conditional Egfr allele was used to elucidate the roles of the receptor in eye development.

Egfrtsla is a tight temperature-sensitive allele which gives rise to structural defects in the adult eye with treatments at the non-permissive temperature as short as 1 hour. Subjecting Egfrtsla flies to the non-permissive temperature for 24 hours (beginning at a time when the furrow is about half way across the eye field) results in structural defects, many of which are observed in external views of the adult eye. The most obvious defect is a physical scar that runs across the eye in a dorsal-to-ventral direction. Since new ommatidia are found anterior to the scar, it is concluded that development can recover following restoration of Egfr function. Young Egfrtsla larvae were temperature shifted when the furrow was not yet initiated and a novel and unexpected Egfr function was found: it is required for the initiation of neural differentiation on the posterior margin. Normally the furrow initiates at the posterior margin and is negatively regulated by Wingless expression on the disc margins, in particular on the dorsal side. In this early EGFR-TS condition, an inhibition of neural differentiation is seen at the posterior margin, but not at the dorsal margin (Kumar, 1998).

To directly visualize RAS/MAPK pathway signaling downstream of Egfr a monoclonal antibody specific to the active, di-phosphorylated form of the MAP kinase (dp-ERK) was used. In the developing eye dp-ERK is found in large clusters of cells in the furrow. The dp-ERK pattern was examined in the eye disc at a high resolution. In addition to the identification of dp-ERK in large clusters of cells in the furrow, followed by smaller clusters in later columns of photoreceptors (note: each column is a dorsal-ventral stack of photoreceptor clusters all born at the same time), dp-ERK is also found in additional positions. There is a low level of cytoplasmic antigen anterior to the furrow, then in the furrow the large clusters develop from the midline. Within one column in the furrow, the dp-ERK staining clusters are initially small, then larger, and then smaller again. As clusters within a column are formed at 15-20 minute intervals, this phase of dp-ERK accumulation corresponds to more than 2 hours in time. The clusters ultimately focus to one or a few cells, in which dp-ERK can then be seen for about two columns (corresponding to 4 hours of development). This development from the eye midline of small clusters, through large clusters and then back to one or a few cells is consistent with the expression of Scabrous and with the proneural focusing of Atonal in the founding R8 cells. Thus the developing dp-ERK pattern appears to correlate with the specification of the ommatidial precluster. The large dp-ERK clusters were positioned relative to the early steps of ommatidial formation by double staining for dp-ERK and cytoplasmic actin. The large clusters dp-ERK correspond to a very early stage, one column anterior to the first clear ommatidial clusters, which appears to be the `rosette' stage (Kumar, 1998).

It is important to note that, contrary to expectations, dp-ERK in the developing eye is primarily cytoplasmic. Wild-type discs were stained to reveal dp-ERK and DNA and then these were colocalized in confocal optical sections. The dp-ERK antigen is clearly mostly cytoplasmic in the large furrow clusters since dp-ERK cannot be detected in cell nuclei in the furrow. At a later stage (more posterior in the same field), dp-ERK can be seen in occasional, apical nuclei. The time series of cluster formation in the furrow shows that detectable nuclear dp-ERK can follow cytoplasmic phosphorylation by 2 hours or more. Later, transient dp-ERK antigen can be detected in cell nuclei, such as the developing R3 and R4 cells, for much shorter times. About nine columns (18 hours) after dp-ERK staining first appears in the furrow, it can be seen in the cytoplasm of the future R7 cell. This dp-ERK persists for about four columns (8 hours), and is genetically dependent on the activity of the sev gene since it is absent in discs derived from sev null mutant larvae. It is interesting to note that dp-ERK is detectable in the cytoplasm of the future R7 for an extended period (about 8 hours). Ectopic activation of the RAS/MAPK pathway increases the level of cytoplasmic but not nuclear dp-ERK (Kumar, 1998).

Historically, the first data to suggest that Egfr functions in Drosophila retinal development came from an analysis of Ellipse mutations. Egfr Ellipse alleles appear to be dominant gain-of-function mutations as they are suppressed in trans to null alleles of Egfr. In Ellipse homozygotes, there are fewer ommatidia than normal and they are separated by increased space]. This led to an attractive model: that the preclusters and/or founder (R8) cells are normally spaced evenly by a short-range diffusible inhibitor (a mechanism known as `lateral inhibition') and that the receptor for this inhibitor is Egfr. Thus in Ellipse, hyperactive Egfr leads to increased space between the clusters. This model led to a testable prediction that loss of Egfr function should reduce the space between ommatidia. This paper shows that in the temperature sensitive Egfr mutants, a complete loss-of-function condition (a normally spaced array of single R8 cells) is formed. This strongly suggests that Egfr has no role in cluster spacing. If there is any role for EGFR in establishing cluster and/or R8 cell spacing, it must be a minimal contribution and/or be highly redundant (Kumar, 1998 and references).

A second model has been proposed for the function of Egfr in the Drosophila retina: that reiterative use of the Egf receptor triggers differentiation of all cell types in the Drosophila eye. This was suggested by the loss-of-function phenotype and overexpression of spitz and from the phenotype of dominant-negative Egfr mutant protein. The fact that a regularly spaced array of R8 cells is formed in the Egfr-TS condition suggests that, at least as far as the R8 cell type is concerned, Egfr cannot be said to trigger the differentiation of all cell types in the eye. It is formally possible that Egfr does normally play a role in R8 cell specification, but that this role is redundant or dispensable (ie other RTKs can specify R8 cells in the absence of Egfr function). It is likely that all subsequent recruitment steps require Egfr however (Freeman, 1996). Egfr may act in combination with other receptors such as Sev to raise the level of RAS/MAPK pathway activity over some critical threshold. The observation that dp-ERK staining initially disappears from the furrow, but later rebounds (without Egfr function) strongly suggests that other receptor tyrosine kinases are present in the furrow and can act there. Thus, Egfr function is necessary for morphogenetic furrow initiation, is not required for establishment of the founder R8 cell in each ommatidium, but is necessary to maintain the differentiated state of founder cells. Egfr is required subsequently for recruitment of all other neuronal cells. The initial Egfr-dependent MAP kinase activation occurs in the furrow, but the active kinase (dp-ERK) is observed only in the cytoplasm for over 2 hours. Similarly, Sevenless-dependent activation results in the cytoplasmic appearance of dp-ERK for 6 hours. These results suggest an additional regulated step in this pathway (Kumar, 1998 and references).

Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity

In the developing eye of Drosophila, Ras performs three temporally separate functions. In dividing cells, it is required for growth but is not essential for cell cycle progression. In postmitotic cells, it promotes survival and subsequent differentiation of ommatidial cells. The different roles of Ras during eye development have been analyzed by using molecularly defined complete and partial loss-of-function mutations of Ras. The three different functions of Ras are mediated by distinct thresholds of MAPK activity. Low MAPK activity prolongs cell survival and permits differentiation of R8 photoreceptor cells while high or persistent MAPK activity is sufficient to precociously induce R1-R7 photoreceptor differentiation in dividing cells (Halfar, 2001).

How does Ras control growth? One possibility is that Ras directly binds and activates PI3K. Clones mutant for components in the insulin receptor/PI3K pathway also have a growth disadvantage compared to wild-type cells. Although in vertebrates, H-RasG12V,Y40C activates PI3K, no evidence was found that the corresponding mutant activates PI3K in Drosophila. Partial loss-of-function mutations in genes coding for Raf and MAPK, respectively, show similar growth defects as Ras mutants. Furthermore, Ras D38E shows a significant rescue of the growth disadvantage of Ras minus clones. Thus, it is proposed that while cell growth depends on the activities of the MAP kinase as well as the PI3K pathway, the activation of the MAP kinase pathway represents the only Ras function. It has recently been proven that cooperation between the Ras/MAP kinase and the PI3K/PKB pathway is required in order to induce growth in cultured cells. In fibroblasts, activation of Raf and PI3K is required for cyclin D1 expression and entry into S-phase. Induction of DNA synthesis by activation of the platelet-derived growth factor (PDGF) receptor requires an early activation of MAPK and a late phase PI3K activity (Halfar, 2001 and references therein).

Ras mutant cells located behind the morphogenetic furrow die by programmed cell death (PCD). Ras controls the PCD inducer Hid, by repressing its expression and by modifying its activity through phosphorylation by MAPK. In mammalian cells, PI3K promotes survival via PKB-mediated phosphorylation of the pro-apoptotic protein Bad. Thus, survival could at least in part be mediated by the activation of PI3K. Indeed, a partial suppression of Hid-induced apoptosis in the eye by the expression of RasG12V,Y40C (providing high levels of Ras activation) has been taken as evidence that PI3K supports survival in the developing eye. This is unlikely in the light of the data presented here, since it has been shown that RasG12V,Y40C is unable to activate PI3K. Furthermore, the RasD38E transgene, providing low levels of Ras activation, rescues Ras minus cells posterior to the morphogenetic furrow from PCD. Thus it appears that the function of Ras in survival is mediated exclusively through the activation of MAPK. In the adult, however, Ras mutant cells were never observed. This may be due to an exclusive role of Ras in promoting cell survival of ommatidial cells at later stages or due to an additional role in cell fate specification. Several lines of evidence argue against an exclusively anti-apoptotic role of Ras during the later stages of eye development: (1) reduced Ras activity in the R7 precursor cell in the absence of the Sev receptor tyrosine kinase results in a change in cell fate rather than death of this progenitor cell; (2) constitutive activation of Ras in cone cell precursors is sufficient to induce R7 differentiation in these cells; (3) ectopic expression of an activated EGF receptor or RasG12V results in precocious induction of photoreceptor cell differentiation anterior to the furrow. Thus in the case of R1-R7 differentiation, high levels of Ras activity are required for a choice in cell fate rather than mere survival of the cells. The differentiation of R8 cells, which depends on Ras activity, however, may be different in that Ras may be required in the R8 photoreceptor for survival only. R8 cell differentiation is rescued by RasD38E, concomitant with the survival of the mutant clones. Therefore it is possible that Ras-mediated survival is sufficient for R8 cell differentiation. Interestingly, loss of EGF receptor function still allows the formation of R8 cells, suggesting that the low levels of Ras activity required for R8 differentiation are achieved by another receptor system (Halfar, 2001).

There are three different models for how specificity of Ras signaling is achieved: specificity may be controlled by (1) the cellular context, (2) the activation of distinct signaling pathways by Ras or (3) by different levels of Ras activity. The experiments presented here support the importance of the cellular context and the different levels of Ras activity but fail to provide evidence for the activation of different signaling pathways by Ras. All aspects of Ras signaling could be rescued by the activation of the Ras/MAPK pathway and no evidence was found (using the Ras effector site mutants) that constitutively active Ras activates the PI3K pathway directly (Halfar, 2001).

The cellular context in which Ras activates MAP kinase is clearly important. Expression of RasG12V in blastoderm cells triggers differentiation of head and tail structures, it triggers vein differentiation in wing disc cells and neuronal differentiation in eye disc cells. Different levels of Ras/MAPK activity appear to control distinct cellular responses within the same tissue. Low levels of Ras activity, provided by the RasD38E mutant, rescue R8 differentiation and survival but not R1-R7 differentiation. High levels of Ras/MAPK activity provided by wild-type Ras or by a combination of RasD38E and rolledSem are required for the differentiation of R1-R7 photoreceptor cells (Halfar, 2001).

There are two possibilities with regard to the nature of the activity thresholds that elicit the different cellular responses. The threshold may be quantitative. Cells could react to different activity levels within the cells. Alternatively, the threshold may be temporal and cells react to the difference in the duration of the signal. Staining of imaginal discs with an antibody that selectively recognizes activated MAPK (dpERK) was not sensitive enough to detect activated MAPK during normal photoreceptor cell recruitment or during ectopic neuronal differentiation in RasG12V-expressing clones anterior to the morphogenetic furrow (Halfar, 2001).

Therefore, it was not possible to distinguish between these two models. In the present case, however, the temporal model is favored because the highest levels of dpERK staining could not be detected behind the morphogenetic furrow during photoreceptor cell recruitment. In response to MAP kinase activation in the developing eye, a number of negative regulators of the pathway are induced. The EGF-related peptide Argos competes with the TGFalpha-like ligand Spitz for EGF receptor binding, and Sprouty, a cytoplasmic protein, associates with the EGF receptor to turn off the signaling pathway. Indeed, neuronal differentiation and ommatidial development in the RasD38E mutant is rescued by the prolonged activity of MAPK caused by the rlSem mutation. It is possible that the reduced activity of RasD38E towards Raf is caused by a more rapid inactivation, owing to increased GTPase activity. The observation that RasG12V,D38E is sufficient to induce neuronal differentiation ahead of the furrow, in conjunction with the G12V substitution, which inactivates the Ras GTPase activity, is consistent with the idea that D38E may stimulate GTP hydrolysis. Thus, neuronal differentiation in Drosophila may depend on the prolonged activation of Ras/MAP kinase, whereas transient activation is sufficient for survival upon exit from the cell cycle and differentiation of R8 photoreceptor cells. Therefore it appears that neuronal differentiation in response to Ras activation in the developing eye of Drosophila is similar to neuronal differentiation in PC12 cells: this also requires prolonged activation of MAPK. The modulation of levels and/or the duration of Ras/MAPK activity levels appear to be important determinants of cellular responses in multicellular organisms (Halfar, 2001).

Nuclear translocation of activated MAP kinase is developmentally regulated in the developing Drosophila eye

In proneural groups of cells in the morphogenetic furrow of the developing Drosophila eye, phosphorylated MAPK antigen is held in the cytoplasm for hours. A reagent has been developed to detect nuclear MAPK non-antigenically. Reported here is the use of this reagent, confirming that MAPK nuclear translocation is regulated by a second mechanism in addition to phosphorylation. This 'cytoplasmic hold' of activated MAPK has not been observed in cell culture systems. MAPK cytoplasmic hold has an essential function in vivo: if it is overcome, developmental patterning in the furrow is disrupted (Kumar, 2003).

Four versions were made of the Drosophila MAPK Rolled for expression in the developing eye: (1) the full-length natural amino acid sequence of Rolled called 'M' (for 'Mapk'); (2) 'NM' ('Nuclear-Mapk'), which adds the strong nuclear localization signal (NLS) from SV40 virus large T antigen fused to the N-terminus of 'M'; (3) 'MG' ('Mapk-Gal4vp16), which contains the entire sequence of Rolled, followed by the yeast GAL4 DNA binding domain (which is not known to contain a nuclear localization signal) with an acidic activation domain from herpes simplex virus protein 16, and (4) 'NMG' ('Nuclear-Mapk-Gal4vp16'), which contains a SV40 NLS on the N terminus of MG. Each version was also engineered to carry a C-terminal epitope tag. These proteins were inserted into two Drosophila transformation vectors to drive their expression under heat induction (hsp70 gene promoter or 'HS') or the eye-specific Glass transcription factor ('Glass-Mediated-Response' or 'GMR') to produce six constructs: HS:M, HS:NM, HS:MG, HS:NMG, GMR:MG and GMR:NMG (Kumar, 2003).

The transcriptional activating activity of the four Gal4-Vp16 constructs was assessed using a UAS:lacZ reporter in third instar eye imaginal discs. In all cases, multiple independently derived transgenic lines for each construct were tested and in each case they gave indistinguishable results. The hsp70 promoter was induced by heat induction. and in the case of HS:MG a subset of cells was observed to express ß-galactosidase antigen (some in the antennal disc, in the peripodial membrane and in the developing retina). In the retina, HS:MG driven reporter gene activity is sporadic on the anterior side, and then is seen in rising numbers of cells posterior to the furrow. By contrast, HS:NMG produces reporter gene expression in many more cells, in all parts of the disc. This difference between HS:MG and HS:NMG strongly suggests that a developmentally regulated nuclear localization signal is present in the MAPK part of the MG fusion protein, and that this is over-ridden by strong dominant NLS activity in the NMG protein (associated with the SV40 NLS placed there). The GMR promoter is active only posterior to the morphogenetic furrow (and in the larval photoreceptor of Bolwig's organ) where the Glass transcription factor is expressed. Reporter gene activity was observed in the GMR:NMG lines immediately posterior to the furrow, but only after a delay in the GMR:MG lines. These data from the GMR constructs are consistent with the same regulated NLS activity as seen in the HS lines: in the absence of the SV40 NLS, the MG protein can only activate the reporter some distance posterior to the furrow in the developing retina (Kumar, 2003).

Consistent with the suggestion that the MAPK pathway signal is blocked in intermediate proneural groups (phase 1), reporter gene expression is seen only later, in the future R8 cells in the last two columns of Atonal expression and later in cells as they are recruited into the assembling ommatidia (phase 2). This is consistent with normal Egfr pathway activity in the downregulation of Atonal at the end of phase 1 and then again at later stages (phase 2), when successive Ras pathway signals recruit each cell type that follows the founding R8 cell (which is specified by other means). Although no MAPK nuclear translocation was detected early in the furrow (in the intermediate groups) with either of the reagents (the transcription factor fusion or the epitope tag) the possibility that there is some lower level of nuclear MAPK at these stages that is below the limits of the two detection systems cannot be formally excluded. Similarly, the possibility that there are cytoplasmic functions for phosphorylated MAPK at these stages cannot be excluded. However, the results are consistent with two Egfr pathway functions in the developing R8 cells at this time (as Atonal expression ends): for the maintained expression of differentiation markers (Boss and Elav) and for later cell survival (Kumar, 2003).

Furthermore, through the addition of a constitutive NLS, MAPK is driven into the nuclei of cells in phase 1, thus overcoming MAPK cytoplasmic hold. This results in a rapid downregulation of Atonal and the precocious neural differentiation of the R8 photoreceptors. Taken together with the observation of the first nuclear translocation of MAPK at the time that Atonal is downregulated in normal development, it is suggested that the Egfr/Ras pathway may normally contribute to the end of phase 1 by ending Atonal expression (Kumar, 2003).

Others have suggested that Egfr pathway loss-of-function normally functions to downregulate Atonal expression at and after the intermediate group stage. This was not observed using the conditional mutation, Egfrtsla; however, since this experiment did not include a clone boundary a short delay (such as one column) could not have been detected. It may be that the pathway functions at this point through a much lower level signal (below the level of detection of the reagents) or it may be that it functions through cytoplasmic targets of phosphorylated MAPK (Kumar, 2003 and references therein).

What is the developmental purpose of this block of MAPK signaling in the furrow? Anterior to the furrow, MAPK cytoplasmic hold cannot function, or it would prevent the MAPK signaling required for the G1/S transition and thus halt cell proliferation. Perhaps this is one reason why all cells in phase 1 exit the cell cycle. However, new data suggest that the Egfr pathway does function in the furrow to maintain G1 arrest (visualized as increased cyclin B expression). This could be mediated through some low level of nuclear MAPK at this stage or possibly through cytoplasmic targets for MAPK signaling. However, although cyclin B expression is elevated posterior to the furrow in all cells other than R8 in Egfr pathway loss-of-function mutant clones, the leading edge of cyclin B expression does not advance (it is not expressed earlier). Thus, it may be that the role of Egfr pathway signals in maintaining G1 arrest is later than the end of Atonal expression (i.e., in phase 2, not in phase 1) (Kumar, 2003).

It is suggested that the founder cells have a special developmental function to fulfill in phase 1: they must act as organizing centers for lateral inhibition to produce the spaced pattern of R8 cells. If the founder cells did not inhibit their neighbors most or all cells in phase 1 might rapidly differentiate as photoreceptors, resulting in disorder. This type of disorder is observed when the Egfr/MAPK pathway is ectopically activated ahead of the furrow, when photoreceptor differentiation becomes independent of Atonal and R8 fate. The model may also explain the loss of ommatidia seen in EgfrElp gain-of-function mutants. Excess Ras/MAPK pathway signals may reduce Atonal expression and thus the number of R8 founder cells. The results led to a prediction that G1 cell-cycle arrest may be found in other cases in which a subset of progenitor cells is selected by lateral inhibition through active Notch pathway signaling and repression of Ras/MAPK signaling. In summary, the data are consistent with a model in which Egfr/MAPK signaling functions in ommatidial assembly but not directly in founder cell specification. It is proposed that MAPK cytoplasmic hold is restricted to the morphogenetic furrow, and does not happen anterior to the furrow (the proliferative phase) or posterior (during ommatidial assembly, or phase 2). It appears to be coincident with the regulated G1 arrest seen in the furrow (Kumar, 2003).

It is interesting to note that the observed pattern of MAPKGal4/VP16 is very different from the observation of the pattern of MAPK phosphorylation (dpErk antigen); indeed, they are almost exclusive. The predominant expression of dpErk in the developing eye is in the intermediate groups in the furrow, and yet little detectable signal function has been shown there. Furthermore, MAPK signaling is absolutely required for ommatidial assembly posterior to the furrow, yet no one can detect much dpErk at that stage. Perhaps MAPK cytoplasmic hold can explain this paradox as well: where MAPK is anchored in the cytoplasm (in the furrow) it can be phosphorylated by MEK but is protected from abundant phosphatase activity that waits in the nucleus. Thus, the pathway is blocked, there is no negative feedback and the antigen builds up to high (and easily detected) levels for several hours. Later (during ommatidial assembly) it is possible that there is no cytoplasmic hold, so MAPK passes rapidly to the nucleus after its phosphorylation by MEK, where the signal is passed, negative feedback is triggered and the antigen is cleared by phosphatase. Thus, in vivo the dpErk stain may actually be a stain not for pathway activity per se, but predominantly for MAPK cytoplasmic hold (Kumar, 2003).

These findings indicate that MAPK nuclear translocation is regulated in vivo by some mechanism in addition to, and regulated separately from, its phosphorylation state. It is not proposed that MAPK phosphorylation is not required for MAPK nuclear translocation, only that phosphorylation is not always sufficient. What might the mechanism for MAPK cytoplasmic hold be? The simplest hypothesis is that some anchoring factor sequesters activated MAPK in the cytoplasm until a second developmental signal permits its release. This could provide for a point of signal transduction pathway integration. However, an alternative model is suggested: activated MAPK cannot translocate to the nucleus in the intermediate groups not because it is held fast by some negative anchoring factor, but because it lacks some specific positive factor, such as an import factor (Kumar, 2003).

In summary, there is the dual regulation of MAPK signal transduction, both through its phosphorylation by MEK and independently through the control of nuclear translocation. Such dual regulation may be important in many developmental events through which a subset of founder cells must first be specified. As such events involve lateral inhibition and cell contact, this mechanism may not be observable in tissue culture systems (Kumar, 2003).

Distinct activation patterns of EGF receptor signaling in the homoplastic evolution of eggshell morphology in genus Drosophila

Homoplasy is a phenomenon in which organisms in different phylogenetic groups independently acquire similar traits. However, it is largely unknown how developmental mechanisms are altered to give rise to homoplasy. In the genus Drosophila, all species of the subgenus Sophophora, including D. melanogaster, have eggshells with two dorsal appendages (DAs); most species in the subgenus Drosophila, including D. virilis, and in the subgenus Dorsilopha (represented by a single species; Drosophila busckii), have four-DAs. D. melanica belongs to the Drosophila subgenus, but has two-DAs, and phylogenetic analyses suggest that it acquired this characteristic independently. The patterning of the DAs is tightly regulated by epidermal growth factor receptor (EGFR) signaling in D. melanogaster. Previous studies suggested that a change in the EGFR signal activation pattern could have led to the divergence in DA number between D. melanogaster and D. virilis. This study compared the patterns of EGFR signal activation across the Drosophila subgenera by immunostaining for anti-activated MAP kinase (MAPK). This analysis revealed distinct patterns of EGFR signal activation in each subgenus that were consistent with their phylogenetic relationship. In addition, the number of DAs always corresponded to the number of EGFR signaling activation domains in two, three, and four-DA species. Despite their common two-DA characteristic, the EGFR signaling activation pattern in D. melanica diverged significantly from that of species in the subgenus Sophophora. These results suggest that acquisition of the homoplastic two-DA characteristic could be explained by modifications of the EGFR signaling system in the genus Drosophila that occurred independently and at least twice during evolution (Kagesawa, 2008).

This study examined the patterns of MAPK activation in the follicle cells during oogenesis in five, eight, and one species belonging to subgenera Sophophora, Drosophila, and Dorsilopha, respectively. The comparative analyses revealed that MAPK was activated in a very similar pattern at the initial stages in all species examined, regardless of the DA numbers that eventually formed on their eggshells. MAPK activation was first detected at the dorsal midline of the follicle cells in all of the examined species of the Drosophila genus at stage 9/10. In D. melanogaster, Grk is specifically localized to the dorsoanterior midline of the oocyte and activates EGFR signaling in the overlying follicle cells at this stage. Therefore, the initial activating mechanism for EGFR signaling in the follicle cells is probably conserved among the species of the Drosophila genus. In addition, a mathematical study predicted that changes in the amount and distribution of Grk protein in the oocyte could account for the formation of zero to four-DAs. Therefore, it remains possible that the level of MAPK activation in the follicle cells at stage 9/10 differs quantitatively with the number of DAs, because this analysis was mostly qualitative and did not provide the relative values of MAPK activation level (Kagesawa, 2008).

Although the activation patterns of MAPK in follicle cells were evolutionarily conserved in the Drosophila genus through stage 10A, they diverged significantly between subgenus Sophophora and the rest of subgenera from stage 10B. That is, Sophophora showed two 'L-shaped' MAPK activation domains, but the domains were 'V-shaped' in the other subgenera. However, among the species of each subgenus, the MAPK activation pattern was very similar, with a few exceptions in subgenus Drosophila exemplified by D. melanica, D. phalerata, and D. guttifera (Kagesawa, 2008).

Molecular phylogenetic analysis revealed that the subgenus Dorsilopha, which has the V-shaped pattern of MAPK activation, diverged from a common ancestor of subgenus Sophophora. This finding suggests that the two-L-shaped-domains pattern of MAPK activation is a trait derived from the V-shaped MAPK activation pattern. Furthermore, the two-DA characteristic and the two-L-shaped-domains MAPK activation pattern probably co-segregated early in the divergence of subgenus Sophophora. Therefore, it is conceivable that the events responsible for the change from the V-shaped to the two-L-shaped pattern of MAPK activation played a crucial role in the morphological evolution from four to two-DAs. However, it remains unclear whether the two-L-shaped pattern of MAPK activation is prerequisite for the activation domain of EGFR signaling to remain unseparated, which consequently results in the formation of two MAPK activation domains. It is also possible that the crucial step for the development of two-DAs is attributable to the lack of a mechanism for separating the MAPK activation domain into two regions in each lateral half at stage 12, instead of the two-L-shaped pattern of MAPK activation at stage 10B (Kagesawa, 2008).

The present results suggest that modifications in EGFR signaling, which independently occurred in different phylogenetic groups, were responsible for the homoplastic evolution of the two-DA eggshell. All of the Sophophora species tested in this study showed two L-shaped MAPK activation domains. In contrast, the species of subgenus Drosophila, except for the three-DA species, showed a V-shaped MAPK activation domain. By stage 10B, the MAPK activation of D. melanica conformed to the typical V-shaped pattern of subgenus Drosophila, as predicted from its phylogenetic status. However, after this stage, the pattern of EGFR signaling activation became distinctive among the species of subgenus Drosophila. In D. melanica, MAPK was not activated in the anterolateral regions at stage 12, which could account for the two-DA phenotype. Based on the comparison of MAPK activation patterns among the species of subgenus Drosophila, it is proposed that at least two alterations of MAPK activation patterns account for the absence of MAPK activation in the anterolateral regions. First, in D. melanica, the two linear domains of MAPK activation were shorter than those of the other subgenus Drosophila species at stage 10B, which could result in the lack of MAPK activation in the anterolateral regions at stage 12. Second, the single large MAPK activation domain failed to be divided into two domains in each lateral set of follicle cells at stage 12. However, it is difficult to determine which changes in EGFR signaling activity played crucial roles in the evolution from the four- to two-DA phenotype in these species. Therefore, it remains unclear whether the modifications of EGFR signaling responsible for the acquisition of the two-DA characteristic are similar between D. melanica and the species of subgenus Sophophora, although phylogenetic analyses indicated that these two modifications occurred independently (Kagesawa, 2008).

It has been shown that aos, a negative regulator of EGFR signaling, is expressed in the regions that are posteriorly juxtaposed to the MAPK activation domains in D. virilis. Similarly, other putative negative regulator(s) of EGFR signaling might be expressed in the triangular region at the dorsal midline, where EGFR signaling is suppressed. Based on these results, it has been proposed that these negative regulators of EGFR signaling might suppress MAPK activation in the middle part of its linear activation domains, resulting in the division of the MAPK activation domain into two in each lateral set of follicle cells. Applying this assumption to the other species of subgenus Drosophila, an absence of these inhibitors could be the reason why the MAPK activation domain is not split into two domains, but remains as a single large domain at stage 12 in D. melanica. Alternatively, these negative regulators of EGFR signaling might suppress the MAPK activation in the anterior part of the linear MAPK activation domains, consequently shortening these domains, at stage 10B in this species. These two possible modifications could result in the acquisition of the two-DA characteristic by D. melanica, although there may be other explanations (Kagesawa, 2008).

Although the developmental basis of homoplastic evolution remains largely unknown, previous studies have provided some insight into the underlying genetic mechanisms. Extensive studies on evolutionary modifications in the pattern of dorsal trichomes in Drosophila larvae revealed that changes in the expression pattern of a single gene, shavenbaby, underlie the observed convergent evolution. Similarly, changes in the cis-regulatory elements of the yellow gene are responsible for independently occurring modifications of similar body-color traits in the subgenus Sophophora. Therefore, evolutionary changes occurring in the same gene could account for the homoplastic evolution of animal morphology. In contrast, it was also reported that the evolution of similar body-color traits can also be achieved by different alterations in the functions or regulation of different genes. Thus, it is important to determine whether the two-DA characteristic was acquired by modifications occurring in the same gene or different genes, or by the same or different modifications of a common gene in D. melanica and the species of subgenus Sophophora. Further comparative studies on the expression patterns of genes that control EGFR signaling activity might provide the information needed to clarify this issue (Kagesawa, 2008).

During the homoplastic evolution of the two-DA characteristic, the expression pattern of rho diverged. However, it has been found that the functions of the rho enhancers were not altered significantly in the divergence of D. virilis and D. melanogaster. In addition, the previous results suggested that the global activity of trans-acting factors that regulates the activation of rho genes did change during the evolution of the four- and two-DA characteristics between these two species. Therefore, it is conceivable that changes in the transcriptional landscape, including divergences in the distributions and/or activation patterns of trans-acting factor(s), could give rise to the independent evolution of similar morphological traits. However, as reported for other examples of homoplastic evolution, it is also possible that modifications of the cis-regulatory elements of a gene that regulates EGFR signaling activity played a crucial role in the evolution of the two-DA phenotype in D. melanica and the species of subgenus Sophophora (Kagesawa, 2008).

Among the Hawaiian species of Drosophila, one, which lays eggs deep within the breeding substrate (e.g., tree or soil fluxes) has very long and thick DAs; the other species, which simply drop their eggs on surfaces, such as leaves, have extremely short and thin DAs. Therefore, the morphologies and numbers of DAs could be adaptations to specific environments. To investigate this issue, it will be important to know whether the homoplastic evolution of the two-DA characteristic is the outcome of adaptation to a common environment. It is interesting to speculate that D. melanica and the species of subgenus Sophophora lay their eggs in similar environments, with the result that these species all acquired a common two-DA phenotype (Kagesawa, 2008).


Heat shock and chemical stress induce activation of heat shock (stress) genes and synthesis of heat shock proteins. It is not yet fully understood which molecular mechanism leads to activation. Probably denatured proteins play an important role in activating the transcription factor (HSF), but there are additional hints that a phosphorylation event is also involved. During a search for a possible signal transduction system in Drosophila, Schneider 2 cells, the response of the mitogen-activated protein (MAP) kinase after stress and its regulation by phosphatases have been analyzed. Stress activates a MAP kinase-specific phosphatase in Drosophila and inhibits MAP kinase activity (Cornelius, 1995).

Heat shock treatment of Drosophila tissue culture cells causes increased tyrosine phosphorylation of several 44 kDa proteins, identified as Drosophila mitogen-activated protein (MAP) kinases. Tyrosine phosphorylation occurs within 5 min, and is maintained at high levels during heat shock. It decreases to basal levels during recovery, concurrent with the repression of heat shock transcription and heat-shock-protein synthesis. The increased MAP kinase tyrosine phosphorylation is parallelled by increased MAP kinase activity. At least two MAP kinases, DmERK-A and DmERK-B, have been identified whose tyrosine phosphorylation increases during heat shock. Thus MAP kinase activation is an immediate early response to heat shock, and its increased activity is maintained throughout heat shock treatment. Protracted MAP kinase activation may contribute to heat shock transcription factor phosphorylation and the numerous metabolic alterations that constitute the heat-shock response (Chen, 1995).

Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila

Epidermal growth factor receptor (EGFR) signaling in the mammalian hypothalamus is important in the circadian regulation of activity. This study examined the role of the EGF pathway in the regulation of sleep in Drosophila. The results demonstrate that rhomboid (Rho)- and Star-mediated activation of EGFR and ERK signaling increases sleep in a dose-dependent manner, and that blockade of rhomboid (rho) expression in the nervous system decreases sleep. The requirement of rho for sleep localized to the pars intercerebralis, a part of the fly brain that is developmentally and functionally analogous to the hypothalamus in vertebrates. These results suggest that sleep and its regulation by EGFR signaling may be ancestral to insects and mammals (Foltenyi, 2007).

The findings reported here show a previously unknown role for EGFR and ERK signaling in sleep regulation and consolidation in Drosophila. In the adult fruit fly, EGFR is expressed ubiquitously throughout the nervous system, where its only known role is in the maintenance and survival of neurons. The current results demonstrate that the overexpression of EGFR pathway signaling components Rho and Star in Drosophila causes an acute, reversible and dose-dependent increase in sleep that tightly parallels an increase in phosphorylated ERK in the head. The ability of a dominant-negative EGFR to block the activation of ERK, as well as the known selectivity of Rho for these ligands, argues that the manipulation is specific to the EGFR pathway. In contrast to the increase in sleep amount after Rho overexpression, inhibiting its expression led to a significant decrease in sleep. Notably, this decrease in sleep was due to a marked shortening of the duration of sleep episodes accompanied by an elevation of sleep bout number. This observation suggests that flies have an increased need for sleep, but are unable to stay asleep, which is perhaps analogous to insomnia in humans. Therefore, it is proposed that the EGFR pathway is essential for sleep maintenance (Foltenyi, 2007).

The brain regions that appear to be involved in the influence of signaling by Rho, EGFR and ERK on sleep are the pars intercerebralis, median bundle and tritocerebrum. The cells of the pars intercerebralis contain Rho and generate EGFR ligand that activates ERK in the receiving cells in the tritocerebrum. The pars intercerebralis was identified as the region that is responsible for EGFR ligand secretion by demonstrating that inhibiting Rho in this region resulted in decreased sleep, and that the cells in that region expressed endogenous Rho. The tritocerebrum was identified though the system-wide overexpression of the EGFR ligand-processing components Rho and Star, which resulted in a localized hyperactivation of ERK. This is presumably because an ectopic presence of Rho and Star will only result in heightened EGFR signaling if the cells contain endogenous ligand precursor (Foltenyi, 2007).

Although the mushroom body is the only region of the Drosophila brain that has been reported to have an effect on sleep, no Rho expression was detected in the mushroom body, nor did inhibiting Rho with UAS-rhoDN in this structure have any effect on sleep levels. However, it is reasonable to expect that the regulation of sleep would involve multiple brain regions and pathways, and that the regulation, versus the function, of sleep could be two distinct, but linked, processes (Foltenyi, 2007).

Cells of the pars intercerebralis send out axonal projections though the median bundle and then bifurcate, innervating the tritocerebrum or running alongside the esophageal canal to innervate the endocrine gland corpora cardiaca. The results indicate that the pars intercerebralis cells innervating the corpora cardiaca are not the ones responsible for the observed decrease in sleep; Gal-4 drivers that are active in these cells did not produce a significant drop in sleep levels when expressing rho RNAi. Developmental studies have led to the postulate that the pars intercerebralis and the corpora cardiaca are the developmental equivalent of the mammalian hypothalamic-pituitary axis. The hypothalamus is a major center in the mammalian brain for the regulation of arousal, and the SCN, which is a part of the hypothalamus, has already been shown to regulate circadian activity through EGFR signaling (Foltenyi, 2007).

Vertebrate studies have only investigated EGFR signaling in the subparaventricular zone, a region located immediately adjacent to the SCN, and this region was shown not to affect total sleep levels, but does alter its timing. In addition, evidence in mammals for a role of EGF in sleep per se is equivocal. These results directly demonstrate that the disruption of EGFR ligand production affects sleep though the pars intercerebralis and not though the circadian control center of the Drosophila brain. It also suggests that the pars intercerebralis shares some functional, as well as developmental, homology with the mammalian hypothalamus through its crucial and conserved involvement in regulating sleep and its maintenance with neural hormones such as the EGFR ligands (Foltenyi, 2007).

In the fly, a single member of the EGFR family binds both the TGF-α-like family of ligands (Spitz, Gurken and Keren) and the neuregulin-like ligand Vein. In vertebrates, these ligands bind to specific ErbB family members, with ErbB-1 (EGFR) binding EGF and TGF-α, whereas ErbB-3 and ErbB-4 bind the neuregulins. In mammalian systems, ErbB-2 and ErbB-4 cofractionate, coimmunoprecipitate and colocalize in cultured rat hippocampal neurons with the postsynaptic density protein PSD-95 (also known as SAP90), and show exclusion from presynaptic terminals. Similarly, ERK colocalizes with, and directly phosphorylates, PSD-95, as is the case with the ErbB receptor-family members. In the fly, EGFR interacts with the postsynaptic density protein Discs Large (Dlg), the Drosophila homolog of PSD-95 (Foltenyi, 2007).

ERK has a role in synaptic plasticity that is conserved among Aplysia, Drosophila and mammals. A recent study shows that ERK directly phosphorylates the pore-forming α subunit of the A-type potassium channel Kv4.2, a member of the Shal-type (Shaker-like) family. This broadens the role of ERK beyond the realm of cell proliferation, differentiation, and even long-term memory consolidation, and suggests that it may also contribute to the more immediate alterations of the electrical properties of the neuronal membrane (Foltenyi, 2007).

On the basis of the current findings and the published reports on the functions of EGFR, the following cellular mechanism is proposed for sleep regulation in Drosophila. Star and Rho in the pars intercerebralis produce and secrete ligand to EGFR located at the postsynaptic membrane of neurons in the tritocerebrum, leading to the activation of ERK in these cells. The difference in staining patterns between inactive ERK clustering near synapses (data not shown) and active ERK located out in the axons indicates that the activated ERK, at least in part, translocates from the postsynaptic membrane and spreads out into the axons that fill out the tritocerebrum and other locations to which these cells project. As a result of a lack of ppERK in the cell bodies of these neurons and the reversible nature of the sleep behavior, it is unlikely that these cells are undergoing long-term synaptic structural changes associated with changes in gene expression. Instead, it is proposed that the action of ppERK occurs at the synapse or in the axon (or both), where it is possibly altering the gating of a neural receptor or channel, and thus changing the membrane properties of the cells. This modification results in an altered brain state that ultimately manifests itself in the sleep behavior of the animal. Such a model is consistent with a previously described mutation in the potassium channel shaker (Kv1.4), which has been shown to be incapable of getting much sleep (Foltenyi, 2007).

The phosphatase SHP2 regulates the spacing effect for long-term memory induction

A property of long-term memory (LTM) induction is the requirement for repeated training sessions spaced over time. This augmentation of memory formation with spaced resting intervals is called the spacing effect. In Drosophila, the duration of resting intervals required for inducing LTM is regulated by activity levels of the protein tyrosine phosphatase corkscrew (Csw). Overexpression of wild-type Csw in mushroom body neurons shortens the inter-trial interval required for LTM induction, whereas overexpression of constitutively active Csw proteins prolongs these resting intervals. These gain-of-function csw mutations are associated with a clinical condition of mental retardation. Biochemical analysis reveals that LTM-inducing training regimens generate repetitive waves of Csw-dependent MAPK activation, the length of which appears to define the duration of the resting interval. Constitutively active Csw proteins prolong the resting interval by altering the MAPK inactivation cycle. This study thus provides insight into the molecular basis of the spacing effect (Pagani, 2009).

This work began with the study of the effects of clinically relevant GOF csw mutations on learning and memory and led to the discovery that Csw plays a critical role in the regulation of the spacing effect for induction of LTM. Several measures were employed to minimize biologic variation, including the use of an isogenic background for all genotypes examined, identical rearing and testing conditions, and batching the analysis for all data presented in the same figure. In addition, multiple mutant alleles were used to support any phenotypes observed. Finally, alternative approaches such as pharmacologic inhibition or RNAi were used when possible to bolster the initial observation (Pagani, 2009).

Among the several functions of Csw, its phosphatase activity seems to be critical for LTM induction. Pharmacological phosphatase inhibition in wild-type fruit flies disrupted LTM, overexpression of phosphatase-dead Csw had no effect on memory formation, and NS- and leukemia-associated Csw mutants share the biochemical feature of having elevated phosphatase activity (Pagani, 2009).

The adverse effects of the GOF Csw on LTM formation are likely mediated through Csw-regulated Ras/MAPK activity. Csw is a key signaling relay in pathways in C. elegans, Drosophila, Xenopus and mammals. The data indicated that GOF Csw deregulated the training-dependent MAPK activation/inactivation. Thus, the most parsimonious interpretation is that csw GOF mutations alter the time course of the activity of the MAPK pathway in such a way that a longer resting period between training sessions is required for promoting normal memory formation (Figure 6D) (Pagani, 2009).

Although the Ras/MAPK pathway is crucial for growth and differentiation, it was interesting to note that the defects in LTM formation associated with GOF Csw were not developmental. Thus, this study together with an increasing body of evidence suggest that the receptor tyrosine kinase-activated Ras/MAPK pathway might be a conserved mechanism from Drosophila to vertebrates and even humans in mediating memory formation (Pagani, 2009).

This study has shown that genetic manipulation can modify the resting interval needed for the induction of LTM. In Drosophila, the spacing effect is well defined phenomenologically and it is used as a behavioral strategy to induce protein synthesis-dependent LTM. It was previously established that LTM can be elicited with 10 repetitive training trials with an optimal spacing of 15 min, and this study showed that LTM is equally well formed as the rest interval is lengthened to 30-40 min. More strikingly, the minimum duration was shortened to 150 sec for transgenic fruit flies overexpressing wild-type csw, but was prolonged to 40 min in transgenic fruit flies with overexpression of GOF Csw mutants. Of note, even though a 150-sec inter-trial was enough to induce LTM in fruit flies overexpressing wild-type csw, a longer interval did not produce more memory as only a small increase in performance was detected by using 30 or 40 min of spacing (Pagani, 2009).

A biochemical correlate of this resting-interval dependence for LTM induction emerged from the analysis of MAPK activation patterns (see Schematic Representations of Training-Regulated MAPK Activity Correlated with Training Protocol and Genotype). For clarity, For wild-type flies subjected to spaced training, MAPK is activated during each 15-min rest interval and is reset to the basal level by the following training cycle. Thus, there is a wave of MAPK activity after each training trial, making for 10 peaks in all. In contrast, in massed training, there is only one peak of MAPK activity, which occurs 15-20 min after finishing the 10th training trial. For fruit flies overexpressing wild-type Csw, however, massed training does create 10 waves of MAPK activation due to the faster MAPK activation combined with a normal post-trial resetting mechanism. Although MAPK may also be activated faster in transgenic fruit flies overexpressing GOF Csw mutants, this activity is not reset by the subsequent training trial, apparently due to the slower kinetics for its decay. Therefore, the standard spaced training protocol with 15 min rest intervals engenders altered MAP activity peaks in these mutant Csw transgenic fruit flies, resulting in an LTM deficit. This is supported by the observation that lengthening the inter-trial interval to 40 min, which presumably provides more time for the decay of the MAPK activity, rescues LTM formation by restoring MAPK activation waves. Taken together, these finding suggests that Csw-dependent MAPK activation is involved in defining the duration of resting intervals necessary for LTM induction (Pagani, 2009).

Hole-in-one mutant phenotypes link EGFR/ERK signaling to epithelial tissue repair in Drosophila

Epithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation. In a previous screen, mutants were isolated with defects in wound healing. This study describes the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that is required for the activation of several Grh and Fos regulated wound response genes at the wound site. Evidence is provided suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, it was shown that wound healing requires the function of EGFR and ERK signaling. Based on these data, it is concluded that holn1 is a novel gene required for a proper wound healing response. A model is proposed whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway (Geiger, 2011).

Holn1 is not required for the initial rapid response to wound infliction, i.e. the formation of the actomyosin cable within minutes of wounding and the phosphorylation of ERK, which is also detectable soon after wounding. This observation is consistent with Holn1 playing an indirect role in the mechanics of wound closure by regulating the mRNA levels of genes required for this process, such as those involved in rapid and productive cable contraction. Interestingly, the actin cable was present in all the wound closure mutants isolated in the previous screen, suggesting that regulatory events downstream of cable formation dominate the wound closure process. In any case, it is clear that Holn1 is required to perform some additional function needed to sustain the closure process, as holn1 mutants take on average 1.5 times longer to close a wound compared to wild type embryos. A similar delay in wound closure was previously reported for rho1 GTPase mutants, which do not form an actin cable, but can still close small wounds, albeit 2 times slower than wild type embryos]. Aside from its possible role in the epithelial hole closure process, Holn1 could also be involved in cuticle repair. Grh and ERK activity are required for the re-establishment of the epithelial permeability barrier after injury. Thus, Holn1 might be involved in this process by regulating the ERK/Grh pathway (Geiger, 2011).

In the future, just as the holn1 mutation uncovered a connection with the EGF/Ras/ERK signaling pathway and wound healing, microarray analysis of wounded holn1 embryos would identify genes that are likely activated downstream of this wound closure pathway. Performing the same experiment using an alternative splicing array as in would further reveal if Holn1 plays a role in wound dependent splicing events (Geiger, 2011).

Automated FRET quantification reveals distinct subcellular ERK activation kinetics in response to graded EGFR signaling in Drosophila

Threshold responses to an activity gradient allow a single signaling pathway to yield multiple outcomes. Extracellular signal-regulated kinase (ERK) is one such signal, which couples receptor tyrosine kinase signaling with multiple cellular responses in various developmental processes. Recent advances in the development of fluorescent biosensors for live imaging have enabled the signaling activities accompanying embryonic development to be monitored in real time. This study used an automated computational program to quantify the signals of a fluorescence resonance energy transfer (FRET) reporter for activated ERK, and this system was used to monitor the spatio-temporal dynamics of ERK during neuroectoderm patterning in Drosophila embryos. The cytoplasmic and nuclear ERK activity gradients were found to exhibit distinct kinetics in response to epidermal growth factor (EGF) receptor activation. The ERK activation patterns implied that the cytoplasmic ERK activity is modulated into a threshold response in the nucleus (Ogura, 2019).

rolled/MAPK: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Effects of Mutation | References

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