Interactive Fly, Drosophila

EGF receptor

Egfr and myogenesis

Inductive interactions between cells of distinct fates underlie the basis for morphogenesis and organogenesis across species. In the Drosophila embryo, somatic myotubes form specific interactions with their epidermal muscle attachment (EMA) cells. The establishment of these interactions is a first step toward further differentiation of the EMA cells into elongated tendon cells containing an organized array of microtubules and microfilaments. The molecular signal for terminal differentiation of tendon cells is the secreted Drosophila neuregulin-like growth factor Vein, produced by the myotubes. Although Vein mRNA is produced by all of the myotubes, Vein protein is secreted and accumulates specifically at the muscle-tendon cell junctional site. In loss-of-function vein mutant embryos, muscle-dependent differentiation of epidermal tendon cells, measured by the level of expression of specific markers (Delilah and beta1 tubulin) is blocked. When Vein is expressed in ectopic ectodermal cells, it induces the ectopic expression of these genes. These results favor the possibility that the Drosophila EGF receptor DER/Egfr expressed by the EMA cells functions as a receptor for Vein. Vein/Egfr binding activates the Ras pathway in the EMA cells leading to the transcription of the tendon-specific genes stripe, delilah, and beta1 tubulin. In Egfr1F26 mutant embryos lacking functional Egfr expression, the levels of Delilah and beta1 Tubulin are very low. The ability of ectopic Vein to induce the expression of Delilah and beta1 Tubulin depends on the presence of functional Egfrs. Activation of the Egfr signaling pathway by either ectopically secreted Spitz, or activated Ras, leads to the ectopic expression of Delilah. These results suggest that inductive interactions between myotubes and their epidermal muscle attachment cells are initiated by the binding of Vein, to the Egfr on the surface of EMA cells (Yarnitzky, 1997).

Muscle development initiates in the Drosophila embryo with the segregation of single progenitor cells, from each of which a complete set of myofibers arises. Each progenitor is assigned a unique fate, characterized by the expression of particular gene identities. The Drosophila Epidermal growth factor receptor (Egfr) provides an inductive signal for the specification of a large subset of muscle progenitors. In the absence of the receptor or its ligand, Spitz, specific progenitors fail to segregate. The resulting unspecified mesodermal cells undergo programmed cell death. In contrast, receptor hyperactivation generates supernumerary progenitors, as well as the duplication of at least one Spitz-dependent myofiber. The requirement for Egfr occurs early in muscle cell specification, as early as five to seven hours after fertilization. The development of individual muscles is differentially sensitive to variations in the level of signaling by the Epidermal growth factor receptor. Such graded myogenic effects can be influenced by alterations in the functions of Star and Rhomboid. In addition, muscle patterning is dependent on the generation of a spatially restricted, activating Spitz signal, a process that may rely on the localized mesodermal expression of Rhomboid. Thus, Epidermal growth factor receptor contributes both to muscle progenitor specification and to the diversification of muscle identities (Buff, 1998).

In a screen for lethal mutations that disrupt the normal embryonic muscle pattern, multiple alleles of two of the Drosophila spitz group genes were identified: Star and spitz. In a strong spi mutant approximately half of the normal myofibers are missing, while those that do develop have morphologies, positions and orientations that allow them to be assigned wild-type identities. For example, all of the lateral transverse muscles form normally in the absence of spi function, whereas gaps are present in the set of ventral longitudinal muscles. Only one of the normal three ventral oblique muscles is present in a spi mutant. Of note, muscle defects are not more severe in ventral regions where the spi group genes are known to be required for ectodermal patterning. Since spi encodes a ligand for the Drosophila Epidermal growth factor receptor, the Egfr loss-of-function phenotype was examined. A temperature-sensitive allele was used to examine the mutant Egfr muscle phenotype in an attempt to bypass the early pleiotropic requirements for Egfr signaling in embryonic development. Many of the spi-dependent muscles were also found to require Egfr function, but this analysis was limited by the finding that the temperature-sensitive period for Egfr involvement in myogenesis overlaps with that of other developmental roles for this receptor. To circumvent this problem, the myogenic function of Egfr was studied in isolation by targeting the expression of a dominant negative form (DNDER) to the mesoderm using the GAL4/UAS expression system. DNDER was constructed by deleting the intracellular domain of the protein, a strategy that has proved effective in other systems for inhibiting full-length RTKs (Buff, 1998).

The spi mutant muscle pattern is phenocopied by mesodermal expression of DNDER. The severity of this phenotype is dependent on the copy number of the DNDER transgene, and the specificity of the response to the truncated receptor is demonstrated by the ability of wild-type Egfr to reverse its effect. This strongly suggests that Spi signaling through Egfr is essential for normal myogenesis. The targeted ectopic expression of DNDER establishes that the receptor functions autonomously in mesodermal cells, as opposed to the known ectodermal abnormalities associated with loss of DER function having an indirect influence on mesoderm development (Buff, 1998).

To determine at what stage of muscle development spi and Egfr are required, the effect of loss-of-function of these genes was examined on the expression of several early myogenic markers. The mature myofibers seen in stage-16 embryos differentiate from muscle precursors that are formed by myoblast fusion starting at stage 12 and continuing through stage 15. Additional dorsal mesodermal cells segregate to become heart precursors during this time. The segmentation genes, even-skipped and Kruppel, are expressed in distinct but partially overlapping subsets of mesodermal precursors. The precursor of muscle DA1, which expresses both Eve and Kr, is missing in the absence of spi and DER functions. Additional Kr-positive muscle precursors, including LL1, VA2 and several other internal ventral precursors, are also spi/Egfr-dependent. However, the Eve-expressing pericardial cell precursors, as well as certain dorsal and lateral Kr-expressing muscle precursors (DO1, LT2 and LT4), form normally in these genetic backgrounds. The presence or absence of these precursors correlates completely with the mature myofiber pattern of spi and Egfr mutant embryos. These results demonstrate that spi and Egfr are required for the formation of some but not all muscle precursors. At an even earlier stage of mesoderm development, mononucleated progenitor cells segregate and divide to generate sibling founder cells, each sibling cell the founder for the formation of one muscle precursor. Progenitors initially express the proneural gene, lethal of scute (l'sc), as well as muscle identity genes such as S59, eve and Kr. The expression of identity genes persists while that of l'sc fades prior to progenitor division. This developmental sequence is illustrated for the two Eve progenitors, P2 and P15. P2 forms first and initially expresses both L'sc and Eve. By the time L'sc disappears from P2, P15 forms and co-expresses L'sc and Eve. Both progenitors then divide, each giving rise to two Eve-positive founder cells (F2 and F15). Eve is retained in only one founder cell of each pair. The F2 founder in which Eve persists divides again, giving rise to a pair of pericardial cells in each hemisegment, while the Eve-expressing F15 contributes to muscle DA1; the subsequent fates of the F2 and F15 founders that lose Eve expression remain unknown. With loss of either Spi or Egfr function, P15 does not develop, whereas P2 and its founders segregate normally. This is consistent with the prior finding that DA1 muscle precursors, but not the Eve pericardial cells, are dependent on spi and Egfr. Additional L'sc-expressing muscle progenitors also are missing from spi mutant embryos. Thus, Spi/Egfr signaling is involved in the earliest step of somatic myogenesis, the specification of muscle progenitors. It is shown that Spi/Egfr signaling specifies particular muscles at different developmental times and that unspecified mesodermal cells undergo programmed cell death in the absence of Spi/Egfr signaling. It is also shown that hyperactivation of Egfr generates supernumerary muscle founders and the duplication of a Egfr-dependent muscle (Buff, 1998).

Star, which is known to interact with Egfr, modifies myogenic signaling by Egfr. Ectopic mesodermal expression of DNDER yields a sensitized background in which to quantitate genetic interactions with Star. One copy of UAS-DNDER caused a partial reduction in the development of DA1 and VA2. This effect is suppressed by co-expression of full-length Egfr or Star. Ectopic expression of Star or full-length Egfr in a wild-type genetic background had no effect on muscle development. The UAS-Star results also indicate that Star is required autonomously for Egfr function in the mesoderm. Star dominantly enhances the effect of DNDER on muscles DA1 and VA2, suggesting that Star is normally limiting for muscle development. Rhomboid is also required for muscle DA1 formation and is expressed in the mesoderm in proximity to the DA1 progenitor. As was found for spi, Star and Egfr, rho is also required for development of the Eve-expressing muscle DA1 precursor but not for formation of the adjacent pericardial cells. Because Rho is a positive regulator of Egfr and its expression is frequently localized to sites where Egfr signaling is active, the expression of rho in the vicinity of DA1 was examined during the course of its development. rho transcripts are found in segmentally repeated dorsal mesodermal cells in stage-11 embryos. These cells are located at the peaks of the mesodermal crests that lie between the tracheal pits, precisely where the Eve-expressing P2 and P15 progenitors and their founders arise. By double-labeling with Rho and Eve antibodies, it was found that Rho is co-expressed with Eve in P2. This is a particularly intriguing finding since the specification of P2 (the pericardial progenitor) precedes that of P15 (the muscle DA1 progenitor): these two cells segregate in very close proximity to each other, and only P15 is Egfr-dependent. Even under conditions where muscle DA1 forms in the absence of Eve pericardial cells, such as with partial inhibition of Heartless activity, Rho is expressed in a mesodermal cell that resembles a normal P2 but lacks Eve. Given the known effects of Rho in modifying Egfr activity in other developmental contexts, the temporal and spatial expression of Rho in the dorsal mesoderm is consistent with a functional role for Rho in the Egfr signaling responsible for P15 induction (Buff, 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).

Of the two clusters of dorsal mesodermal cells that express the pair-rule gene even-skipped that arise sequentially from a single precluster, one cluster gives rise to a single Eve-positive progenitor that divides to give rise to a pair of pericardial cells (P2). The second cluster (P15, arising from the same C2 precluster that gives rise to P2) gives rise to at least one dorsal somatic muscle and forms from a second Eve progenitor. The two L'sc clusters from which these progenitors arise are prefigured by a broader domain of L'sc expression (the precluster) that is dependent on the combined activities of Wg and Dpp. The corresponding equivalence groups (clusters) are formed within this prepatterned mesodermal region via localized activation of the Ras1 pathway by two receptor tyrosine kinases (RTKs), Htl, and Egfr. Whereas Htl is required for the Eve cardiac equivalence group, both Egfr and Htl are involved in the Eve muscle cell cluster. These findings demonstrate how positional information initially establishes a mesodermal prepattern, and establish that individual progenitors are progressively determined by unique combinations of intercellular signals. It is concluded that distinct cellular identity codes are generated by the combinatorial activities of Wg, Dpp, Egf, and Fgf signals in the progressive determination of embryonic mesodermal cells (Carmena, 1998).

To examine the question of how Egf and Fgf signals are involved in precluster or cluster development, expression of dominant-negative forms of both receptors were targeted to the embryonic mesoderm. Neither dominant-negative Htl nor dominant-negative Egfr affect preC2 development. However, inhibition of Htl, but not Egfr, signaling leads to loss of C2. This requirement for Htl reflects a direct involvement in cell fate specification because mesoderm migration is completely normal when dominant-negative Htl is expressed under the present conditions. After the singling out of P2 from the C2 precluster, C15 forms at the position occupied previously by C2, a process that requires both Egfr and Htl, in agreement with the demonstration that both RTKs are required for muscle DA1 development. Htl (but not Egfr) also contributes to the formation of other dorsal L'sc clusters. In summary, establishment of the L'sc prepattern does not require the activity of either Egfr or Htl. However, both RTKs are essential for the subsequent organization of L'sc equivalence groups within the mesodermal territory prepatterned by Wg and Dpp (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).

Constitutive activity of DER, Htl, or Ras1 is associated with the development of supernumerary Eve-expressing mesodermal cells. However, it has not been established whether this response is due to activated Ras1-induced proliferation of normal Eve cells or is due to the recruitment of additional cells to an Eve-positive fate. Activated Ras1 does not increase the sizes of L'sc clusters, suggesting that Ras1 is not simply stimulating the division of mesodermal cells. However, to definitively address this question, the effects of constitutive Ras1 activity were examined in string (stg) mutant embryos. Because strong alleles of stg prevent all post-blastoderm cell divisions, a potential cell-proliferation effect of activated Ras1 should be blocked in this genetic background. However, stg should not inhibit the overproduction of Eve-expressing cells if Ras1 promotes their determination. In a stg mutant, only one to two Eve-positive cells are present in each hemisegment. These cells correspond to the Eve progenitors that form in wild-type embryos. The presence of only one Eve progenitor in some segments of stg mutant embryos presumably is due to the smaller number of mesodermal cells that contribute to L'sc clusters when zygotic cell divisions do not occur. Significantly, activated Ras1 generates more Eve progenitors in a stg mutant than are seen in the mutant alone. It is concluded that Ras1 promotes the formation of additional Eve progenitors by inducing more mesodermal cells to assume this fate and not by stimulating the normal progenitors to divide. Next, an assessment was carried out to determine if the Ras1 pathway is sufficient to induce progenitor differentiation by examining the myogenic effects of Ras1 at later developmental stages in both wild-type and myoblast city (mbc) mutant embryos. In the absence of mbc function, muscle fusion does not occur and differentiated muscle founders appear as spindle-shaped myoblasts that express myosin and founder cell markers, such as Eve. In contrast to the mbc mutant in which one Eve-expressing muscle DA1 founder is present in each hemisegment, multiple occurrences of such cells form in mbc embryos under the influence of activated Ras1. All of the Eve plus myosin-positive myoblasts seen in these embryos have the elongated morphology of muscle founders, as opposed to the round shape of neighboring Eve-negative myoblasts. Large syncytia containing many Eve-positive nuclei are present when Ras1 is activated in a wild-type background. Although extra Eve pericardial cells initially appear under the influence of activated Ras1, Eve expression in such cells is lost by later stages. It is concluded that ectopic activation of the Ras1 pathway not only stimulates Eve expression in additional progenitors, but also promotes the formation and differentiation of supernumerary muscle founders (Carmena, 1998).

A salient feature of these findings is that equivalence groups form in response to the localized functions of two RTKs acting within the larger prepatterned region. The mesodermal diphospho-MAPK expression pattern not only provides strong support for this hypothesis, but also raises the question of how the upstream receptors are activated in discrete subsets of competent cells. For C15, this may occur through induction by P2, which expresses Rhomboid, a factor that can nonautonomously stimulate Egfr in adjacent cells. The localized activation of Htl could result from restricted expression of its as yet unidentified ligand. Htl itself is enriched in groups of Eve-positive mesodermal cells, a process that might also contribute to the Htl-dependent formation of these clusters. RTK signaling in the Drosophila embryonic mesoderm is important for cell migration and cell fate specification. The latter role can be divided into several distinct functions: (1) Htl and DER promote the formation of L'sc clusters or equivalence groups; (2) Ras1 signaling activates identity gene expression in the entire group of equivalent cells; (3) activated MAPK becomes restricted to progenitors, suggesting a role for RTK signaling in progenitor selection, and (4) MAPK is reactivated in one of the sibling founders derived from a single progenitor, suggesting that RTK activity helps to establish or maintain the founder identity that is initiated by the asymmetric division of its progenitor. Alternatively, RTK function in some founder cells could promote their differentiation. The ability of activated Ras1 to generate supernumerary Eve founders in mbc mutant embryos is consistent with this last possibility. Interestingly, the RTK/Ras1 pathway is similarly utilized in a sequential manner during development of the Drosophila compound eye (Carmena, 1998 and references).

Activation of the Drosophila Egfreceptor (Egfr) is spatially and temporally controlled by the release of its various ligands. Egfr and its ligand Spitz mediate the formation of specific somatic muscle precursors. A second Egfr ligand, Vein, complements the activity of Spitz in the development of various somatic muscle precursors. In vn mutant embryos, the Egfr-dependent muscle precursors do not form in some of the segments. Double labeling with anti-Vein and anti-Kruppel antibodies reveal that the Kruppel-positive muscle precursors overlap. In vein mutant embryos at stage 12-13 of embryonic development DA1 muscle precursors are missing in one (or occasionally two) segments of mutant embryos. The Kruppel-positive precursors LL1 and VA2 are also missing in vein mutant embryos in similar frequency as that observed for DA1. It is concluded that the loss of the various muscle precursor cells observed in vein mutant embryos is in line with the expression of Vein in these cells. This phenotype is significantly enhanced in embryos carrying only one copy of wild type spitz. This analysis suggests that Vein activation of Egfr differs qualitatively from that of Spitz in that it does not lead to the expression of the inhibitory protein Argos, possibly leading to a continuous activation of the Egfr signaling pathway. The results support the idea that the role of Vein in tissues where Spitz is the major ligand is to complement Spitz activity. A model of synergistic activation by the two ligands is not favored. This explains the extremely weak vein phenotype observed in comparison to a significant and measurable phenotype obtained in tissues where Vein functions as a single ligand (Yarnitzky, 1998).

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