Interactive Fly, Drosophila

Effects of Mutation or Deletion (part 1/2)

Mutations in the genes spitz, Star, single-minded, pointed , rhomboid (all zygotic), and sichel (maternal), collectively called the spitz group, cause similar pattern alterations in ventral ectodermal derivatives of the Drosophila embryo. The cuticle structures lacking in mutant embryos normally derive from longitudinal strips of the ventro-lateral blastoderm. Defects in these mutants are found in the median part of the central nervous system. Individual members of the spitz group can produce different effects. As shown by pole cell transplantations, spi and S are also required for normal development of the female germ line, while sim, and pnt appear to be exclusively zygotically expressed (Mayer, 1988).

Examination of mutant spitz embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. Mutants show a reduction in external sensory neurons and chordotonal neurons (Rutledge, 1992).

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called 'head midline') that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene lísc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ëcyclopsí like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

The spitz class genes, pointed (pnt), rhomboid (rho), single-minded (sim), spitz (spi) and Star (S), as well as the Drosophila Epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis shows that the expression of all the spitz class and Egfr signaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. Overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. In contrast, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the Egfr signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling Egfr activity through the regulation of the expression of spitz class genes and Egfr signaling genes (Chang, 2001).

CrebA is thought to be epistatic to known dorsal/ventral patterning genes. Epistasis tests were done with the decapentaplegic gene and the spitz gene. In dpp/CrebA double mutants the entire cuticle is lateralized, while in dpp mutants the cuticle is ventralized. In spitz/CrebA double mutants narrower denticle bands are seen with some fusion of denticles between segments; however the denticles have the same morphology found in CrebA mutants alone. It is thought that near the end of both the Dpp- and Spi-signaling cascades, CrebA functions to translate the corresponding extracellular signals into changes in gene expression. The only determinant tested that shows altered expression patterns in CrebA mutants was Dsc73, a secreted protein expressed at late embryonic stages in the epidermal cells that produce denticles and hairs. There was a decrease in the levels of Dsc73 on the dorsal and ventral surfaces compared to levels of Dsc73 in lateral positions, which appear unchanged (Andrew, 1997).

The spitz-group mutants (spitz, rhomboid, and pointed) are embryonic lethal and have similar cuticle phenotypes; they are shorter than wild type and have deletions of ventral cuticle. vein mutant are shorter and the Keilin's organs and ventral black dots are closer together than in wild-type. Ventral cuticle is deleted between Keilin's organs. The deletions occur in a similar region in spitz-group mutants; spitz and rhomboid have a larger portion of ventral cuticle deleted than vein mutants, but pointed embryos have similar deletions. In vein mutants sensory hairs surrounding the pit structure of Keilin's organs are missing. Unlike the spi-group genes, vein is not critical for embryonic survival and head skeleton and sense organs are normal. Most vein mutants die either as embryos or as larvae, but a small number do pupariate. Individuals that survive to pupariate secrete a pupal case with pattern abnormalities (Schnepp, 1996).

vein and spitz show a strong genetic interaction suggesting a molecular interdependence. Reducing vein dose in a spi null genotype dramatically worsens the phenotype to produce a collapse embryo with an extruded head skeleton. However, the double mutants are not as severly affected as are Egf-R null mutants. Genetic interactions are also observed between vn, Egf-R and rolled. The gain of function alleles Egf-R-Ellipse and rolled-Sevenmaker rescue proliferation defects in strong and null vein mutants. These defects include a small wing disc and the size of the pupal case (Schnepp, 1996).

Signaling through the Drosophila EGF receptor (DER) is important for the growth and differentiation of the wing. These processes may be mediated by different DER ligands including Spitz and Vein . The roles of these ligands and other DER pathway components in wing disc development were investigated using in vivo culture to produce mutant discs from genotypes that are normally embryonic lethal. No role for spi in wing disc growth was found, whereas vn is essential. spi mutant wing discs are morphologically normal as judged by expression of the vein marker rhomboid and analysis of the differentiated wing tissue. spi embryos produce mature-size wing discs patterned with vein and intervein territories like wild-type discs and differentiated wing structures look normal. rho, Star and argos, all known to be involved in Spi/DER signaling are likewise not required for wing growth, whereas pointed, which acts at the end of the intracellular pathway, is required. The results suggest different ligands and molecular mechanisms control EGF-R signaling in wing growth and differentiation (Simcox, 1997).

Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes

Genes of the spalt family encode nuclear zinc finger proteins. In Drosophila melanogaster, they are necessary for the establishment of head/trunk identity, correct tracheal migration and patterning of the wing imaginal disc. Spalt proteins display a predominant pattern of expression in the nervous system, not only in Drosophila but also in species of fish, mouse, frog and human, suggesting an evolutionarily conserved role for these proteins in nervous system development. Spalt works as a cell fate switch between two EGFR-induced cell types, the oenocytes and the precursors of the pentascolopodial organ in the embryonic peripheral nervous system. Removal of spalt increases the number of scolopodia, as a result of extra secondary recruitment of precursor cells at the expense of the oenocytes. In addition, the absence of spalt causes defects in the normal migration of the pentascolopodial organ. The dual function of spalt in the development of this organ, recruitment of precursors and migration, is reminiscent of its role in tracheal formation and of the role of a spalt homolog, sem-4, in the C. elegans nervous system (Rusten, 2001).

By analogy with the developing lch5, it was hypothesized that the oenocytes require Egfr signalling for proper development. Embryos mutants for Star and spitz were examined at different stages of development. Interestingly, in stage 11 embryos the sal pattern of expression remains unaltered in the cells surrounding the C1 precursor, as well as in the epidermis. However, later on, the development of the oenocytes is inhibited. These results indicate that sal regulation is independent of the Egfr pathway and that the oenocytes development depends on both sal and Egfr signaling activity. Furthermore, if the signaling arises from the precursor C1, the formation of oenocytes would be restrained in the absence of SOPs. Indeed, in ato mutant embryos oenocytes originate only in the segments where remnant SOPs develop (Rusten, 2001).

In conclusion, the results are consistent with a model where sal restricts the ability of C1-surrounding cells, receiving Egfr signaling, to adopt sensory organ precursor cell fate; these cells then develop as oenocytes rather than chordotonal organs (Rusten, 2001).

The Egfr pathway is implicated in the development of the chordotonal organs in Drosophila. The pathway is necessary for the second step of recruitment of SOPs from ectodermal precursors, and for the consequent increase of number of scolopodia in the lch5 and in the vchA/B organs. Thus, during development of the lch5 organ, where two secondary SOPs are recruited, removal of positive Egfr pathway components like rho, S, spi, pnt, sos, Drk, or Egfr itself, reduces the number of scolopodia in the lch5 from five to three. Conversely, mutations in negative regulators of Egfr signaling like argos, gap1 or spry result in an increase of secondary recruited SOPs in the thorax as well as in the abdominal segments (Rusten, 2001 and references therein).

Sal plays a role in the formation of the lch5 in parallel with the Egfr signaling pathway: the absence of sal generates supernumerary scolopodia, while the overexpression of Sal reduces the number of scolopodia from five to three. These results are consistent with the idea that under wild-type conditions, sal modifies the Egfr signaling output in the cells surrounding the primary precursor C1, which instead of becoming secondary SOPs adopt the oenocytes cell fate. Five lines of evidence support this idea. (1) Supernumerary support cells accompany the supernumerary neurons observed in sal mutants. Thus, the phenotype is not caused by cell fate transformation within the SOP lineage. (2) The C1-surrounding cells receive the Egfr signal (shown by the antibody staining for activated Rolled/MAPK) and, therefore, are capable of becoming secondary precursors. These cells are sal positive while the other potential secondary precursors, also showing activated Rolled and overlying the more ventrally located C2-C5, are not. Given that the number of cells receiving the Egfr signal is larger than the number of cells that become secondary SOPs (two for lch5 and one for vchA/B), the output of the Egfr pathway must be modified in the rest of the cells receiving the signal. (3) The analysis of allelic combinations between sal and Egfr pathway mutants reveals that the supernumerary neuronal phenotype observed in the absence of sal is Egfr dependent. (4) The oenocyte precursors depend on sal and Egfr signaling to develop, and (5) in the absence of primary precursors, oenocytes do not develop, as shown in ato mutants (Rusten, 2001).

The effects of sal loss- and gain-of-function are similar, but not identical, to the ones exhibited by corresponding changes in negative regulators of Egfr signaling. There are at least two important differences between the role of these regulators and sal. (1) aos, pnt and spry are expressed in all the cells receiving the Egfr signal from the primary SOPs, while sal is expressed only in a subset of them. Consistent with this, the loss of function of these regulators affects the secondary recruitment of SOPs to other chordotonal organs, like vchA/B and v'ch1, while sal seems to modify only lch5. (2) The increase of scolopodia numbers in lch5 is moderate in the spry and aos mutants, while in sal mutants, up to eight scolopodia are observed. In conclusion, sal is involved specifically in the formation of lch5 in a manner different from that of the Egfr pathway regulators that are involved in the development of all the chordotonal organs (Rusten, 2001).

The cells surrounding C1 migrate along the dorsoventral axis, closely associated with the pentascolopodial organ. These cells are easy to recognize by the elongated shape of their nuclei and the strong sal expression that they display. These cells occupy the location of oenocytes in late embryonic stages. It is then likely that sal plays a role in deciding the fate of the Egfr responding cells surrounding the C1 precursor. In the presence of sal these cells will become oenocytes while in the absence of sal (as is true for the presumptive secondary precursors overlying C2, C3, C4 and C5), the cells will become sensory organ precursors. Since the putative precursors of the oenocyte cells need Egfr signaling to accomplish some aspects of their development, sal is thought to act as a selector gene being necessary to direct them to their correct fate (Rusten, 2001).

spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor

Signaling from the EGF receptor can trigger the differentiation of a wide variety of cell types in many animal species. The mechanisms that generate this diversity have been explored using the Drosophila peripheral nervous system. In this context, Spitz ligand can induce two alternative cell fates from the dorsolateral ectoderm: chordotonal sensory organs and non-neural oenocytes. The overall number of both cell types that are induced is controlled by the degree of Egfr signaling. In addition, the spalt gene is identified as a critical component of the oenocyte/chordotonal fate switch. Genetic and expression analyses indicate that the Sal zinc-finger protein promotes oenocyte formation and supresses chordotonal organ induction by acting both downstream of and in parallel to the Egfr. To explain these findings, a prime-and-respond model is proposed. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to raise the apparent threshold for induction by Spi. Subsequently, sal-dependent Sal upregulation is triggered as part of the oenocyte-specific Egfr response. Thus, a combination of Sal in the responding nucleus and increased Spi ligand production sets the binary cell-fate switch in favour of oenocytes. Together, these studies help to explain how one generic signaling pathway can trigger the differentiation of two distinct cell types (Elstob, 2001).

Oenocytes are induced from the ectoderm by an inductive signal that is generated in the developing PNS. The production of active Spi by the C1 precursor cell, under the control of ato and rho, triggers Egfr activation and thus oenocyte induction in adjacent ectoderm. Oenocyte induction by the PNS appears to be a short-range event with only the cells immediately surrounding C1 switching on oenocyte markers. In argos mutants, however, the range of the response is increased from one to two concentric rings of cells. Hence, as in photoreceptor recruitment, Spi ligand is not intrinsically limited to immediate neighbors but the response is nevertheless kept short-range by argos-mediated feedback inhibition of the receptor (Elstob, 2001).

In wild-type embryos and in all of the mutant backgrounds examined, the number of cells in the whorl at any one time is less than the final number of mature oenocytes. For example, a wild-type whorl contains 3-4 cells with sickle-shaped nuclei but the final oenocyte cluster contains on average 6 cells. The basis for this difference is not yet understood but it might be explained by cell division or by sequential delamination of oenocyte precursors (Elstob, 2001).

The specification of secondary COP and oenocyte fates requires the Egf pathway. In ato, rho, spi and Egfr^DN backgrounds, where signaling is compromised, the induction of both cell types is blocked. Conversely, when the Egfr is hyperactivated, both cell types become more numerous. These results indicate that the number of recruited cells is controlled by the amount of Egf pathway signal. It is important to realize, however, that the level, duration and spatial extent of ligand production are all being altered in these experiments. More sophisticated methods would be needed to clearly distinguish which of these three signaling parameters is critical for controlling cell number (Elstob, 2001).

Surprisingly, there is no parity between the numbers of excess oenocytes and lateral chordotonal organs that are produced by Egfr hyperactivation. Thus for a given increase in ligand, more oenocyte precursors than COPs are recruited. This implies the existence of an additional tier of control that restricts neural but not oenocyte induction. Such a selective inhibition process would ensure that the number of chordotonal organs is more tightly controlled than that of oenocytes, as is observed in wild-type embryos (Elstob, 2001).

The sal gene plays opposite roles in oenocyte and chordotonal induction. It is both necessary and sufficient for repressing secondary COP induction and it is necessary but not sufficient for promoting oenocyte formation. The lack of sufficiency for oenocyte induction is revealed when sal is misexpressed using the en-GAL4 driver. Oenocytes are not ectopically induced in ventral regions, even in the presence of excess Spitz. It is likely that other factors are required, together with Sal, to promote the oenocyte induction process (Elstob, 2001).

Using epistasis tests and gene expression analysis the regulatory relationship between sal and the Egf pathway in oenocyte and COP formation has been elucidated. These data allow the exclusion of the possibility that sal acts upstream of spi in the signaling cell. Importantly, the results indicate that sal functions in the responding ectoderm, either downstream of the Egfr or in a parallel pathway leading to oenocyte induction and secondary COP repression (Elstob, 2001).

In fact, it is probable that sal plays a dual role that is downstream and also in parallel to the Egfr. In rho and spi mutants, the normal upregulation of Sal in the vicinity of C1 is abolished. Conversely, Spitz misexpression produces ectopic Sal upregulation in dorsal locations. Both results indicate that sal lies downstream of the Egfr and that Sal protein levels are controlled by receptor activation. However, Sal is also expressed at moderate levels in presumptive oenocyte precursors prior to Egf pathway activation and this expression remains normal in rho and spi mutants. For these reasons, it is likely that at least part of the function of sal lies in a parallel pathway that, in conjunction with the Egf signal, promotes oenocyte induction and inhibits COP recruitment (Elstob, 2001).

A prime-and-respond model is presented to integrate the dual roles of sal downstream and also in parallel to the Egfr. In this model, sal functions in the parallel pathway as a competence switch. Thus, Sal prepatterns the dorsal ectoderm so that, on receipt of the Egf signal, oenocytes rather than COPs are induced. One consequence of the Sal oenocyte prepattern is to increase the apparent induction threshold in responding cells. This makes the prediction that the signaling cell inducing oenocytes needs to express more ligand than those that recruit secondary COPs, and this is indeed the case. C1 is known to express high levels of rho for longer than any of the other primary COPs. Thus, the Egf pathway does contribute to the cell-type specificity of the induction event in the sense that more Spi ligand is required to induce oenocytes than to recruit chordotonal organs (Elstob, 2001).

One of the early oenocyte-specific responses to the Sal prepattern is the subsequent upregulation of Sal itself. This, in turn, stimulates the expression of the sal target gene svp, one member of the repertoire of oenocyte early differentiation genes. A key feature of the prime-and-respond model is that moderate levels of sal expression serve to prime the responding cells to further upregulate Sal when they receive Spi ligand. In support of this priming mechanism, it has been demonstrated that upregulation in response to constitutive Spitz expression is restricted to those cells that have already expressed sal. Hence, Sal proteins provide a molecular link between the prepattern and the Egfr response (Elstob, 2001).

In the prime-and-respond model, it is implicit that the early and late phases of sal expression produce distinct effects on the responding cell. As the levels of Sal are different in the two phases, it may be that there are at least two different concentration-dependent effects for this transcription factor. In agreement with this, it has been shown that strong expression of the sal target gene, svp, correlates with the domain of sal upregulation and not with the lower-level prepattern. In another system, wing vein development, there is a very extreme example of a concentration difference, with low and high levels of Sal producing completely opposite transcriptional effects on the knirps target gene (Elstob, 2001 and references therein).

Regulation of cell number by MAPK-dependent control of apoptosis: A mechanism for trophic survival signaling

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).

The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).

The spi gene is required for MG survival and encodes a candidate trophic factor for MG survival. To prove that spi function is required to suppress hid activity, the fate of the MG in spi;hid double mutant embryos was determined. The MG survive in spi embryos if hid is removed as well, and it is concluded that the survival function of spi is mediated through suppression of hid-induced apoptosis (Bergmann, 2002).

The question arises as to which cells process mSPI and provide a source of sSPI for MG survival. Since spi is ubiquitously expressed, it is difficult to determine histochemically where sSPI, the active ligand, is generated. Therefore, a genetic approach was used; whether the loss of MG in spi mutant embryos can be rescued by expression of the membrane bound inactive precursor (UAS-mSPI) either in the MG (using the sim-GAL4 driver) or in neuronal axons (using the elav-GAL4 driver) was examined. It was reasoned that the MG would be rescued in spi mutant embryos only if mSPI is presented in the location where it is normally processed for MG survival in wild-type embryos. Presentation of mSPI by the MG itself does not result in rescue of the MG in spi mutant embryos, ruling out an autocrine mechanism. In contrast, expression of mSPI in neuronal axons appears to be sufficient for MG survival in spi embryos. This argues in favor of a paracrine mechanism. In control experiments, sSPI was examined using these two Gal4 drivers in wild-type embryos. With both GAL4 drivers an increase in the number of MG cells is detected, indicating that they are expressed at the right time and that the MG does not fail to secrete Spi once it has been processed (Bergmann, 2002).

A key regulator of Spi activation is rhomboid, a gene encoding a cell surface, seven-pass transmembrane protein that appears to function as a serine protease directly cleaving mSPI. rhomboid has been implicated in suppression of MG apoptosis. Ectopic expression of Rho in neurons (elav-Gal4/UAS-Rhomboid) promotes an excess of MG, suggesting that neurons have the capacity to process endogenous mSPI. Another essential protein for Spi processing is Star. Star mutants display an MG phenotype similar to spi. STAR regulates intracellular trafficking of mSPI. Expression of Star from the neurons but not from the MG rescues the Star phenotype in the MG. Thus, this analysis clearly demonstrates that the sSPI signal for MG survival is generated and secreted by neurons (Bergmann, 2002).

In vivo analysis of argos structure-function. Sequence requirements for inhibition of the Drosophila epidermal growth factor receptor

The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, since it is a secreted protein with a single epidermal growth factor (EGF) domain. An investigation was carried out to determine which regions of the Argos protein are essential for inhibition. A series of deletions were made and tested in vivo; analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-alpha-like factor), would disclose what makes one protein inhibitory and the other activating (Howes, 1998).

In one set of chimeras, the entire EGF domains of Spitz and Argos were swapped. The SAS (Spitz N-terminal; Argos EGF domain; Spitz C-terminal) and ASA pair (in which only the EGF domains themselves were swapped) were designed to test the possibility that the EGF domain is solely an EGFR binding domain, with no role in activation or inhibition. If this were the case, the EGF domains would be interchangeable and SAS might behave like Spitz, and ASA like Argos. In the SA and AS pair, the complete C termini of Spitz and Argos were swapped, including the EGF domains. If the Argos EGF domain and the adjacent C terminus were sufficient to confer the Argos protein's inhibitory function, the SA chimera (Spitz N terminus with the Argos EGF domain and C terminus) might be expected to behave like Argos. Similarly, since the N terminus of Argos appears not to contain sufficient information to confer inhibitory function, the AS chimera (Argos N terminus with the Spitz EGF domain and C terminus of the secreted form) might retain Spitz activity. However, neither SAS nor ASA has any activity; they neither suppress nor enhance the argos mutant phenotype. This suggests that the EGF domain is more than simply an EGFR binding domain; instead, it participates in specifying whether the receptor is activated or inhibited. SA also has no Argos activity (nor, as expected, is it Spitz-like). This implies that sequences N-terminal to the Argos EGF domain are necessary for its function. The AS chimera activates the EGF receptor, suggesting its function is similar to Spitz, not Argos. When overexpressed in the wing, it produces the characteristic extra vein phenotype of EGFR activation. In this same assay, overexpression of Argos causes the opposite phenotype, namely loss of wing veins. These results indicate that the large N-terminal region of Argos is not sufficient to convert Spitz into an inhibitor. AS, however, rescues spitz eye mutants only partially, suggesting that Spitz's potency is reduced by the addition of 355 amino acids of the Argos N terminus (Howes, 1998).

The spacing between the six essential cysteines in EGF domains varies only slightly. In particular, between C3 and C4 (the B-loop), there are usually 10 amino acids in EGF-like ligands; the range is 8-13 in all known EGF repeats. In Argos, there are 20 residues between C3 and C4, and this extension might account for Argos's unusual inhibitory function. Indeed, although the function of this B-loop remains uncertain, many studies have implicated it in EGF binding and activation of the receptor. The C-loop of EGF domains, between cysteines 5 and 6, is also critical for the function of EGF-like ligands. In Argos, the C-loop is also atypical. It is only five residues long, compared with eight in Spitz and all the mammalian activating ligands, and it has two extra positive charges. To examine the functional importance of the Argos B- and C-loops, four chimeras were constructed between Argos and Spitz in which the B- and C-loops were exchanged, and tested for whether they activate or inhibit the EGFR. The extended B-loop is necessary for Argos function, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting this inhibitory function. Comparison of the argos genes from Drosophila and the housefly, Musca domestica, supports this structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors (Howes, 1998).

Spitz and Eye Development

Recessive spitz loss-of-function mutations affect compound eye development. In mosaic clones, spitz function is required in the differentiation of the R8 photoreceptor cells, the first of eight photoreceptor cells to differentiate in each ommatidium. spitz loss-of-function mutations are dominant suppressors of EgfrE gain-of-function mutations of the epidermal growth factor-receptor gene. These data suggest that the spitz product is a precluster promoting factor. spitz transcription increases abruptly in the morphogenetic furrow, the obverse of Egfr expression (Tio, 1994).

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homolog (Egfr) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. This phenotype suggests a possible role for Egfr in specifying the founder R8 photoreceptor cells for each ommatidium. Ellipse mutations have been used to probe the role of Egfr in eye development: Elp mutations result from a single amino acid substitution in the kinase domain, which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirm that the mutation is hypermorphic in vivo, but the Egfr function is elevated less than by ectopic expression of the ligand Spitz. Ectopic Spi promotes photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of Egfr activation were assessed to explore the basis of these distinct outcomes. Elp mutations cause overexpression of the Notch target gene E(spl) mdelta and require the function of Notch to suppress ommatidium formation. E(spl) mdelta is known to be expressed in cell clusters in the morphogenetic furrow of wild type eyes. The Elp phenotype also depends on the secreted protein Argos and therefore, in Elp;aos double mutants, the phenotype is reverted. Complete loss of Egfr function in clones of null mutant cells leads to delay in R8 specification and subsequently to the loss of mutant cells. The Egfr null phenotype is distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands is required for aspects of Egfr function. No delay in ato refinement is found for spi mutant eyes, and vn mutants have vestigial eye discs that usually fail to differentiate any type of eye cell. In normal development, Egfr protein is expressed in most retinal cells, but at distinct levels. Antibody specific for diphospho-ERK as well as expression of the Egfr target gene argos was used to assess the pattern of Egfr activity; highest activity was found in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggest that this activity may not be required for normal ommatidium development. Since distinct phenotypic effects for four different levels of Egfr activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function are seen, it is proposed that multiple thresholds separate several aspects of Egfr function. These include activation of N signaling to repress R8 specification; turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation. It is suggested that Egfr activation by Spi is the only signal that the R8 cell needs to provide for most photoreceptor cells and that ubiquitous targeted Spi expression converts every retinal cell to an R1-R7 photoreceptor, at the expense of R8 and other cell types. Argos is required for loss of ommatidia in Elp. Nonautonomy of Elp mutations suggests that aos acts nonautonomously to inhibit ommatidium formation (Lesokhin, 1999).

Receptor tyrosine kinases such as the EGF receptor transduce extracellular signals into multiple cellular responses. In the developing Drosophila eye, Egfr activity triggers cell differentiation. This study focuses on three additional cell autonomous aspects of Egfr function and their coordination with differentiation, namely, withdrawal from the cell cycle, mitosis, and cell survival. Whereas differentiation requires intense signaling, dependent on multiple reinforcing ligands, lesser Egfr activity maintains cell cycle arrest, promotes mitosis, and protects against cell death. Each response requires the same Ras, Raf, MAPK, and Pnt signal transduction pathway. Mitotic and survival responses also involve Pnt-independent branches, perhaps explaining how survival and mitosis can occur independently. These results suggest that, rather than triggering all or none responses, Egfr coordinates partially independent processes as the eye differentiates (Yang, 2003).

Focus was placed on three aspects of Egfr function that occur at two developmental stages. These are the withdrawal from the cell cycle that accompanies the first fate specifications and the mitosis and survival of cells that pass unspecified through a 'second mitotic wave' (SMW) before later recruitment to retinal cell fates. The onset of advancing differentiation is defined by a morphogenetic furrow, which sweeps anteriorly from the posterior part of the eye imaginal disc. Just anterior to the morphogenetic furrow, cells arrest in G1 of the cell cycle. Within the morphogenetic furrow some of the arrested cells are specified as individual R8 photoreceptor cells, the founders of each ommatidium. Each R8 cell then produces ligands that activate Egfr in four neighboring cells. These neighbors are recruited to become the R2, R3, R4, and R5 photoreceptor cells of each ommatidium. While these five cells maintain their G1 arrest and differentiate in response to Egfr activity, the surrounding cells in which Egfr is inactive reenter the cell cycle and begin S phase DNA synthesis. Entry into this SMW occurs around retinal column 1, also the stage at which 'preclusters' of R8, R2, R3, R4, and R5 cells first become morphologically recognizable. The SMW produces unspecified cells that will be recruited to take the remaining 14 retinal cell fates. Egfr is required for survival and G2/M progression of SMW cells as well as later cell fate specification (Yang, 2003).

R2-5 differentiation requires the Egfr ligand Spitz (Spi) and the Ras/Raf/MAPK pathway. Spitz is not required to maintain R2-5 in G1, even though experiments with a temperature-sensitive allele show that Egfr is required simultaneously to maintain G1 arrest and to initiate differentiation. Any function of the neuregulin-like Egfr ligand Vein (Vn) in cell cycle withdrawal was ruled out from the study of spi; vn double mutants. The role of another zygotic ligand, Keren (Krn), was evaluated through mutations in rhomboid1 (rho) and rhomboid3 (a.k.a. roughened [ru]), two proteases required to cleave transmembrane precursors of the Keren and Spitz proteins. Cell cycle progression was assessed in clones of homozygous ru; rho1 double mutant cells with Cyclin B as a marker. Cyclin B protein accumulates in cells that have passed the G1/S checkpoint but not yet divided. All cells except R8s reentered the cell cycle in ru; rho1 double mutant clones. It is inferred that Krn must be necessary and sufficient to maintain R2-5 cells in G1 in the absence of spi (Yang, 2003).

To investigate the difference between Egfr activation by Spi and by Krn, whether the Ras/Raf/MAPK/Pnt pathway is required to maintain G1 arrest was examined. Mosaic analysis of null alleles was used. For Ras and Raf the result was the same as that for Egfr. All these genes are required to prevent cells from reentering the cell cycle. It was not possible to generate clones of cells lacking MAPK using the FLP/FRT system for mosaic analysis because of the centromeric location of the MAPK gene rolled proximal to all FRT transgene insertion sites. The transcription factor Pnt is the next component downstream in photoreceptor differentiation. Only R8 cells remain in G1 in cells deleted for the entire pnt gene. Thus, keeping R2-5 in G1 requires Egfr signaling through the same Ras/Raf/Pnt pathway as taken during differentiation (Yang, 2003).

Since spi activates the Ras/Raf/MAPK/Pnt cascade, it was asked whether Spitz could also maintain cells in G1. A secreted Spi protein (sSpi) that does not require Rho function was overexpressed to test this. The GMRGal4 driver was used to target expression posterior to column 1 of the eye disc. The GMR>sSpi eye discs lacked any secondary mitotic wave (SMW). Neither mitotic figures nor cells reentering the cell cycle were seen. Thus, not only does maintaining R2-5 cells in G1 require the same effectors as differentiation, but the differentiation ligand Spi can also maintain G1 arrest (Yang, 2003).

Spitz and Mesoderm Development

Continued: Spitz Effects of Mutation part 2/2

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