Star

Effects of Mutation or Deletion

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 l’sc, 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 ‘cyclops’ 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 Drosophila Malpighian tubules (MTs), form a simple excretory epithelium comparable in function to kidneys in vertebrates. MTs function as the insect kidney both in the larva and the adult. They consist of two pairs of blind ending tubes that are composed of a single cell-layered epithelium made up of a tightly controlled number of cells. The tubules float in the hemolymph from where they take up nitrogenous waste that is excreted as uric acid. During embryogenesis, MTs evert as four protuberances from the hindgut primordium, the proctodeum. The everting tubules grow by cell proliferation, which takes place in a few cells along the tubules and extensively in a distal proliferation domain located in the tip region of the tubules. Cell ablation experiments and studies on the pattern of cell division have shown that a single large cell at the distal end of each tubule, termed the tip cell, is decisive for controlling the proliferation of its neighboring cells. The tip cell that differentiates into a cell with neuronal characteristics during later stages of development arises by division of a tip mother cell that is selected in the tubule primordium by lateral inhibition involving the Notch signaling pathway and the transcription factor Krüppel (Kr). It has been suggested that the tip cell sends a mitogenic signal to adjacent cells in the distal proliferation zone. It has remained elusive, however, what the signal is or what its target molecules in the signal-receiving cells could be and how cell proliferation during MT morphogenesis is regulated. Seven-up is shown to be a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up (Svp) in turn is capable of regulating the transcription of cell cycle regulators (Kerber, 1998).

To identify the nature of the mitogenic tip cell signal a screen was carried out for genes specifically active in the tip cells. The genes rhomboid (rho) and Star (S), which encode transmembrane proteins involved in epidermal growth factor receptor (EGFR) signaling, are expressed in the tip cells and both are required for MT growth. When the tubules start to evert, rho and S are expressed in the tip mother cell; subsequently rho is strongly expressed in the tip cell and S in the tip cell and its former sister cell. An analysis of the MTs in the corresponding amorphic mutants reveals a strong decrease of cells in rho mutants and a weaker decrease in S mutants. In a rho;S double mutant, the tubules are barely detectable, indicating that rho and S activities are essential (albeit redundant) components controlling MT growth. The tubule phenotype of rho;S double mutants is very similar to that of EGFR mutants, which also show a drastic decrease in the tubule cell number. As in svp mutants, the allocation and the differentiation of the tip cells are normal in the receptor mutants, indicating that receptor activity is not required for tip cell determination and differentiation. The reduction of the tubule cell number in EGFR mutants is due to a failure of proper cell divisions. No BrdU incorporation occurs in EGFR mutants in the outbudding tubules at the time when cells divide in wild-type embryos. However, BrdU incorporation occurs again much later during the endomitotic cycles, indicating that in EGFR muants, a specific defect in DNA replication exists in cells that would normally divide (Kerber, 1998).

Rho and S process a membrane-bound form of the activating ligand of the receptor, the TGFalpha-like Spi protein, to generate the secreted form of Spi (sSpi). sSpi is then proposed to diffuse to neighboring cells, bind to the receptor, and activate target genes via the Ras/Raf signaling cassette; these include the primary target gene pointedP1 (pntP1), encoding an ETS domain transcription factor, and the secondary target gene argos (aos), encoding a negatively acting ligand of the receptor. These downstream components of the pathway are also active during tubule development. pntP1 and aos are expressed during stage 10 in six to eight cells on one side of the MTs overlapping the rho and S expression domains and later, weakly in several cells in the tip region. In amorphic aos mutants a slightly larger number of tubule cells are observed, whereas amorphic pnt mutants show a decrease of tubule cells. These results indicate that for controlling cell proliferation and cell determination, the same key components of the EGFR cascade are required (Kerber, 1998).

These findings suggest that the EGFR pathway provides the mitogenic tip cell signal that activates svp expression and regulates cell division. To test this hypothesis, svp expression was analyzed in EGFR mutants and ectopic expression studies were performed with various members of the pathway using the UAS-Gal4 system. svp is absent in mutants for the Egfr. It is still expressed, however, in amorphic pnt mutants, suggesting that Svp is a transcriptional regulator that is likely to be activated in parallel to the primary transcription factor PntP1 in the signaling cascade. If sSpi activity is provided ectopically in all of the tubule cells, the svp expression domain becomes dramatically expanded and an increase of the tubule cell number is observed. Similar, although slightly weaker effects on svp transcription and the number of tubule cells could be observed upon ubiquitous expression of other components of the EGFR pathway, like Rho, activated Ras, or Raf. Conversely, when a dominant-negative Ras allele is ectopically expressed in all of the tubule cells, svp transcription became strongly reduced. Ectopic expression of svp in an Egfr mutant background restores the tubule cell number to a considerable extent. These results provide strong evidence that svp is a downstream target gene of EGFR signaling in the tubules (Kerber, 1998).

Loss-of-function mutations in Star impart a dominant rough eye phenotype. When homozygous they are embryonic lethal, producing ventrolateral cuticular defects. Star interacts genetically with mutation in Spitz/EGFR and sevenless, both of which function in the eye (Kolodkin, 1994).

In fused commissure mutants (rhomboid and Star) neuron number is reduced in the ventral unpaired median neuron lineage and the median neuroblast lineage before commissure formation (stage 12). Subsequent to these neuronal defects, the midline glia die by apoptosis (stage 13). Commissure fusion and glial apoptosis seems to be triggered by the earlier perturbations in neuronal lineages (Sonnenfeld, 1994).

Star interacts with Ras1 in both eye and wing morphogenesis (Heberlein, 1993). The onset of pattern formation in the developing Drosophila eye is marked by the simultaneous synchronization of all cells in the G1 phase of the cell cycle. These cells will then either commit to another round of cell division or differentiate into neurons. roughex functions as a negative regulator of G1 progression in the developing eye. rux is suppressed by mutations in genes that promote cell cycle progression (i.e., cyclin A and string) and enhanced by mutations in genes that promote differentiation (i.e., Ras1 and Star) (Thomas, 1994).

The results of mutation to any one of the spitz group genes (zygotic genes spitz, Star, single-minded, pointed, and rhomboid , and maternal gene sichel ), compared to any other in the group, yields similarly distinctive pattern alterations in embryonic ventral ectodermal derivatives. The cuticle structures lacking in mutant Drosophila embryos normally derive from longitudinal stripes of the ventro-lateral blastoderm. Defects are found in the median part of the central nervous system. These results suggest that groups of cells from the same region, including both epidermal and neural precursor cells, require spitz-group gene activity for normal development. The members of the spitz group differ from one another: sim affects a more median stripe of the ventral ectoderm than the other zygotic genes and pnt causes separation rather than deletion of pattern elements. As shown by pole cell transplantations, spi and S are also required for normal development of the female germ line (Mayer, 1988).

Star encodes a putative transmembrane protein that is a critical component of the Epidermal growth factor receptor tyrosine kinase signaling pathway. A new dominant allele of Star, termed Star^Kojak, is described that alters Epidermal growth factor receptor tyrosine kinase signaling in the follicle cells and in the eyes in Drosophila. Star^Kojak was isolated in a screen for follicle-cell-dependent dominant female sterile mutations. In addition to an egg and eye phenotype, Star^Kojak mutants produce a bristle phenotype. Interommatidial bristles are mis-aligned and do not point in the same direction. Also, the long bristles, such as the dorsocentral and scutellar, are short and bent in these flies. Because the short-bristle phenotype is the most readily apparent, the mutation was named Kojak, after the bald title character in a television series. It was renamed Star^Kojak after determining that it is allelic to Star. Star^Kojak and revertants of Star^Kojak do not complement Star loss-of-function mutations. It is proposed that Star^Kojak has both dominant gain-of-function phenotypes early in development and dominant loss-of-function phenotypes later in development. Early in oogenesis, STAR mRNA expression is higher in Star^Kojak egg chambers than in wild-type egg chambers, consistent with its gain-of-function phenotype. Later in oogenesis, STAR mRNA expression is lower in Star^Kojak follicle cells than in wild-type follicle cells, consistent with its loss-of-function phenotype. By genetically analyzing Star^Kojak and its revertants, evidence is presented that Star is involved in anterior-posterior axis formation both in the female germline cells and in the somatic follicle cells. At least part of the dominant female sterile phenotype of Star^Kojak is restricted to the posterior-pole follicle cells. It is proposed that Star functions by processing pro-Gurken to mature Gurken, which is thereby released in the region between the oocyte and the follicle cells and binds to the Epidermal growth factor receptor in the follicle cells (Ruden, 1999).

Based on the results presented in this paper, a model is proposed to explain how Star protein both in the follicle cells (FC) and in the oocyte activates Egfr signaling in wild-type egg chambers. In this model, the presence of Star protein in the cytoplasmic membrane in both the posterior of the oocyte and the posterior FCs is required to process pro-Grk from an inactive transmembrane form to an active secreted form in stage 5 egg chambers. The later expression of Star protein in the dorsal anterior FCs could be required to activate Grk in the dorsal-anterior FCs in a similar manner. In Star^Kojak/+ germaria and stage 1 egg chambers, Star protein is overexpressed in both the oocyte and the FCs, thus, it is proposed, causing Grk to be processed and to activate the Egfr in all of the FCs by diffusion. The stage 1 egg chamber is very small compared with the stage 10 egg chamber, and it is possible that complete processing of pro-Grk to Grk at the posterior pole of stage 1 egg chambers will allow sufficient Grk to activate Egfr signaling in all of the FCs (Ruden, 1999).

In the eye, Star^Kojak allows photoreceptor R7 to development in the absence of Sevenless receptor tyrosine kinase. It is possible that Star^Kojak constitutively activates the pro-Spitz ligand, thus activating Egfr in the R7 photoreceptor cells and suppressing the sevenless-mutant phenotype. Intriguingly, when eyes that express a dominant negative allele of Ras are also mutant for Star^Kojak, there are often as many as three of four extra R7 photoreceptor cells in each ommatidium. However, Raf^HM7 (a temperature sensitive hypomorphic allele of Raf) and Star^Kojak double-mutant eyes have the opposite phenotype, namely, very few of the ommatidia have any R7 photoreceptor cells. It is believed that these opposite phenotype reflect the early gain-of-function phenotype of Star^Kojak and the late loss-of-function phenotype of Star^Kojak. Consistent with this idea, reiterative use of the Egfr is thought to trigger differentiation of all cell types in the eye (Ruden, 1999).

Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila Egf-Receptor; spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid and Star (S) encode membrane proteins. This study shows that rho-mediated hyperactivation of the Egfr/MAPK pathway is required for vein formation throughout late larval and early pupal development. Consistent with this observation, rho activity is necessary and sufficient to activate MAPK in vein primordium during late larval and early pupal stages. Epistasis studies using a dominant negative version of Egfr and a ligand-independent activated form of Egfr suggest that rho acts upstream of the receptor. rho and S function in a common aspect of vein development since loss-of-function clones of rho or S result in nearly identical non-autonomous loss-of-vein phenotypes. Furthermore, mis-expression of rho and S in wild-type and mutant backgrounds reveals that these genes function in a synergistic and co-dependent manner. In contrast, spi does not play an essential role in the wing. These data indicate that rho and S act in concert, but independent of spi, to promote vein development through the Egfr/MAPK signaling pathway (Guichard, 1999).

rho and S were initially identified based on similar embryonic loss-of-function phenotypes, suggesting that they are involved in a common molecular process. Moreover, S is the most potent known dominant suppressor of rho-induced extra-vein phenotypes. Further support for a close partnership between these two genes has been provided in this study. In the wing, loss-of-vein phenotypes caused by rho- or S- clones are very similar, but different from phenotypes associated with clones of mutants in the Egfr signaling cassette. For example rho- and S- clones exhibit local cell non-autonomy, in contrast to Egfr - clones. Furthermore, cells in rho - or S - clones have normal viability and size, whereas mutant clones lacking Egfr or downstream components have reduced cell size and viability. Thus, loss-of-function analysis reveals that rho and S define a subgroup of genetic functions required for strong EGFR signaling, distinct from activities in the EGFR/MAPK pathway proper (Guichard, 1999).

The relationship between rho and S was addressed in series of epistasis experiments. Overexpression of S, which alone is unable to cause any phenotype in the wing, generates a strong ectopic vein phenotype only when ectopic rho is provided, showing that S needs rho to function in intervein regions. Clonal analysis indicates that S also depends on endogenous rho expression in veins, since overexpression of S in the absence of rho cannot rescue vein formation. Reciprocally, strong ectopic rho expression cannot generate any phenotype in wing clones lacking S. Collectively, these data indicate that rho and S function co-dependently, and collaborate to activate Egfr signaling by a common molecular mechanism (Guichard, 1999).

Although rho and S mutant phenotypes are similar during many stages of development, the co-dependence of rho and S does not seem to apply to the eye. In the eye imaginal disc, S is required for cell viability and generates dominant morphological defects in the heterozygous condition, while clones lacking rho have no obvious phenotype. This difference between rho and S function in the embryo and wing, versus in the eye, could be explained by the possible existence of other Rho-like proteins interacting with S during eye development, or might reflect the ability of S to function in the absence of Rho in certain cellular contexts (Guichard, 1999 and references).

Two classes of models have been proposed to explain the activity of Rho at the molecular level. In the first type of model, Rho activates a separate signaling pathway that ultimately converges on the RAS/MAPK pathway. This class of models accounts for the fact that in most situations, rho is required in the cells in which it is expressed. An exception to this rule is in embryonic chordotonal organs, where rho is expressed in the sensory organ precursor cell, but activates MAPK only in surrounding epidermal cells. In a second class of models, Rho produces an extracellular signal that activates Egfr. The current data are consistent with aspects of both models, but do not support the specific proposal that Rho promotes the processing of an m-Spi precursor into a diffusible active form. According to this latter model, spi- loss-of-function clones would be expected to induce phenotypes equivalent to or stronger than those observed in rho- or S- clones, particularly in a vn¹ mutant background. Since spi- clones have no detectable effect, the data argue against the 'Spi processing' model in the context of wing vein development. Even if Spi produced in wild-type cells were able to diffuse and rescue the vein-loss phenotype of spi- clones, the fact that small rho - or S - clones induces vein-loss phenotypes argues strongly against the possibility that Rho acts through the processing or the activation of m-Spi. In contrast, the ability of m-Spi to enhance and sharpen the vein phenotype caused by ectopic Rho and Star proteins suggests that Rho and Star can collaborate with m-Spi to generate a stronger and more localized signal. The inability of m-Spi to induce any phenotype in the absence of ectopic Rho and Star proteins suggests that m-Spi may require a prior action of Rho and Star to activate the Egfr in this artificial situation, whereas s-Spi or Vein do not. It is also very unlikely that Rho promotes the processing of Vein, since the Vein ligand is not transmembrane bound and does not require a cleavage to function. Nevertheless, it is possible that Rho and S support the processing or facilitate the presentation of an unknown Egf ligand in the wing, or promote transcytosis of Egf ligands or ligand-receptor complexes. The drawback to these last two hypotheses is that they involve an as yet unidentified molecule(s). Alternatively, S may constitute the missing link between Rho and Egfr. S could be a precursor for a diffusible factor, which ultimately activates Egfr, either as a ligand, or as a co-ligand. Such a co-ligand would reinforce or coordinate the effect of EGF ligands, possibly by promoting the formation of receptor oligomers. According to this scenario, Rho might act by promoting the processing of a membrane tethered S precursor into an active diffusible form. Finally, Rho and S could be acting directly on the Egfr (i.e. on the extracellular domain of the receptor itself) of the cells expressing Rho, and sometimes also of the adjacent cells (e.g. Rho could promote receptor dimerization or aggregation and thereby enhance the Egfr signal). Although this last hypothesis would not account for rho action over more than one cell diameter, it could explain why rho generally has a greater effect and ability to promote MAPK activation in cells expressing rho. Additional biochemistry experiments will be required to understand the basis for the non-autonomous action of Rho, and its predominant action in cells in which it is expressed (Guichard, 1999 and references).

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).

The synthesis of dorsal eggshell structures in Drosophila requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. Advantage of this process was taken to identify genes that link patterning and morphogenesis; lethal mutations on the second chromosome were screened for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. These genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61ß, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway: Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a 'neurospecific' receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype (Schnorr, 2001).

Ras1 signaling downstream of the Egfr, Torso, and Sev RTKs has been studied extensively in the fly. Consequently, the recovery of mutations in second chromosomal genes known to function in Ras1 signal transduction was expected, as well as genes necessary for the D/V patterning of the eggshell. Indeed, l(2)k05115, an allele of Egfr was recovered. Another Ras1 pathway member identified is Star, a member of the Spitz group of genes that functions during Egfr-mediated formation of the embryonic ventral midline. Star mutations appear repeatedly in screens for RTK-related eye phenotypes and these Star alleles suppress gain-of-function mutations affecting Egfr and sev signaling pathways. In addition, a dominant female sterile allele, Star^Kojak, produces phenotypes that suggest a dual function for Star in A/P and D/V patterning in oogenesis. Finally, Star encodes a single-pass transmembrane protein; recent evidence supports the hypothesis that Star is involved in processing the Egfr ligands Spitz and Gurken. Both the recovery of Star and epistasis tests with lambdatop (which place Star upstream of or in parallel with Egfr) support these previous findings (Schnorr, 2001).

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 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).

The specification of bract cells in Drosophila legs has been analyzed. Mechanosensory bristles induce bract fate in neighboring epidermal cells, and the RAS/MAPK pathway mediates this induction. Spitz and EGF receptor have been identified as the ligand and the receptor of this signaling; by ubiquitous expression of constitutively activated forms of components of the pathway it has been found that the acquisition of bract fate is temporally and spatially restricted. The role of the poxn gene in the inhibition of bract induction in chemosensory bristles has also been studied (del Álamo, 2002).

Drosophila legs are covered by a constant and leg-specific pattern of different types of external sensory organs, mainly mechanosensory (MB) or chemosensory (ChB) bristles. Bristles on the legs can be classified by the presence of bracts. Bracts are small epidermal structures that appear associated to MB in specific places on adult femur, tibia and the tarsal segments of all legs. Bracts appear on the proximal side of the bristles, and share the same polarity. Bracts are also present in the proximal costa of the wing, showing the same morphology as in the leg (del Álamo, 2002).

Are bristle and bract related by lineage? Sensory organ precursors (SOPs) undergo a specific pattern of cell divisions that give rise to four cells: two epidermal cells, the shaft and socket, and two neural cells, a neuron and a sheath cell. Previous clonal analyses of leg disc have suggested a lack of lineage relationship between bristles and bracts. These results were confirmed by labelling clones of cells induced in early third instar larvae; the bract cell does not belong to the SOP lineage (del Álamo, 2002).

How is bract fate specified? The results indicate that the acquisition of bract fate is controlled at three levels. One level of control takes place in the receptor cell, where the competence to acquire bract fate is spatially and temporally controlled. Ubiquitous expression of activated Raf provided in short pulses of time indicated that the competence to acquire bract fate is spatially restricted to specific regions of imaginal discs. There is also a temporal restriction to early pupal development, with peak competence between 8-12 hours APF. These results are consistent with there being a temporally and spatially restricted expression of a tissue-specific regulator that gives the receptor cell the competence to activate bract fate (del Álamo, 2002).

Another level of control occurs in the bristle cell that sends the inductive signal. Spi protein requires the functions of rho1 and S genes to be processed into a soluble, activated form. S and spi are ubiquitously expressed, and rho1 is expressed in SOP cells. The phenotype of rho1 mutant cells in clones indicates that rho1 is required for the induction of bract fate. Nevertheless, rho1 is also expressed in bract-less ChBs, and ubiquitous overexpression of S and rho1 results in a mild phenotype of extra bracts in wild-type positions. Together these results suggest that another component, whose expression must be restricted to the SOP of MBs, is required for bract induction (del Álamo, 2002).

A long-standing mystery in Drosophila has been how certain bristles induce adjacent cells to make bracts (a type of thick hair) on their proximal side. The apparent answer, based on loss- and gain-of-function studies, is that these bristles emit a signal that neighbors then transduce via the epidermal growth factor receptor pathway. Suppressing this pathway removes bracts, while hyperactivating it evokes bracts indiscriminately on distal leg segments. Misexpression of the diffusible ligand Spitz (but not its membrane-bound precursor) elicits extra bracts at normal sites. What remains unclear is how a secreted signal can have effects in one specific direction (Held, 2002).

The EGFR pathway is involved in the development of ommatidia of the fly eye via dosage-sensitive interactions between loss-of-function (LOF) alleles of Star and Ras1. If the EGFR pathway were instrumental in bract development, then those same alleles might be expected to also manifest dosage effects on the frequencies of bracts. Indeed, they do. Star⁵⁶⁷¹/+ heterozygotes have a missing-bract phenotype (31%Ti and 89%Ba), which is aggravated slightly in deficiency heterozygotes such as Df(2L)ast4/+ (17%Ti and 78%Ba). In contrast, Ras1^e1B/+ heterozygotes look nearly wild-type (96%Ti and 100%Ba). The double heterozygote shows synergistic effects: Star⁵⁶⁷¹/+; Ras1^e1B/+ flies have fewer bracts than either heterozygote alone (2%Ti and 60%Ba). In each of the above genotypes, the Ti was more strongly affected than the Ba. This disparity was seen in other contexts as well. Another trend in differential sensitivity was found among the basitarsal bristle rows: dorsal bristles tend to lose bracts more readily than ventral ones (Held, 2002).

If all epidermal cells are competent to make bracts in response to EGFR stimulation, then it should be possible to fool them into 'thinking' that they have 'heard' a signal (when in fact they have not) by activating the pathway downstream of the receptor. For this purpose, a constitutively active Ras1 transgene was used under the control of a heat shock promoter. When hs-Ras1*^M11.2 males are heat-shocked at any time from 5 to 27 h AP, their legs acquire extra bracts. On the Ti, these excess bracts are patchily distributed. On the Ba, the bracts are also patchy (mainly found near bristles) for shocks between 11 and 27 h AP, but earlier shocks (5-10 h AP) typically yield a confluent lawn of unpigmented bracts (Held, 2002).

Since Star acts upstream of the EGF receptor, the missing-bract defect of Star⁵⁶⁷¹/+; Ras1^e1B/+ heterozygotes should be rescueable by hyperactivating Ras1. When the hs-Ras1*^M11.2 transgene is introduced and the resulting Star⁵⁶⁷¹/hs-Ras1*^M11.2; Ras1^e1B/+ pupae are heat-shocked during the extra-bract sensitive period (24 h AP), a partial rescue is indeed observed. The shocked flies have significantly more bracts (30%Ti and 59%Ba) than their unshocked control siblings (0%Ti and 40%Ba) (Held, 2002).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Star, rhomboid, and spitz belong to the 'spitz' group of genes and encode an essential function necessary for ventral midline development. In addition to the recessive lethal embryonic phenotype, S mutations are haplo-insufficient and show a dominant, rough eye phenotype. During development, S is required in a wide variety of tissues and S mutations show genetic interactions with genes from multiple signaling pathways. S encodes a putative membrane protein that, in combination with Rhomboid (rho), participates in the processing of the EGFR ligand, Spitz. In the modifier screen an S deficiency was identified as a strong enhancer of the rugose rough phenotype. A number of S alleles have been tested for their interactions with multiple alleles of rugose. Mutations at the S locus act as strong enhancers of the rugose eye phenotype and conversely heat-shock promoter-driven overexpression of the wild-type Star protein acts as a dominant suppressor of the rugose mutant phenotype. Consistent with these results, rhomboid (rho) mutations act as dominant enhancers of the rough eye phenotype of rugose mutants. rho encodes a novel intramembrane serine protease and is involved in the proteolytic processing of the EGFR ligand Spitz. A single copy of the rho mutation acts as an enhancer of the rg eye phenotype and a single copy of the hs-rho acts as a weak suppressor (Shamloula, 2002).

Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90°, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Epidermal growth factor receptor (Egfr) signalling is required for rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. It is proposed that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, there is an error-correction mechanism operating during pupal stages to reposition inappropriately oriented ommatidia. These results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling (Brown, 2003).

The Egfr ligand Keren was misexpressed in developing photoreceptors and cone cells under the control of sev-Gal4. Surprisingly, this caused a disruption in the orientation of ommatidia relative to WT, a phenotype not previously associated with excess Egfr signalling. In the WT adult eye, all ommatidia are oriented at 90° relative to the equator. By contrast, when Keren is misexpressed, many ommatidia are abnormally oriented, with some ommatidia having rotated more than 90° and some less than 90°. In general, excess Egfr signalling leads to over-recruitment of cells in the eye, but photoreceptor recruitment is not affected when Keren is expressed at these levels. However, analysis of the pupal retina shows that Keren misexpression causes over-recruitment of cone cells, consistent with it acting through the Egfr. Previous work has shown that recruitment of cone cells is more sensitive than photoreceptors to Egfr overactivation; these results support this view, and also suggest that rotation is more sensitive than photoreceptor recruitment to perturbation of Egfr signalling (Brown, 2003).

Further examination of the adult phenotype indicates that it is rotation specifically that is disrupted on overexpressing Keren; the chirality (i.e. the correct specification of R3 and R4) of the ommatidia remains unaffected. This distinguishes the UAS-Keren phenotype from disruption of PCP components, which can cause both rotational and chiral defects (Brown, 2003).

Is Egfr activity normally required for correct rotation? Several conditions were examined that decrease Egfr signalling, including a haploinsufficient Star allele (which has slight rotational defects), rho3/roughoid mutants, and expression of dominant-negative Egfr under the control of heatshock HS-Gal4. In all these cases, rotational defects are clearly seen in correctly specified ommatidia. In order to quantify and compare the rotational defects further, the rotation angles of approximately 600 ommatidia each were measured in WT, UAS-keren and ru¹ eyes. Strikingly, defects caused by too little or too much Egfr activity are very similar -- ommatidia are over- or under-rotated, although in both cases there is a bias towards rotation angles of greater than 90°. The similarity of the rotational defects caused by increasing and decreasing pathway activity is reminiscent of some PCP mutations (Brown, 2003).

The rotational phenotypes caused by perturbation of Egfr signalling are very similar to the published phenotype of the roulette mutation, one of the few mutations reported to specifically disrupt rotation and not chirality. Interestingly, roulette turns out to be allelic to argos. The roulette mutation is now referred to as argos^rlt (Brown, 2003).

There are four ligands that activate the Drosophila Egfr: Spitz, Gurken and Keren (which resemble mammalian TGFalpha), and Vein, a neuregulin-like molecule. Spitz is thought to mediate most of the Egfr functions in eye development, although spitz clones do not phenocopy Egfr clones in all respects. Specifically, spitz clones do not show defects in cell survival or ommatidial spacing, which are seen in Egfr loss-of-function clones. spitz hypomorphic eyes were examined to determine whether these show rotational defects. Under-recruited ommatidia are very common in the spi^scp1 hypomorph, indicating that Egfr activity is substantially impaired – to beneath the threshold for photoreceptor recruitment. Despite this, very few misrotated ommatidia are seen. In comparison, ru¹ eyes show only minor recruitment defects, indicating a less dramatic reduction of Egfr activity than spi^scp1. ru¹ eyes, however, show severe rotational defects. These data suggest that Spitz is not essential for normal rotation. They do not, however, rule out the possibility that Spitz acts redundantly with another ligand. To test this, a genetic interaction between Star and a spitz hypomorph was tested. As expected, heterozygosity for spitz enhances the recruitment defects in the S/+ eye. A significant enhancement of rotational defects is observed, implying that Spitz does function in ommatidial orientation. Together, these results suggest that Spitz acts redundantly with another Egfr ligand to control rotation. The fact that loss of Rho3/ru, a protease that activates Egfr ligands, results in rotational defects, whereas spitz mutants do not, implies the involvement of another cleaved ligand. Gurken is restricted to the germline. By elimination, it is therefore tentatively concluded that Keren also acts in the Egfr-dependent regulation of ommatidial rotation. Note, however, that keren expression is too low to detect by in situ hybridisation in any tissue so it is not possible to tell whether keren is transcribed appropriately. Confirmation of this hypothesis awaits the identification of a keren mutant (Brown, 2003).

These results demonstrate that Egfr signalling is required for the maintenance through eye development of the correct orientation of ommatidia. It was speculated that rotation may rely at least partly on the adhesive properties of the cells. In an initial attempt to examine this hypothesis, genetic interactions between components of the Egfr pathway and various adhesion molecules were sought. A Star heterozygote, in which Egfr signalling is slightly reduced, was used as a background in which to look for interactions, because this phenotype is very weak, allowing any enhancement of rotational defects to be easily recognized. Halving the dose of alpha-laminin (wing blister) and the integrin ß subunit (myospheroid) does not modify the Star/+ phenotype. In contrast, alleles of E-cadherin (shotgun) shows a significant interaction with Star, with many more misrotated ommatidia. Under the strongest condition, there is also an enhancement of the rare misrecruitment defects seen in Star/+ eyes, but the enhancement of the rotational defect is independent of this by two criteria. First, the rotational defects were only measured in correctly specified ommatidia; and second, the weaker alleles of shotgun affected rotation without enhancing recruitment. On the basis of these results, it is concluded that the control of rotation by Egfr signalling is linked to cadherin-based adhesion (Brown, 2003).

A model that might account for these results is proposed that suggests that the role of Egfr signalling is to establish a 'locking' mechanism that ensures that ommatidia remain in their final orientation. Such a mechanism might be necessary to protect the ommatidia against positional disruption during later events in eye development. Signalling would therefore be required during or at the end of normal rotation in order to set in place this hypothetical 'lock', although defects might not arise until significantly later than this, when processes occur that would cause ommatidia to reorient in the absence of such a lock (Brown, 2003).

The jing and ras1 pathways are functionally related during CNS midline and tracheal development as revealed by genetic interations with spitz and Star

The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. There is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells (Sonnenfeld, 2004).

The effect of a reduction in EGFR signaling on the jing gain-of-function phenotype was examined in the midline glia. sim-Gal4 and sli-Gal4 drivers were used to over-express jing specifically in the CNS midline in heterozygous and homozygous spi and S mutant backgrounds. The number of sli-lacZ-expressing midline glia in each nerve cord segment was quantified during stage 13 and compared to that in wild-type embryos over-expressing jing. Expression of two copies of the UAS-jing transgene in the midline glia of wild-type or heterozygous spi and S embryos resulted in an average of 12 midline glia instead of the normal 8 during stage 13. In contrast, UAS-jing transgene expression was unable to induce 12 midline glia in homozygous spi and S mutant backgrounds. In these embryos, there was an average of 1.5 midline glia in each nerve cord segment after jing over-expression; this is similar to the number of midline glia present in homozygous spi and S mutant embryos during stage 13 (Sonnenfeld, 2004).

The jing ectopic expression phenotype was dominantly suppressed by a 50% reduction in the levels of spi(spi¹) and Df(2L)TW50 or Egfr deficiency [Df(2R)Egfr5]. After spi reduction, ommatidia were more organized and more abundant, although the position of the photoreceptors was not like that in controls. This interaction was not influenced by activation of the glass promoter in the heterozygous spi background (P[GMR-Gal4]/spi¹). These results suggest that there is a dosage-sensitive interaction between the Egfr pathway and jing function in the eye, where increased jing activity can be suppressed by a reduction in downstream components such as spi and Egfr. Given that sim and trh are not expressed in third instar larval eye discs, these experiments suggest that jing can have an effect on the Egfr pathway in the absence of sim or trh and support the model that jing works as an independent regulator in bHLH-PAS pathways (Sonnenfeld, 2004).

Gene dosage experiments were used to determine the effects of simultaneously altering the levels of jing and genes of the Egfr pathway. Mutations in spi and its regulator Star, have been characterized for their midline and tracheal phenotypes. To determine whether jing and Egfr function is inter-dependent, the development of the CNS midline and trachea was analyzed in double heterozygotes of jing and S or spi. The basis for this experiment is that if the Egfr and jing pathways are inter-dependent then simultaneous reduction of only one copy of each gene should alter CNS midline and tracheal function. Multiple jing alleles balanced with wg-lacZ Cyo were crossed to S^IIN23/wg-lacZ Cyo or spi¹/wg-lacZ Cyo flies and their progeny were double stained with anti-Sim or anti-Trh and anti-β-Gal (Sonnenfeld, 2004).

The number of CNS midline cells was reduced from wild-type in embryos homozygous and double heterozygous for jing, spi or S and stained with anti-Sim. Since some of the Sim-positive nuclei appeared to be fragmenting, their fate was determined by TUNEL labeling to identify apoptotic cells. In wild-type embryos, cell death is uncommon in the CNS midline during stage 12 with an average of 6(±2) Sim-positive apoptotic nuclei per embryo. In contrast, in homozygous jing stage 12 mutant embryos, there was an average of 35(±3) Sim-positive apoptotic nuclei per embryo, therefore, displaying a significant increase over that in wild-type embryos. In embryos double heterozygous for mutations in jing and S or spi there was an average of 25(±2) and 30(±3) SIM-positive apoptotic nuclei per embryo during stage 12, respectively. This is consistent with the time period for the requirement of Egfr function in CNS midline glia. Embryos heterozygous for either jing, spi or S mutations did not alter the normal events of midline cell apoptosis. In summary, these results suggest that proper dosage of both jing and spi group gene function is required for midline cell survival (Sonnenfeld, 2004).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.