small wing



The mRNA of Small wing, Drosophila Plc gamma, is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole mount in situ hybridization, reveals that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes, mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation (Emori, 1994).

Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP

Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. This study used insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. A Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, this study describes mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection (Tsuzuki, 2014).

Effects of Mutation or Deletion

All three sl alleles are recessive, homozygous viable mutations that are classically described as having a modest reduction in wing length and a mildly 'rough' eye (disturbance to the crystalline array of facets in the compound eye) (Lindsley, 1992). In addition to these phenotypes, animals homozygous for any of the three sl alleles have ectopic wing veins, most frequently adjacent to vein L2, but sometimes also connected or adjacent to the posterior crossvein or within the posterior cell. Heteroallelic combinations of all three alleles fail to complement with respect to the rough eye or wing phenotypes. This combination of ectopic wing veins and rough eyes resembles defects previously observed in mutations that activate the Ras/MAPK cassette, such as the Ellipse alleles of Epidermal growth factor receptor (DERElpB1), the Sevenmaker alleles of rolled (rlSem) and mutations of Gap1. Because these mutations disrupt photoreceptor cell fate, the compound eye was examined from each of the sl alleles for photoreceptor defects that might underlie the observed roughness in the eye (Thackeray, 1998).

The wild-type Drosophila compound eye consists of about 800 hexagonal units, or ommatidia, packed in a crystalline array. In flies homozygous for any of the three sl alleles, this regular array is disrupted and many ommatidia are abnormally shaped. In addition, the sl eyes have interommatidial bristle defects: some bristles are duplicated, some are missing and some appear at additional vertices. Each wild-type ommatidium contains a precise assembly of eight photoreceptor cells (R1-R8) capped by four lens-secreting cone cells and surrounded by a sheath of pigment and bristle cells. The rhabdomeres (photosensitive organelles) of each of the six outer photoreceptor cells (R1-R6) can be seen in tangential sections, arranged in a trapezoidal ring around a central, smaller rhabdomere. In the distal part of the retina, the central rhabdomere corresponds to photoreceptor R7; in more proximal parts, it corresponds to the R8 cell. Tangential sections through the distal part of sl homozygous mutant eyes indicate that many ommatidia (51%) contain extra inner photoreceptors (i.e. with centrally located rhabdomeres). In addition, a small fraction of mutant ommatidia have one or more extra (2%) or missing (10%) outer photoreceptors. The distal position, central location and small rhabdomere size of the extra inner photoreceptors in the mutants suggests that they are R7 cells. To confirm their identity, the expression of an Rh4-lacZ fusion construct was examined in an sl mutant background. In wild-type, expression of the Rh4 opsin is limited to a random subset (~70%) of R7 cells. In sl mutant eyes, the majority of the supernumerary cells express the Rh4- lacZ gene, demonstrating that they have both the molecular and morphological characteristics of R7 photoreceptors (Thackeray, 1998).

Since the C2 and PH domains, which constitute potential regions of interaction with Ca2 + and IP4, are required for GTPase-activating protein 1 in vivo activity, it became of interest to see whether genes involved in Ca2+ and phosphoinositide signaling regulate Gap1 function in vivo. One such candidate is the Drosophila gene small wing, which encodes a phospholipaseCgamma (PLCgamma) homolog. PLCgamma comprises a catalytic core, two PH, two SH2, and one SH3 domains and a conserved tyrosine phosphorylation site, all of which act as signal-dependent regulators of enzymatic activity. Subsequent to RTK stimulation, PLCgamma is recruited to the receptor through its SH2 domains and then itself activated by tyrosine phosphorylation. Upon activation, PLCgamma converts phosphatidylinositol-4,5-bisphosphate into diacylglycerol and IP3; IP3, in turn, serves as a precursor for IP4, both of which act as signals for mobilization of intracellular Ca2+. Loss-of-function mutations in the sl locus display a phenotype that is consistent with PLCgamma playing a role as a negative regulator of Egfr signaling, similar to Gap1 (Thackeray, 1998). In the eye, sl complete loss-of-function mutations cause a supernumerary R7 phenotype in approximately 30% of ommatidia; this is qualitatively similar to the mutation caused by Gap1, but much less severe. In hemizygous sl1 mutants, the average number of R7s is 1.3. Thus, the sl complete loss-of-function phenotype is extremely mild compared to that of Gap1 partial or complete loss-of-function mutations. To probe the relationship between PLCgamma and Gap1, a test was performed to determine whether they genetically interact. In a wildtype background, Gap1/+ flies are phenotypically wildtype, i.e. all ommatidia have only one R7 cell (Gaul, 1992; Lai, 1992), while flies hemizygous for sl1 and heterozygous for Gap1 have a supernumerary R7 phenotype greatly enhanced over that seen in hemizygous sl1 flies; greater than 80% of ommatidia have multiple R7 cells. Thus, the phenotype of mutants hemizygous for sl and heterozygous for Gap1 closely resembles that of the partial loss-of-function Gap1S1-11 allele, demonstrating a synergistic positive genetic interaction between sl and Gap1. Given PLCgamma’s role in Ca2+ and phosphoinositide signaling and Gap1’s requirement for its Ca 2+ and IP4-sensitive domains, this result suggests that Gap1 activity is positively regulated by PLCgamma (Powe, 1999).

Calcineurin is a Ca2+-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca2+ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca2+-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin is activated by a sustained increase in intracellular Ca2+ levels that can result from the opening of intracellular Ca2+ channels in response to phosphoinositide (PI) signaling. PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol 1,4,5-trisphosphate (InsP3), which then activates the InsP3 receptor Ca2+ channel (Sullivan, 2002).

GPCRs and RTKs activate an integrated signaling network that includes the Ras/mitogen-activated protein (MAP) kinase cascade, PI3-kinase, and the small GTPase Rho. Depending upon the cellular context, these pathways can either antagonize or cooperate with each other and with PI signaling. For example, T-cell activation requires the activation of both NFAT, which is transduced to the nucleus upon dephosphorylation by calcineurin, and AP1, which acts downstream of Ras and MAP kinase. Conversely, PI signaling has been found to antagonize the Ras pathway in Drosophila. The Egf receptor and Ras/MAP kinase cascade are essential for formation of wing veins and photoreceptor (R) cells in the eye. Mutations in the single phospholipase Cgamma gene, small wing (sl), cause the formation of extra R7 cells and wing vein material and also genetically interact with Egf-receptor-signaling components. A recently proposed model for sl-mediated repression of Egf receptor signaling was based on the identification of the GTPase-activating protein Gap1 as an InsP4 receptor. PLCgamma-generated InsP3 is converted to InsP4, which then activates Gap. Gap converts the active form of Ras, Ras-GTP, to the inactive form, Ras-GDP (Sullivan, 2002 and references therein).

An activated form of Pp2B-14D, canAact, was made by deleting the autoinhibitory and calmodulin-binding domains. The canAact construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

The screen yielded 11 complementation groups, 9 of which failed to modify rough eyes caused by other glass-induced transgenes. This demonstrates that the majority of the modifier groups do not act through the glass enhancer. The nine specific modifiers were then divided into class I genes, which act downstream of calcineurin, and class II genes, which act at the level of CanB (Sullivan, 2002).

The hypermorphic allele EgfrE1 inhibits Ras signaling; thus it might be expected to enhance the effects of activated calcineurin. However, low levels of inappropriate Egf receptor activity in eye development are thought to increase secretion of the Egf receptor antagonist Argos. The Argos protein inhibits subsequent Egf receptor signaling that is required for photoreceptor determination. Thus, suppression of the EgfrE1 rough eye by may be the result of activated calcineurin inhibiting inappropriate Egf receptor signaling (Sullivan, 2002).

Consistent with these findings, PLCgamma is a negative regulator of Egf receptor/Ras signaling in eye and wing development. However, PLCgamma was identified in this study as a strong suppressor of activated calcineurin, although biochemically PLCgamma has been placed upstream of calcineurin in the PI signaling pathway. One explanation is that PLCgamma acts on one of the other canA genes. Another possibility is that the signaling pathways activated by PLCgamma parallel to calcineurin are required for calcineurin function (Sullivan, 2002).

In a recent model, PLCgamma has been proposed to inhibit Egf receptor/Ras signaling via the activation of Gap1 by InsP4. The results presented in this study suggest that PLCgamma is also acting through calcineurin. The genetic evidence presented indicates that calcineurin intersects with the Ras pathway at roughly the same point that PLCgamma does, and thus a modified model is proposed for the function of PI signaling in Drosophila development. Additionally, the fact that calcineurin can be activated by any sustained Ca2+ flux suggests a mechanism by which other signaling pathways, such as GPCRs acting via PLCß, can modulate Egf receptor signaling (Sullivan, 2002).

A simplified schematic is presented that illustrates upstream Egf receptor signaling components in an eye disc cell. PLCgamma is activated by the Egf receptor and cleaves PIP2 to yield InsP3. PLCgamma is proposed to negatively regulate Egf receptor signaling through InsP4, which is generated from InsP3 by an InsP3-3 kinase. Gap1 is then activated by InsP4, which results in the inhibition of Ras. Sprouty, which may be linked to the Egf receptor by the adaptor protein Drk, may facilitate the inactivation of Ras by Gap. In this model, it is proposed that PLCgamma also acts via Ca2+ and calcineurin. Genetic evidence suggests that calcineurin acts at the level of sty and Gap1, although it should be noted that calcineurin may act further upstream, e.g., at the level of InsP4. In addition, it is possible that calcineurin is activated by other Ca2+ signaling pathways (Sullivan, 2002).

The Drosophila genome contains a single phospholipase C-gamma (PLC-gamma) homolog, encoded by small wing (sl), that acts as an inhibitor of receptor tyrosine kinase (RTK) signaling during photoreceptor R7 development. Although the existing sl alleles behave genetically as nulls, they may still produce truncated Sl products that could in theory still provide limited PLC-gamma function. Both to identify a true null allele and to probe structure-function relationships in Sl, an F1 screen was carried out for new sl mutations and seven new alleles were identified. Flies homozygous for any of these alleles are viable, with the same short-wing phenotype described previously; however, two of the alleles differ from any of those previously isolated in the severity of the eye phenotype: sl9 homozygotes have a slightly more extreme extra-R7 phenotype, whereas sl7 homozygotes have an almost wild-type eye. The mutant defect was determined in all seven alleles, revealing that sl9 is a molecular null due to a very early stop codon, while sl7 has a missense mutation in the highly conserved Y catalytic domain. Together with in vitro mutagenesis of the residue affected by the sl7 mutation, these results confirm the role of Sl in RTK signaling and provide evidence for two genetically separable PLC-gamma-dependent pathways affecting the development of the eye and the wing (Mankidy, 2003).

One of the major goals of this study was to determine whether the previously characterized sl alleles indeed represent the loss-of-function phenotype, because the three previously characterized alleles could produce a truncated protein with several domains intact. Five of the seven new alleles showed a homozygous phenotype indistinguishable from that previously described for sl1, sl2, or sl3 homozygotes. The molecular defects in the new alleles vary widely in location within the protein, occurring at both N and C termini with different degrees of truncation and including an in-frame deletion and a single amino acid substitution among them. The most telling allele of all in this regard is sl9, which contains a nonsense codon at amino acid 54. Because mRNAs containing premature stop codons tend to be degraded by the nonsense-mediated decay pathway, it is unlikely that many sl9 transcripts would survive to be translated; however, even if some sl9 mRNAs escaped degradation, Sl products translated from them would have no recognized domains remaining. Therefore, the sl9 mutation clearly represents a true null allele. The sl9 homozygous phenotype is qualitatively the same as the previously characterized sl alleles (i.e., with extra-R7 cells and ectopic wing veins), which confirms previous results showing that Sl is a negative regulator of the RTK pathways involved in cell-fate decisions during ommatidial and wing-vein development (Mankidy, 2003).

The phenotype of sl9 homozygotes is identical to that of sl1,2,3,4,5,6,8,10, except that it is slightly more extreme in the eye. Why sl9 homozygotes should show a more extreme phenotype than that of the other alleles is uncertain; one possibility that cannot be ruled out is that a closely linked enhancer of the extra-R7 phenotype is present in the sl9 background. However, this allele is the only one unlikely to produce any protein; every other allele could produce either a mutated full-length protein or a truncated protein containing intact copies of the N-terminal PH domain, EF hand region, region X, and the N-terminal SH2 domain. Because mammalian PLC-gamma does not depend on its lipase function for its role in mitogenesis, truncated Sl proteins lacking intact X and Y catalytic domains (such as those that could be generated by the alleles other than sl9) might still be able to participate at some level in certain aspects of PLC-gamma function. For example, the intact N-terminal SH2 domain in each of the truncated Sl proteins would likely still be able to bind to an activated RTK at the membrane, where either its N-terminal PH domain or its EF hands might have an effect on signaling, perhaps by binding phospholipids or Ca2+ (Mankidy, 2003).

Three of the new alleles, sl5, sl7, and sl8, contain either a missense mutation or an in-frame deletion, identifying functionally important residues. In the case of sl5, a five-amino-acid deletion at the N-terminal end of the C2 domain removes five conserved residues within ß-strand 2/1 of the eight ß-strand C2 structure. C2 motifs typically bind calcium and phospholipids, but have proven to be capable of interacting with a wide range of other molecules, sometimes independently of calcium binding. The C2 domains of PLC-gamma proteins appear to lack several residues shown to be necessary for calcium binding in the PLC-delta1 C2 domain, suggesting that the PLC-gamma C2 domain has another (as yet unidentified) function. Combined with the fact that the C2 domain is well conserved in all PLC-gamma proteins, including within the genus Drosophila, the near-null phenotype observed in sl5 homozygotes indicates that the C2 domain does retain an indispensible function in Sl. The sl8 mutation replaces Gly385 with aspartate within the region X catalytic domain. There is a glycine at the homologous position in all of the described PLC proteins, and the immediately adjacent histidine on the N-terminal side of this glycine is required for PIP2 hydrolysis in both PLC-delta and PLC-gamma, playing a direct role in catalysis by acting as a proton donor. It is therefore not surprising that the introduction of an aspartate at this critical location would disable the phospholipase activity of the enzyme (Mankidy, 2003).

By far the most intriguing of the new alleles is sl7, because flies homozygous for this allele show the null wing-length phenotype, but have an almost wild-type eye. A trivial explanation of this phenotype might be the presence of a closely linked modifier that suppresses the extra-R7 phenotype, but not the wing-length phenotype. However, the fact that the X10-P1035A construct produced an almost identical phenotype in the eye, in an unmutagenized background, suggests that the sl7 phenotype is a genuine reflection of altered PLC-gamma activity rather than an artifact produced by an interacting mutation. Furthermore, there is a precedent for an sl allele with an sl7-like phenotype: sl34 was isolated by Gottschewski in 1934 (cited in Mankidy, 2003) and was described as having short wings, but normal eyes; unfortunately, sl34 has been lost. The fact that an independently isolated allele shows this same combination of eye and wing phenotypes tends to suggest that the sl7 mutation is not simply a rare hypomorph with threshold activity, but may in fact be a member of a class of sl mutations that are able to separate the extra-R7/ectopic wing-vein and wing-length phenotypes. This model is also consistent with evidence for two distinct Sl-mediated pathways in the eye and wing: a partial loss-of-function mutation of the rl-encoded MAPK, rl1, is able to suppress both the extra-R7 and ectopic wing-vein phenotypes of sl homozygotes, but does not suppress the short-wing phenotype (Mankidy, 2003).

The sl7 mutation is a replacement of Pro1035 by leucine within the region Y catalytic domain. An alanine at the same position recapitulates the phenotype of sl7 in the eye, but not in the wing, in which P1035A rescues the sl wing phenotype. By contrast, an sl construct containing valine at position 1035 is equivalent to one containing the wild-type proline, rescuing both the eye and wing defects of either sl1 or sl9. First of all, the ability of the X10-P1035V construct to rescue flies lacking sl function is very surprising, because this proline is one of a small number of absolutely conserved sites across all PLC isoforms in region Y. In PLC-delta1 the homologous proline is predicted to form a turn leading into one loop of a hydrophobic ridge that lines the active site, a role that might be expected to depend on a proline at this position. A possible explanation is that because valine has a side chain slightly more hydrophobic and compact than that of leucine it is drawn into the ridge by hydrophobic clustering, thereby partially overcoming the loss of proline. The slightly lower hydrophobicity and longer side chain of leucine may be unable to achieve this effect, possibly because of steric clashes due to its length. In contrast, alanine is much less hydrophobic than either valine or leucine and so may be unable to overcome the loss of the turn resulting from replacement of the proline. In any event, the fact that three different amino acids at position 1035 can produce three different combinations of eye and wing phenotypes clearly indicates that Pro1035 has a key modulatory role in Sl signaling (Mankidy, 2003).

Pro1035 of Sl is homologous to Pro552 of human PLC-delta in a region close to the active site where several mutations have been made and tested in vitro. One of the most interesting of these PLC-delta mutations was a replacement of Arg549 by alanine; this change dramatically reduces PIP2 hydrolysis, but has little effect on hydrolysis of PI, demonstrating that changes in this part of region Y can alter the substrate specificity of PLC-delta. All PLC enzymes are thought to be able to hydrolyze PIP2, PIP, and PI, although the relative physiological importance of the different substrates is unknown for any PLC. If the sl7 mutation has an effect similar to the Arg549 mutation of human PLC-delta1, hydrolysis of one substrate necessary for Sl-mediated signaling in the wing might be reduced, whereas the hydrolysis of a different substrate more important for signaling during photoreceptor development is less affected. An alternative explanation is that in sl7 homozygotes Sl is partially functional at a threshold of activity that is almost sufficient for wild-type function in the eye, but is not quite enough in the wing. However, the phenotype of sl7/sl8 and sl7/sl9 trans-heterozygotes suggests that there is a qualitative difference between these alleles, consistent with the sl7-encoded PLC-gamma protein lacking a function needed in the wing, but retaining a function needed in the eye. Direct assays of phospholipid hydrolysis will be required to determine whether differential substrate use by Sl occurs in different physiological contexts (Mankidy, 2003).

Small Wing PLCγ is required for ER retention of cleaved Spitz during eye development in Drosophila

The Drosophila EGF receptor ligand Spitz is cleaved by Rhomboid to generate an active secreted molecule. Surprisingly, when a cleaved variant of Spitz (cSpi) was expressed, it accumulated in the ER, both in embryos and in cell culture. A cell-based RNAi screen for loss-of-function phenotypes that alleviate ER accumulation of cSpi identified several genes, including the small wing (sl) gene encoding a PLCsγ. sl mutants compromised ER accumulation of cSpi in embryos, yet they exhibit EGFR hyperactivation phenotypes predominantly in the eye. Spi processing in the eye is carried out primarily by Rhomboid-3/Roughoid, which cleaves Spi in the ER, en route to the Golgi. The sl mutant phenotype is consistent with decreased cSpi retention in the R8 cells. Retention of cSpi in the ER provides a novel mechanism for restricting active ligand levels and hence the range of EGFR activation in the developing eye (Schlesinger, 2004).

The accumulation of cSpi in the ER appears to reflect a novel mechanism for ER retention. In contrast to ER retention of the full-length Spi precursor, cSpi remains in the ER when the retrograde trafficking machinery from the Golgi to the ER is compromised following incubation with dsRNA for COPI. Utilization of a novel ER retention and export machinery has been identified for the SREBP protein-regulating cholesterol synthesis (Schlesinger, 2004).

To identify the mechanism responsible for cSpi retention and assess its biological significance, a screen was conducted for dsRNAs that would compromise this property. The analysis focussed on the sl gene, in view of previous observations demonstrating that it is a negative regulator of EGFR signaling in the eye. Sl is broadly expressed. Compromising the levels of Sl in embryos, either by dsRNA injection or in a sl mutant background, led to efficient release of cSpi. Thus, Sl is also required in vivo for the retention of cSpi. The actual retention mechanism remains unknown. Sl is a cytoplasmic protein, while cSpi is a secreted protein that is retained within the ER lumen. Additional proteins must participate to form a physical link. While sl encodes a PLCγ, it is believed that its enzymatic activity is not necessary for the retention process. sl interacts genetically with EGFR signaling only in the eye. sl mutants that were defective in the catalytic domain did not give rise to an eye phenotype. In addition to the catalytic domain, Sl also contains several motifs that may mediate protein-protein interactions, including SH2, SH3, and PH domains. It is thus possible that in addition to its enzymatic role, Sl serves as a scaffold protein in other contexts. In mammalian cells, PLCγ has been shown to function in the cells receiving the signal, downstream to receptor-tyrosine kinases (Rhee, 2001). The implicated role of Sl/PLCγ in the cells producing the signal points to a novel function of this protein (Schlesinger, 2004).

While Sl is broadly expressed, the EGFR hyperactivation phenotype of sl null flies is manifested only in the eye. This phenotype entails recruitment of extra R7 photoreceptor cells and misrotation of ommatidia. The restricted effect led to an examination of the possibility that cSpi is normally generated in the ER only in the eye. The cleavage of EGFR ligands depends upon a distinct family of serine proteases that carry out intramembrane proteolysis. Rhomboid-1 is the primary player, and hence mutations in this gene give rise to embryonic phenotypes that are similar to spi or Star (Schlesinger, 2004).

Two additional members of the family, Rhomboid-2/Brho and Rhomboid-3/Roughoid (Ru), are expressed in the oocyte and the eye, respectively. Recently, expression of Ru was also detected in the embryonic VUM neurons, where it plays a role in guidance of tracheal migration in the CNS. Homozygous null mutations for ru demonstrated that it is essential for normal eye development, but its role is partially redundant with Rhomboid-1, since some photoreceptor cells are recruited in ru mutants. The question is whether there are properties of Ru that are distinct from those of Rhomboid-1 and may account for the generation of cSpi in the ER during eye development (Schlesinger, 2004).

As far as substrate specificity is concerned, the Rhomboid 1-3 proteins are all capable of cleaving the membrane precursors of the EGFR ligands Spi, Keren, and Gurken. In addition, all three proteins are enriched in the Golgi when expressed in mammalian cells. However, functional assays in cell culture, including both Drosophila and mammalian cells, suggest that in contrast to Rhomboid-1, Ru may be capable of cleaving the Spi precursor already in the ER. However, cSpi is secreted only upon coexpression of Star. While Ru is located primarily in the Golgi, this cleavage may take place en route to the Golgi. The failure to secrete cSpi in the absence of Star likely represents the property of ER retention that was uncovered in this work (Schlesinger, 2004).

ru and sl give rise to opposite phenotypes in the eye. It is assumed that they act in a sequential manner, i.e., Ru generates cSpi in the ER and Sl mediates the retention of this ligand, to avoid excessive secretion. The genetic interaction experiments between ru and sl can be interpreted within this context. Indeed, the eye phenotype of hypomorphic mutations in ru could be efficiently rescued by mutations in sl. While the level of cSpi in the ER was compromised, more efficient secretion was facilitated in the sl background, thus compensating for the initial defect. Surprisingly, even null mutations in ru were partially rescued by sl mutants. In a ru null background, Rhomboid-1 is the only other known Rhomboid family protease that is functional in the eye. It is suggested that residual levels of cSpi may also be generated in the ER by Rhomboid-1. An efficient release of these low levels in the absence of Sl may lead to the partial rescue that was observed in ru null mutants (Schlesinger, 2004).

Finally, the requirement for Sl specifically in the cells processing the ligand was demonstrated by the capacity to rescue sl null flies by expressing Sl in the R8 cells. Normally, expression of Rhomboid-1 and Ru in the differentiating photoreceptor cells is induced by EGFR activation, thus making these cells a source for subsequent rounds of photoreceptor cell recruitment. Incomplete rescue by expression of Sl in R8 cells may be explained by the failure to restore ER retention of cSpi in the other photoreceptor cells expressing Ru (Schlesinger, 2004).

In conclusion, it has been demonstrated that the cleaved form of Spi is efficiently retained in the ER through a novel mechanism. This retention is significant only in the developing eye, where the Rhomboid-3/Ru protein may normally generate the cleaved ligand in the ER. Thus, in spite of efficient cleavage of mSpi in the ER, only the molecules that will overcome retention by association with Star will be secreted to activate EGFR in the neighboring cells. small wing, encoding a PLCγ, provides a link to the retention mechanism, and sl mutants exhibit EGFR hyperactivation phenotypes mainly in the eye. The eye is a tissue where the restricted range of EGFR activation is particularly crucial. The number of undifferentiated precursor cells is limited. EGFR activation is responsible for sequential inductions of the different cell types comprising the mature ommatidia. It is thus imperative to restrict the number of cells that are induced at every cycle. Negative feedback loops that are transcriptionally induced by EGFR activation in the cells receiving the signal were previously shown to be central to this restriction. This study demonstrates that fine tuning the level of ligand that is released by the cells providing the signal represents another cardinal tier of regulation (Schlesinger, 2004).


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Interactive Fly, Drosophila small wing: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

date revised: 25 November 2014

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