InteractiveFly: GeneBrief

Keren: Biological Overview | References

Spitz (Spi) is the most prominent ligand of the Drosophila EGF receptor. It is produced as an inactive membrane precursor that is retained in the endoplasmic reticulum (ER). To allow cleavage, Star transports Spi to the Golgi, where it undergoes cleavage by Rhomboid. Since some Egfr phenotypes are not mimicked by any of its known activating ligands, an additional ligand (Keren) was identified by database searches. Krn is a functional homolog of Spi since it can rescue the spi mutant phenotype in a Rho- and Star-dependent manner. In contrast to Spi, however, Krn also possesses a Rho/Star-independent ability to undergo low-level cleavage and activate Egfr, as evident both in cell culture and in flies. The difference in basal activity correlates with the cellular localization of the two ligands. While Spi is retained in the ER, the retention of Krn is only partial. Examining Spi/Krn chimeric and deletion constructs implicates the Spi cytoplasmic domain in inhibiting its basal activity. Low-level activity of Krn calls for tightly regulated expression of the Krn precursor (Reich, 2002).

In order to identify additional Egfr ligand(s), databases searches were performed for new Spi homologs. Two expressed sequence tags (ESTs) representing the same gene were found to be highly homologous to Spi (LD34470 and LD34429), mapping to 74E/F. The complete sequence of both ESTs revealed an open reading frame of 217 amino acids (Reich, 2002).

In the three activating Egfr ligands identified to date, the homology was restricted to the EGF domain. In contrast, the similarity between Spi and the new putative ligand, extending throughout the coding region, is 56%. The length and domain organization of both proteins are similar. Homologous stretches are concentrated mainly in the extracellular domain, with significant identity in the EGF domain (65%), while limited homology can be found in the cytoplasmic domain. Because of the similarity to Spi, this protein was named Keren, which is the Hebrew word for an antler, or a sharp object (Reich, 2002).

The ESTs of Krn are comprised of two exons, where the entire coding region is located in the second exon. Interestingly, the 5' non-coding exon is also found in ESTs of a coding sequence located more 3' to the gene encoding Krn. This coding sequence is homologous to novel mouse and human proteins. The splicing of the 5' non-coding exon to the TGFα transcript was confirmed by RT-PCR of RNAs extracted from adult flies and S2 cells. According to the RT-PCR, Krn is expressed during the stages of embryonic and larval development, and in adults. Attempts were made to examine the Krn expression pattern using RNA in situ hybridization and antibody stainings on wild-type embryos or imaginal discs, but the signal was below detection level (Reich, 2002).

Spi is active only as a cleaved moiety (sSpi), which diffuses to neighboring cells and activates the receptor. Following this paradigm, the secreted form of Krn (sKrn) was overexpressed in various tissues. Indeed, in all tissues, the phenotypes of sKrn resembled the ectopic phenotype of sSpi. In the wing, MS1096-Gal4 driving sKrn gave rise to lethality, but several survivors had short bloated stumps for wings. In the ovary, expression of sKrn in the anterior follicle cells using 55B-Gal4 resulted in the formation of extra dorsal appendages. Ectopic sKrn driven by 69B-Gal4 in the embryonic ectodermal cells causes lethality, with the cuticle showing wider and square-edged denticle belts characteristic of Egfr hyperactivation. It is evident that sKrn can activate MAPK (as revealed by dpERK antibody) wherever it is expressed in the embryo, or in the wing disc. These results show that sKrn can mimic sSpi activity and activate the Egfr pathway (Reich, 2002).

Having demonstrated the biological activity of sKrn, it was of interest to determine whether its processing is regulated in a similar manner to Spi. Triggering of Egfr by Spi at stage 10 generates two prominent domains of activation, in the tracheal placodes and ventral ectoderm. These domains correspond to the sites of Rho expression in the tracheal placodes and midline, respectively. dpERK can be readily detected in these domains, while in spi mutant embryos no dpERK is observed at this stage. The capacity of the Krn precursor to rescue spi mutant embryos was examined. When Krn was ubiquitously expressed in the ectoderm of spi^- embryos (using the 69B-Gal4 driver), complete rescue of the dpERK pattern was observed. Induction of the pathway by Krn at the sites of Rho expression in the midline and tracheal placodes indicates that, like Spi, processing of Krn is dependent upon Rho. To test whether Krn cleavage requires Star, Krn was expressed in Star mutant embryos. No rescue of the phenotype was observed, as monitored by dpERK. It is thus concluded that, like Spi, processing of Krn is Rho and Star dependent (Reich, 2002).

While Spi is an extremely potent ligand in its secreted form, when expressed in the precursor form, even at high levels, it shows no ectopic activity. Following this paradigm, the rescue of spi mutant embryos by high levels of expression of Krn shows detectable levels of dpERK only at the tissues where Spi cleavage normally takes place. Surprisingly, in contrast to Spi, Krn is capable of eliciting phenotypes in a variety of tissues following overexpression. For example, in the wing, Krn overexpression gives rise to bloated wings, similar to those obtained upon moderate activation of Egfr with the lambda-top construct. Over-expression in the eye leads to rough eyes and in the follicle cells to excess dorsal appendage material. In the embryo, expression of Krn gives rise to embryonic lethality, with cuticles displaying expansion of head structures, but otherwise normal in appearance (Reich, 2002).

A clearer understanding of Spi cleavage has been gained by studies in cells. Efficient cleavage of Spi occurs only in cells in which both Star and Rho are expressed. Spi is retained in the ER through its intracellular domain. Star binds Spi and translocates it from the ER to the Golgi, where Rho functions as a protease and cleaves Spi. Krn-GFP in Drosophila S2 cells shows partial release from retention, manifested by a vesicular distribution, indicating exit from the ER. The functional implication of this was observed through the ectopic phenotypes in various tissues. For instance, whereas ectopic Spi driven by ubiquitous GAL4 in embryos does not prevent hatching of larvae, ectopic Krn leads to lethality. This highlights the importance of retention, since it allows expression of high amounts of Spi protein in the cell, yet controls Spi activity by preventing Spi from reaching further compartments where cleavage occurs (Reich, 2002).

Chimeric and deletion constructs identify the cytoplasmic domains of Spi and Krn as the domains responsible for their different cleavage profiles. This is a result of different levels of retention in the ER. The mechanism of Spi and Krn retention is not yet clear. Spi has also been shown to be retained in a heterologous system of mammalian cells, implicating the action of conserved molecules or an intrinsic property of the protein. In one model, association of the Spi cytoplasmic domain with an additional protein(s) could mediate retention. In that case, it would be expected that Krn would have lower affinity to this protein(s). In another model, the Spi C-terminus itself could have an intrinsic inhibitory capability through protein folding that sterically prohibits association to proteins -- this would carry Spi further in the secretory pathway. In this case, Krn would be expected to possess a higher affinity to such chaperones, that would allow it to exit the ER without total dependence on Star (Reich, 2002).

Compared with Spi or Krn, the cytoplasmic domain of Grk, the third Egfr ligand with a transmembrane domain, is shorter (only 24 amino acids). Deletion of its cytoplasmic domain did not influence signaling by Grk. Like Krn, overexpression of the full-length Grk protein causes ectopic wing phenotypes. It would be interesting to see to what extent Grk is retained in the ER of S2 cells (Reich, 2002).

Expression of Krn in S2 cells allows the mechanism of low-level cleavage, which is Star and Rho independent, to be followed. What is the protease responsible for this cleavage? The sensitivity of Krn cleavage to inhibitors of serine proteases indicates that cleavage may be mediated by a protease of this family. Unlike Rho, which is expressed in a spatially and temporally regulated manner, the protease is expected to be ubiquitously expressed, since ectopic Krn causes abnormal phenotypes wherever it was expressed. This further elaborates the need for tight transcriptional control on Krn expression (Reich, 2002).

Co-expression of Star with Krn in S2 cells raises the amounts of secreted sKrn in the medium. This is a result of the efficient export of Krn from the ER by Star. Since higher levels of cleavage can be obtained by co-expressing both Rho and Star, it would seem to indicate that the protease involved in low-level cleavage is less efficient than Rho (Reich, 2002).

High-level cleavage of Krn was followed in embryos through the detection of dpERK. The activation profile followed the restricted expression of Rho, since Star is broadly expressed. Only in cell culture could Rho enhance cleavage of Krn without co-expression of Star. This probably occurs because Krn can 'leak' out of the ER, reach compartments where Rho is present and undergo cleavage by Rho independent of Star (Reich, 2002).

Sequence conservation within the transmembrane domains of the Rho protein have suggested that the cleavage site of Spi would reside within the transmembrane domain. There is also evidence to indicate that Rho cleaves Spi within the membrane. The transmembrane regions of Spi and Krn are conserved, and show 50% sequence identity. The transmembrane domain of Krn may thus possess the same recognition sites as that of Spi (Reich, 2002).

In view of the two modes of Krn cleavage, where could it have biological roles? The capacity of Krn to undergo Rho- and Star-dependent high-level cleavage suggests that it could provide the missing ligand in tissues where the rho phenotype is more severe than that of spi. This includes the formation of veins in the wing, generation of correct R8 spacing and inhibition of apoptosis after the morphogenetic furrow in the eye disc. In the embryo, the spi phenotypes are similar to those of rho or Star mutants, indicating that Krn is not likely to be required at this stage (Reich, 2002).

Could the low-level cleavage of Krn also play a physiological role in activating Egfr? In the eye imaginal disc, generation of clones of Egfr mutants either anterior or posterior to the morphogenetic furrow was not possible, due to cell lethality. The known rho family genes are not expressed anterior to the furrow. It is thus possible that the low-level cleavage of Krn may provide the residual levels of secreted ligand necessary to trigger Egfr anterior to the furrow, and allow cell survival. The low levels of Egfr activation anterior to the furrow are consistent with the non-detectable levels of dpERK in this domain. Sufficient levels of secreted ligand would need to be produced to elicit a biological response, in spite of the low level of krn expression. In the Krn misexpression assays presented in this work, high levels of expression were required to detect this low activity (Reich, 2002).

In order to examine the biological roles of Krn, krn double-stranded (ds) RNA was carried out. Efforts were focussed in the eye and wing imaginal discs, where Krn activity is expected. Uniform expression of krn dsRNA in these tissues, even when combined with spi dsRNA, did not yield a noticeable phenotype. The possible roles of Krn in these tissues may be confirmed only when mutations for krn become available and their phenotypes are tested, alone or in combination with mutants for spi (Reich, 2002).

In conclusion, Krn was found to be the functional homolog of Spi. Unlike Spi, Krn is capable of undergoing inefficient Star- and Rho-independent cleavage in flies and in cell culture. This is due to differences between the intracellular domains of Krn and Spi, which allow Krn to evade retention in the ER and reach further along in the secretory pathway. This calls for tight transcriptional control of Krn expression, in contrast to Spi, which can be ubiquitously and abundantly expressed (Reich, 2002).

EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

In holometabolous insects, which undergo development through metamorphosis involving muliple stages, the adult appendages and internal organs form anew from larval progenitor cells during metamorphosis. The adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgut progenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs first disperse, but later proliferate within distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium. Signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceral muscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz and Keren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during late larval stages (Jiang, 2009).

The insect midgut, like the vertebrate intestine, is an endoderm-derived organ. Both the larval and adult Drosophila midguts are composed of a single layer of epithelial cells with two layers of visceral muscle (VM) wrapped outside. Inside the gut lumen, a peritrophic membrane separates the food from the intestinal epithelium. During both mammalian and insect embryonic development, Forkhead and GATA transcription factors play evolutionary conserved roles in the specification and subsequent morphogenesis of the digestive tract. Similarly, multiple signaling pathways, including the EGF, Wingless (Wnt), Dpp (TGFβ), Notch and Hedgehog pathways, are involved in the embryonic development of the Drosophila midgut and mammalian intestine. In both systems, cross-talk between mesodermal cells and endoderm-derived epithelial cells in the gut primordium plays important roles during embryonic gut development (Jiang, 2009).

Starting from embryonic development stage 11, the Drosophila midgut epithelium consists of two distinct cell populations: differentiating midgut epithelial cells (larval enterocytes, ECs) and undifferentiated adult midgut progenitors (AMPs, also referred to as midgut histoblast islets or midgut imaginal islets). In Drosophila embryos, AMPs can be marked by expression of asense or by one of several lacZ- or Gal4-expressing enhancer-trap insertions. AMPs first appear as spindle-shaped cells localized to the apical surface of the midgut epithelium, but later migrate to the basal surface of the epithelium where they remain throughout larval development. Notch signaling has been shown to be involved in the development of Drosophila AMPs. In Notch mutant embryos, the number of AMPs in the midgut rudiment is strongly increased at the expense of differentiated larval ECs (Hartenstein, 1992). During larval development, the ECs grow in both size and ploidy by undergoing several endocycles, reaching 64C (DNA content) by the wandering L3 stage. The AMPs remain diploid throughout larval development and appear as scattered islets of cells (hence the term 'midgut histoblast islets') in late-stage larval midguts. During pupal development, the ECs histolyze and a new adult midgut epithelium forms from the AMPs (Bender, 1997; Jiang, 1997; Li, 2003). Similar midgut progenitor cells have also been found in other insect species (Jiang, 2009).

The adult Drosophila midgut has been shown to undergo dynamic self-renewal, a process similar to that found in the mammalian intestine/colon. Fly and mammalian gut homeostasis are both powered by intestinal stem cells (ISCs), and Notch signaling plays similar roles in regulating their differentiation into mature gut cells (Fre, 2005; Micchelli, 2006; Ohlstein, 2006; Ohlstein, 2007; van Es, 2005). Thus, the Drosophila midgut may serve as a model to study gut homeostasis and the development of cancers, such as colorectal carcinoma, that are directly associated with this dynamic process in humans (Jiang, 2009).

This study describes the development of the AMPs in Drosophila larvae and pupae. Drosophila AMPs are shown to divide extensively throughout larval development, and their proliferation can be separated into two distinct phases. During early larval stages, the AMPs divide and disperse to form islets throughout the midgut, but during late larval development the dividing AMPs are contained within these islets. Furthermore, this study revealed that Drosophila EGFR signaling is both necessary and sufficient to induce the proliferation of AMPs during larval development (Jiang, 2009).

Drosophila AMPs were previously thought to be relatively quiescent during larval development, dividing just once or twice, and not initiating rapid proliferation until the onset of metamorphosis. This is the case for several other larval progenitor/imaginal cell types, such as the abdominal histoblasts and cells in the salivary gland, foregut and hindgut imaginal rings. Various studies have suggested that AMP proliferation might precede the onset of metamorphosis. However, these studies did not report the extensive proliferation of the AMPs that is describe in this study, and failed to recognize the early larval proliferative phase when the AMPs divide and disperse. The extensive proliferation of the AMPs is similar to that of the larval imaginal disc cells, which also proliferate throughout larval development, dividing about ten times (Jiang, 2009).

Lineage analysis revealed that the proliferation of the Drosophila AMPs occurs in two distinct phases. In early larvae, the AMPs divide and disperse throughout the midgut to form individual islets. During later larval development, the AMPs continue to divide but do so within these islets, forming large cell clusters. It is speculated that in the early larva, secretion of Vn from the midgut visceral muscle (VM) cells results in low-level activation of EGFR signaling in the AMPs, which is sufficient for their proliferation and might also promote their dispersal. No proliferation defects were detected in AMPs defective in shot function, suggesting that the mechanism of EGFR activation used by tendon cells during muscle/tendon development is probably not the same as in the larval midgut. Specifically, it is unlikely that the Short stop-mediated concentration of Vn on AMPs activates EGFR signaling in the AMPs during early larval development. Consistent with this, dpERK staining was only observed in AMP clusters and not in the isolated AMPs present at early larval stages (Jiang, 2009).

The mechanisms that regulate the transition between these two proliferation phases remain unclear. Fewer AMP clusters are observed when sSpi, sKrn, lambdaTOP (activated Egfr) or Ras^V12 were induced in the AMPs starting from early larval stages, suggesting that EGFR signaling, in addition to its crucial role as an AMP mitogen, might also play a role in AMP cluster formation. In the late larval midgut (96-120 hours AED), high-level EGFR activation, resulting from expression of spi and Krn in the AMPs themselves, might not only promote AMP proliferation, but might also suppress AMP dispersal and thus promote formation of the AMP clusters. How the timing and location of Spi- or Krn-mediated EGFR activation are regulated during larval development is also unclear. It is noted, however, that the pro-ligand form of Krn acted similarly to sKrn, and no functions were uncovered for the Rho-like gene products that regulate Spi and Krn function by proteolytic cleavage in other tissues. This suggests that the localized expression of these ligands in the AMP clusters might be the critical parameter that controls their effects. Consistent with this, Rho-independent cleavage and function of Krn have been documented (Jiang, 2009).

In the developing Drosophila wing, EGFR/RAS/MAPK signaling promotes the expression and controls the localization of the cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin). Ras^V12-expressing clones generated in the wing imaginal disc are round, much like the AMP clusters described in this study, owing to increased adhesive junctions. In developing Drosophila trachea, EGFR activity upregulates shg expression to maintain epithelial integrity in the elongating tracheal tubes. In the eye, EGFR activity leads to increased levels of Shg and adhesion between photoreceptors. Given these precedents, it seems reasonable to suggest that high-level EGFR activity in the AMP islets upregulates Shg and promotes the homotypic adhesion of the AMPs. Alternatively, changes in the differentiated cells of the midgut epithelium might promote AMP clustering. In either case, the dispersal of early AMPs and subsequent formation of late AMP clusters facilitate the formation of the adult midgut epithelium during metamorphosis (Jiang, 2009).

This study confirms previous reports that Drosophila AMPs replace larval midgut epithelial cells to form the adult midgut epithelium during metamorphosis (Bender, 1997; Jiang, 1997; Li, 2003). Furthermore, this study shows that the majority of AMPs lose esgGal4-driven GFP expression as they differentiate to form the new adult midgut epithelium. These cells lacked Prospero, which marks enteroendocrine cells in both the larval and adult midgut. They to through several rounds of endoreplication during late pupal development, and thus probably all differentiated into adult enterocytes (ECs). During early metamorphosis, some cells in the new midgut epithelium remain small and diploid and maintained strong esgGal4 expression. For several reasons, it is believed that these esg-positive cells are the future adult intestinal stem cells (ISCs). First, esgGal4 expression marks AMPs, including adult ISCs and enteroblasts. Second, mitoses in the adult midgut are observed only in ISCs, and mitoses were observed only in the esg-positive cells during metamorphosis. Third, esg-positive cells migrate to the basal side of the midgut epithelium, the location of adult ISCs. Fourth, AMP clones generated during early larval development contained just a few esg-positive cells when the new adult midgut first formed (24 hours APF), but when such clones were scored in newly eclosed adults, they contain large numbers of ECs, as well as cells positive for the enteroendocrine marker Prospero and the ISC marker Delta. This suggests that a small fraction of AMPs differentiate into adult ISCs. However, esg-positive cells in the new pupal midgut lack Delta expression until eclosion, suggesting that they are probably not mature adult ISCs (Jiang, 2009).

How a small fraction of AMPs are selected to become adult ISCs in the newly formed pupal midgut epithelium is not known. One possibility is that the adult ISCs are determined during larval development, long before the formation of the adult midgut. Another is that they are specified during early metamorphosis. This second hypothesis is preferred for several reasons. First, in lineage analysis, it was found that all AMP clones induced during early larval stages formed multiple clusters. This suggests that there are no quiescent AMPs in the larval midgut. Second, when AMP clones are induced at mid-third instar, the mosaic clusters always contains multiple GFP-positive cells, suggesting that all AMPs in the mid-third instar midgut remain equally proliferative. Third, during larval development, differentiation of the AMPs was never observed, as judged by their ploidy (diploid) and lack of expression of the enteroendocrine marker Prospero (not shown). Fourth, all AMPs appeared to express esgGal4 throughout larval development. Given the crucial role that Notch signaling plays in regulating AMPs during embryonic midgut development (Hartenstein, 1992) and ISCs in adult midgut homeostasis, it is expected that Notch might also function to specify adult ISCs during metamorphosis (Jiang, 2009).

EGFR signaling is both required and sufficient to promote AMP proliferation. Hyperactivation of EGFR signaling, such as by expression of activated Ras (Ras^V12), promoted massive AMP overproliferation and generated hyperplastic midguts that were clearly dysfunctional. In contrast, inhibiting EGFR/RAS/MAPK signaling dramatically reduced AMP proliferation. Furthermore, the ability of EGFR signaling to induce ectopic AMP proliferation is almost unique. With the exception of larval hemocytes, activated EGFR signaling does not promote cell proliferation in the imaginal discs, salivary gland imaginal rings, abdominal histoblasts, foregut and hindgut imaginal rings. This suggests that the regulation of AMP proliferation is different from that in other imaginal cells (Jiang, 2009).

Despite the obvious differences between adult ISCs and their larval progenitors, the AMPs, there are also similarities. First, when the new adult midgut epithelium forms, larval AMPs give rise to the new adult midgut including the adult ISCs. Many genes, such as esg, that are specifically expressed in the larval AMPs are also expressed in the adult ISCs. Second, the structure of the midgut epithelium with basal AMPs or ISCs is similar in larval and adult stages. Third, vn expression in larval VM persists in the adult midgut, suggesting that Vn from the adult VM might also regulate the ISCs (Jiang, 2009).

In two Drosophila stem cell models, the testis and ovary, stem cells reside in special niches comprising other supporting cell types. These niches maintain the stem cells and provide them with proliferative cues. For example, in the testis, germ stem cells attach to the niche that comprises cap cells. The cap cells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp], which maintain the stem cells and induce their proliferation. Whether Drosophila ISCs utilize supporting cells that constitute a niche remains unclear. This study shows that multiple EGFR ligands are involved in the regulation of Drosophila AMP proliferation. During early larval development, the midgut VM expresses the EGFR ligand vn, which is required for AMP proliferation. Thus, the early AMPs might be considered to require a niche comprising non-epithelial VM. Later in larval development, however, the AMPs express two other EGFR ligands, spi and Krn, which are capable of autonomously promoting their proliferation and may render vn dispensable. It was found, however, that depleting spi and Krn in the AMPs did not affect AMP proliferation, suggesting that vn or another trigger of EGFR/RAS/MAPK activity might complement spi and Krn in late-stage larvae (Jiang, 2009).

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. Two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1 is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. An assay was developed to detect the ability of secreted factors to reroute migrating border cells in vivo, and the activity of EGFR ligands was tested compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, do not. In addition, only Keren and Spitz are expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte (McDonald, 2006).

Ectopic expression of ligands for the Drosophila EGFR can redirect migratory cells in vivo. The ability of s-SPI and s-KRN to reroute border cells, together with the endogenous expression of spi and Krn mRNA in the oocyte and the strong border cell migration phenotype following reduction of both EGFR and PVR signaling, indicate that signaling through EGFR normally contributes to guiding border cells to the oocyte. VN is unlikely to contribute significantly because VN is ineffective in the misguidance assay and endogenous expression is not detected in the germline. The question then arises as to whether GRK contributes to guiding border cells. It seems unlikely to contribute significantly to their posterior migration because it is ineffective in the misguidance assay, even when the ectopic expression equals or exceeds the endogenous expression and the ectopic expression is closer to border cells. Furthermore, GRK protein is highly localized in a dorsal/anterior crescent within the oocyte, whereas there is no discernable dorsal bias to the path that border cells take to the oocyte, even in the absence of Pvf1 (McDonald, 2006).

Spitz acts redundantly with another Egfr ligand to control rotation 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).

A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. Four of the rhomboid family members have been examined: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signaling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. These data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands (Urban, 2002).

Within the seven Rhomboid-like molecules in Drosophila, Rhomboids 1-4 are more closely related to each other than they are to any of the remaining three Rhomboids. Rhomboids 6 and 7 are more divergent, although they both retain the residues required for the serine protease activity of Rhomboid-1. Rhomboid-5 has similarities to the others but does not contain the catalytic residues. Whether Rhomboids 1-4 all have proteolytic activity against Spitz, the known substrate for Rhomboid-1, was examined. Spitz is cleaved efficiently by all Rhomboids tested, albeit with some significant differences. A cell culture assay allowed the distinguishing of Spitz cleaved in cell lysates from that which had been secreted into the medium. The amount of cleaved Spitz detected in cells varies with different Rhomboids; no or very little intracellular cleaved Spitz is detected in cells with Rhomboid-1, while cleaved intracellular Spitz is readily detected with Rhomboids 2-4. This is most apparent by comparing the relative levels of full-length and cleaved Spitz in cell lysates. In the presence of Star, the amount of secreted Spitz present in the medium is the same for all core Rhomboids, even when they are made limiting by reducing their levels of expression, indicating that all four Rhomboids have similar levels of proteolytic activity against Spitz (Urban, 2002).

Star regulates Spitz cleavage by Rhomboid-1 by transporting Spitz to the Golgi apparatus. Strikingly, although Star was essential for ligand secretion into the culture medium in each case, it does not affect the ability of Rhomboids 2, 3 and 4 to catalyse Spitz cleavage. The extensive O-linked glycosylation that is diagnostic of transit through the Golgi apparatus (and which increases the apparent molecular weight of Spitz) was not present in cell lysates. Therefore, in contrast to Rhomboid-1, Rhomboids 2, 3 and 4 causes the accumulation of an intracellular cleaved Spitz that is not transported past the trans-Golgi network and thus not secreted (Urban, 2002).

The ability of Rhomboids 1-4 to catalyse Spitz cleavage in the tissue culture assay suggested that all may be involved in activating the EGF receptor in vivo. This has been clearly demonstrated for Rhomboid-1, was genetically determined in the case of Rhomboid-3, and has been proposed for Rhomboid-2. To investigate this further, the potential activity of Rhomboids 2-4 in vivo was compared by overexpressing them in developing Drosophila tissues. In all cases examined, Rhomboids 2-4 cause similar phenotypes to Rhomboid-1, consistent only with EGF receptor hyperactivation. When expressed in the developing wing, for example, all core Rhomboids produce ectopic and thickened vein phenotypes similar to those observed for Rhomboid-1. This phenotype was modified predictably by mutations in other members of the EGF receptor pathway. Furthermore, as in cell culture assays, all four Rhomboids are synergistic with the co-expression of Star. In all cases, UAS Rhomboids 1 and 3 produce consistently strong wing phenotypes, whereas Rhomboids 2 and 4 are weaker. Similar results were obtained in the eye, follicle cells of the ovary and the embryo. Importantly, no other phenotypes were observed in eyes, wings or embryos expressing Rhomboids, suggesting that they do not affect any other pathways. If, for example, the previously uncharacterized Rhomboids 2 or 4 caused the activation of other signaling pathways, their ectopic expression would lead to additional phenotypes. These observations confirm that Rhomboids 2-4 contain the same proteolytic activity as Rhomboid-1; furthermore, the absence of phenotypes associated with other pathways strongly suggests that Rhomboids 1-4 are all dedicated to regulating EGF receptor signaling. These data demonstrate that Rhomboids 1-4 all share proteolytic activity against the ligand Spitz (Urban, 2002).

A new Spitz-like gene was identified as a cDNA submitted to GenBank by the Berkeley Drosophila cDNA sequencing project. With the subsequent completion of the Drosophila genome sequence, this gene has been annotated as Keren and is the only previously unknown membrane-tethered EGF-like molecule identified by the Drosophila genome project. Keren has been referred to previously as Spitz-2 and Gritz. Like Spitz and Gurken, Keren has a single extracellular EGF repeat and a single transmembrane domain. The amino acid sequence of Keren is more closely related to Spitz than to Gurken (49% identity, 55% similarity to Spitz; 30% identity, 37% similarity to Gurken). While all three ligands were predicted to have N- and O-linked glycosylation signals, Spitz contains a 10 residue insert in its N-terminus, which contains an additional O-linked glycosylation site. Consistent with this, Spitz is the only ligand to be hyperglycosylated in the presence of Star, although deletion of the insert does not fully abolish hyperglycosylation. Rhomboids 1-4 all cleaved Keren in a mammalian tissue culture assay. There was, however, an interesting distinction between Keren and Spitz: Star was not essential for Keren secretion in every case. A significant amount of Keren was secreted in the presence of Rhomboids 3 and 4 alone. Despite this, Star always enhanced the secretion of cleaved Keren, implying that it can interact with Keren (Urban, 2002).

A key to understanding the regulation of Spitz activation by Rhomboid-1 and Star was the observation that the ligand was restricted to the ER in the absence of Star. The intracellular localization of Keren was examined in COS cells. Like Spitz, Keren was only detectable in the ER; it exhibited characteristic perinuclear and reticular staining and co-localized with the ER marker protein disulfide isomerase (PDI) (Urban, 2002).

Star's role in Spitz activation is to export Spitz from the ER to the Golgi apparatus where it encounters the proteolytic activity of Rhomboid-1. Star also promotes the release of Keren into the medium, suggesting its role is similar to that in Spitz processing. This relocalization of Keren is very similar to the relocalization observed for Spitz, suggesting that Keren also needs to be relocalized to the Golgi apparatus for efficient processing and secretion (Urban, 2002).

To test the prediction that Keren is a genuine EGF receptor ligand, it was misexpressed in developing Drosophila tissues. By analogy to similar experiments with Spitz, either full-length, membrane-tethered Keren (mKeren) was expressed, or a truncated form that corresponds to the extracellular, secreted form of Keren (sKeren). In most contexts, full-length Spitz is unable to signal when misexpressed because Star and Rhomboid-1 activity limit its activation, while a truncated form of Spitz, missing its transmembrane domain and C-terminus, signals in a Rhomboid-1- and Star-independent manner. In contrast to Spitz, ectopic expression of mKeren activates the EGF receptor pathway in both eyes and wings. For example, when expressed in the developing wing, mKeren produced wing phenotypes similar to misexpression of other positively acting members of the EGF receptor pathway, ranging from thickened and ectopic wing veins to blistering. In many cases, the activation was so strong that the entire wing was converted to vein-like material. These results indicate that either Keren has membrane-tethered, juxtacrine activity or that it is processed and secreted, possibly by Rhomboids 3 or 4, which would be consistent with the results obtained in the cell culture assay (Urban, 2002).

To test whether the activity of mKeren represents the full potential phenotype of ectopic Keren, or whether proteolytic activation has the potential to activate it further, the effects of sKeren were examined. This form is even more potent than mKeren, causing lethality even when driven by tissue-specific drivers. However, in the embryo, where mKeren has a weak effect, ubiquitous misexpression of sKeren causes lethality and results in significantly widened denticle belts and a reduction in naked cuticle in the ventral epidermis, identical to that caused by the misexpression of sSpitz. The greater potency of sKeren therefore suggests that Keren is proteolytically activated in vivo (Urban, 2002).

Together, these results indicate that Keren is a genuine ligand for the EGF receptor, being able to activate the receptor pathway in vivo. They also suggest that Keren is activated by Rhomboid proteases and Star, although it remains possible that the membrane-tethered form of the ligand has some juxtacrine activity (Urban, 2002).

Gurken function is restricted to oogenesis where it is required to polarize both major axes of the egg. Recent evidence strongly suggests that Gurken undergoes proteolytic processing in vivo: Gurken is released from the oocyte and is internalized by follicle cells, exists exclusively in a cleaved form in oocytes, and an uncleavable mutant form is inactive. Gurken can be processed directly by Rhomboid proteases 1-4. In the tissue culture assay, Rhomboid protease activity is required for Gurken cleavage and secretion. Although Rhomboid-1 is not required in the female germline, the specific expression of Rhomboid-2 in the early oocyte suggests that a Rhomboid might have a role in Gurken processing. Star can translocate Gurken from the ER to the Golgi apparatus in cell culture and, in some cases, enhance Gurken secretion. Together, these results strongly suggest that Gurken activity, like that of Spitz, is at least partially regulated by Star-dependent ER to Golgi transport. The regulation of Gurken activity, however, also depends on the transmembrane protein Cornichon. Recent evidence in yeast and Drosophila suggests that Cornichon is an ER export factor, raising the question of the relative roles and significance of Star and Cornichon (Urban, 2002).

Current data suggest two possible explanations for the multiplicity of Drosophila Rhomboids. First, several members appear to have tissue-specific functions. For example, Rhomboid-3 acts specifically in the developing eye. This duplication of function may reflect the complexity of regulation of EGF receptor activation. Rhomboid-1 is the principal determinant of EGF receptor activity and is regulated transcriptionally. The full gamut of transcriptional control may be too difficult to achieve in a single rhomboid gene. Partitioning this transcriptional regulation among multiple genes of similar biochemical activity could solve this problem. Note that although there is also a requirement for Star, which exists only as a single gene in Drosophila; its expression is not as restricted as that of Rhomboid-1, and its requirement is not absolute, as certain Rhomboids have the ability to release EGF receptor ligands in the absence of Star (Urban, 2002).

A second reason for the multiplicity of Rhomboids is suggested by their distinct characteristics: although all four cleave EGF ligands, they have different abilities to elicit cleavage and secretion of ligands in the absence of Star. As such, multiple Rhomboid-ligand combinations may result in distinct signaling characteristics such as intensity, duration or range (Urban, 2002).

Finally, the present analysis has been limited to Rhomboids 1-4. Rhomboid-5 lacks residues necessary for proteolysis, but the sequences of Rhomboids 6 and 7 suggest that they are proteases, and a key question will be whether these more distant members of the family are also dedicated to the EGF receptor pathway or whether they have distinct functions (Urban, 2002).

Search PubMed for articles about Drosophila Keren

Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B. and Hogness, D. S. (1997). Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. Cell 91: 777-788. PubMed ID: 9413987

Brown, K. E. and Freeman, M. (2003). Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye. Development 130: 5401-5412. 14507785

Fre, S., Huyghe, M., Mourikis, P., Robine, S., Louvard, D. and Artavanis-Tsakonas, S. (2005). Notch signals control the fate of immature progenitor cells in the intestine. Nature 435: 964-968. PubMed ID: 15959516

Hartenstein, A. Y., Rugendorff, A., Tepass, U. and Hartenstein, V. (1992). The function of the neurogenic genes during epithelial development in the Drosophila embryo. Development 116: 1203-1220. PubMed ID: 1295737

Jiang, C., Baehrecke, E. H. and Thummel, C. S. (1997). Steroid regulated programmed cell death during Drosophila metamorphosis. Development 124: 4673-4683. PubMed ID: 9409683

Jiang, H. and Edgar, B. A. (2009). EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors. Development 136(3): 483-93. PubMed ID: 19141677

McDonald, J. A., Pinheiro, E. M., Kadlec, L., Schupbach, T. and Montell, D. J. (2006). Multiple EGFR ligands participate in guiding migrating border cells. Dev. Biol. 296(1): 94-103. PubMed ID: 16712835

Li, T. R. and White, K. P. (2003). Tissue-specific gene expression and ecdysone-regulated genomic networks in Drosophila. Dev. Cell 5: 59-72. PubMed ID: 12852852

Micchelli, C. A. and Perrimon, N. (2006). Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439: 475-479. PubMed ID: 16340959

Ohlstein, B. and Spradling, A. (2006). The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439: 470-474. PubMed ID: 16340960

Ohlstein, B. and Spradling, A. (2007). Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling. Science 315: 988-992. PubMed ID: 17303754

Reich, A. and Shilo, B.-Z. (2002). Keren, a new ligand of the Drosophila epidermal growth factor receptor, undergoes two modes of cleavage. EMBO J. 21: 4287-4296. 12169631

Urban, S., Lee, J. R. and Freeman, M. (2002a). A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands. EMBO J. 21(16): 4277-86. PubMed ID: 12169630

van Es, J. H., et al. (2005). Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435: 959-963. PubMed ID: 15959515

date revised: 20 May 2009

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