hbs expression was analyzed by in situ hybridization in embryos and in imaginal discs. hbs transcripts were first detected at stage 8 in precursors of the amnioserosa and in the mesectoderm, where they are maintained in progeny of these midline cells throughout embryogenesis. By stage 10, hbs was expressed in the visceral mesoderm in a pattern reminiscent of bagpipe. These cells maintained hbs expression while forming the visceral mesoderm surrounding the gut. By late stage 11, hbs is expressed in somatic mesoderm where its expression peaks between stage 12 and early stage 13, and subsequently declines, disappearing by stage 14. By stage 12 there is expression in the precursors of the heart, where hbs is maintained throughout the remainder of embryogenesis. Late hbs expression is detected in the epidermis at putative muscle attachment sites, hindgut and pharyngeal muscles (Artero, 2001).
Immunohistochemical staining reiterated the same embryonic expression pattern of the Hbs protein. Notably, Hbs localization at the cell membrane is consistent with the protein structure predicted by the sequence data. Hbs is detected weakly in the somatic mesoderm by late stage 11. This expression increases around stage 12 and 13, yet declines thereafter, and mature muscles are completely devoid of Hbs. Hbs is expressed at specific points in the myoblast membrane, as described for its paralog, Sns (Bour, 2000). This localized expression is detected also in the hindgut, at putative epidermal attachment sites and in the midline (Artero, 2001).
Transgenic flies carrying a 4 kb fragment of hbs genomic sequence fused to a lacZ reporter reproduce aspects of hbs expression and provide an additional tool with which to study hbs. Embryos containing this reporter inserted in the hbs locus (P[w+]36.1) show expression in visceral and somatic mesoderm, mesectoderm, pharyngeal muscles and hindgut in a pattern similar to that found with the anti-Hbs antibody. To reinforce further that P[w+]36.1 recapitulates hbs expression in the mesoderm, ß-galactosidase expression and hbs transcription in P[w+]36.1 embryos were simultaneously detected, confirming there is overlap between both signals. Because of the high degree of conservation between Hbs and the kidney protein Nephrin, hbs expression was analyzed in Malpighian tubules in the reporter P[w+]36.1. Interestingly, weak expression was detected at stage 12/13 in a subset of tubule cells that, based on their morphology, appear to be stellate cells (Artero, 2001).
Since these results suggested that hbs was excluded from founder cells and was present in fusion-competent cells, several additional experiments were performed to confirm this possibility. Co-localization experiments using either P[w+]36.1 that reiterates Hbs expression in the mesoderm or Hbs antibody and various founder cell markers reveal that Hbs is absent from founder cells. These experiments also suggest that Hbs is not expressed in all fusion-competent cells during myoblast fusion. It was therefore asked whether Hbs and Sns, a protein known to be expressed exclusively in fusion-competent cells (Bour, 2000), co-localize in fusion-competent cells. Wild-type embryos double labeled with antisera against Sns and Hbs show several examples of co-localization of these proteins. During stage 11, when muscle progenitors are being specified and the fusion process has not begun, Hbs expression is initially more widespread in somatic and visceral mesoderm than that of Sns. Subsequently, as the process of muscle fusion begins, Hbs expression is less widespread than Sns. It is noted that at this stage, although overlap was detected in Hbs and Sns expression, cases of Sns and Hbs-specific expression are found. At later stages of the fusion process, Hbs protein is no longer detected in the remaining fusion-competent cells, in contrast to the continued presence of Sns. Co-localization of both Hbs and Sns in the visceral mesoderm is also found. It is concluded that Hbs expression initially precedes Sns, but then largely overlaps with Sns, suggesting that Hbs could modulate the fusion-promoting properties of Sns in fusion-competent cells during the course of myoblast fusion (Artero, 2001).
hbs is expressed in a highly dynamic manner across tissue types and life stages. At stage 5, along the dorsal surface of the cellularized embryo, a strong narrow band of hbs expression is present that extends along approximately two-thirds the length of the embryo. This band of expression broadens laterally, decreases in length, and becomes confined to the dorsal furrows. By stage 8, dorsal expression is still present at the furrows, and hbs expression also begins ventrally, where it is associated with the mesectodermal cells. Expression strengthens in the mesectodermal cells as they move into close juxtaposition with one another at the ventral midline, forming neighboring columns (across stages 9 and 10). Expression continues during stage 11 as the mesectodermal cells intermingle, divide and move internally. By stage 12, as the midline axonal scaffold is forming, a subset of midline cells posterior to the developing posterior commissure continue to express hbs. The number of hbs-expressing cells at the midline decreases so that by late stage 14 there are two to three hbs-expressing cells below the posterior commissure. Expression in these cells is absent by stage 15. Double-labeling of glial cells with anti-Repo mAb reveals that a subset of the exit glia at the edge of the CNS are hbs positive from stages 12 to 15 (Dworak, 2001).
In the periphery during stage 10, laterally located clusters of cells begin expressing hbs and are distinct patches at stage 11. These cells are visceral mesoderm, as their nuclei co-label with the anti-myocyte enhancer factor 2 polyclonal antibody (anti-MEF2). By late stage 11, the somatic and visceral mesoderm expresses hbs. During stage 12, hbs is clearly present in the fusion competent myoblasts. This expression is truly restricted to fusion competent myoblasts. In Notch mutant embryos, all myoblasts adopt a founder cell fate and in NXK11 mutant embryos, there are no hbs-positive myoblasts. Similarly, co-labeling with anti-Kruppel antibody reveal that the fusion-competent myoblasts, but not the founder cells, are hbs positive. By stage 14, expression in the somatic mesoderm has ceased. As mesodermal expression decreases, epidermal expression begins. At stage 12, this expression is several cells broad and occurs at the segment boundary and in lateral patches. It becomes confined to the muscle attachment sites by stage 14. Along the dorsal edges of the embryo lie some hbs-positive cells. These cells are MEF2 negative, identifying them as the pericardial cells (Dworak, 2001).
In third instar larvae, in the eye/antennal disc, hbs expression is strong behind the morphogenetic furrow, and also as clusters within the furrow. In the larval brain there is hbs expression in the optic lobes. Expression in the larval wing disc consists of a striking cruciform pattern, corresponding to the regions that abut the presumptive wing margin, and those areas destined to be wing veins L3 and L4. More proximally expression is found in the region destined to be wing veins L0 and L1. There is also light expression in the presumptive notum region. In leg discs expression is seen in concentric circles (Dworak, 2001).
hbs is expressed in imaginal tissues, most notably in the eye-antennal, wing, leg and haltere discs (Artero, 2001).
The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the 'garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function (Zhuang, 2009).
In Drosophila, the Immunoglobulin superfamily (IgSF) proteins encoded by kin of irre [kirre; also known as dumbfounded (duf)], roughest (rst), sticks and stones (sns) and hibris (hbs) function as ligand-receptor pairs on the surface of founder cells and fusion competent myoblasts. These proteins mediate recognition, adhesion and fusion to form multinucleate syncitia through direct interaction at sites of myoblast contact. However, neither their action nor their expression is exclusive to the musculature, and previous studies noted their role in cell recognition and adhesion in the Drosophila eye. Moreover, multiple studies have confirmed the presence of the kirre transcript and sns transcript in the binucleate garland cell nephrocytes (GCNs). These nephrocytes possess a structure visible by transmission electron microscopy (TEM) reminiscent of the slit diaphragm (SD) in the vertebrate kidney, and process waste products from the hemolymph. It is therefore compelling that the fly detoxification machinery may have similarities to that in mammals, and that Sns and Kirre play roles similar to those of their vertebrate counterparts (Zhuang, 2009).
Removal of waste products from the closed circulatory system of vertebrates takes place in the kidney glomerulus. Podocytes, kidney epithelial cells that surround the capillary blood vessels, extend foot processes that contact the surface of these vessels. Filtration then occurs as molecules flow out of the bloodstream through slits between adjacent foot processes into the urine. Neph1, vertebrate orthologs of the above Drosophila IgSF proteins, localize to this filter and appear to be an important determinant of glomerular permeability (Hamano, 2002; Liu, 2003). Mutations in nephrin and neph1 are associated with congenital nephrotic syndrome as a consequence of defects in this filtration diaphragm. Lack of either nephrin or neph1 leads to podocyte foot process effacement and detachment of podocytes from the glomerular basement membrane, loss of SDs, and proteinuria (Donoviel, 2001; Putaala, 2001; Zhuang, 2009 and references therein).
In addition to their high degree of homology, Nephrin and Neph1 have other features in common with Sns and Kirre. Heterophilic interactions occur in trans between the extracellular domains of Nephrin and Neph1, and Sns and Kirre. Studies have suggested that, in addition to serving as a scaffold onto which other proteins in the SD assemble, Nephrin and Neph1 function as signaling molecules to direct downstream cytoplasmic events (Benzing, 2004). They cooperate to transduce a signal that directs actin polymerization (Garg, 2007), and activation of this pathway occurs through interaction of phosphorylated tyrosines in the cytoplasmic domains of Nephrin and Neph1 to adaptor proteins (Jones, 2006; Verma, 2006). These adaptor proteins recruit components of the actin polymerization machinery that include N-WASp and Arp2/3. Similar phosphotyrosine modifications are important for Sns function and studies have shown that the WASp and Arp2/3 actin polymerization machinery functions in Drosophila myoblast fusion, probably downstream of Sns and Kirre (Zhuang, 2009).
The pericardial cells and garland cells comprise two subpopulations of Drosophila nephrocytes that, along with Malpighian tubules, form the excretory system. Approximately 25-30 tightly associated binucleate GCNs encircle the anterior end of the proventriculus in a 'garland' at its junction with the esophagus. The cortical region of the cytoplasm includes elaborate channels that are generated by invagination of the plasma membrane during embryogenesis and early larval instar stages. The initial invagination is associated with formation of a junction between two sites on the plasma membrane that are visible by TEM. Through a mechanism that is not entirely clear, this initial invagination expands into an extensive array of labyrinthine channels by the third-instar larval stage. The GCNs are very active in endocytosis via coated vesicles at sites deep within these labyrinthine channels. Thus, molecules to be eliminated must gain access to the endocytic machinery deep in these channels. These studies also identified a thin bridge spanning the channel opening that is visually similar to the vertebrate SD. The presence of Sns and Kirre and a slit diaphragm-like structure in these binucleate cells raised the possibility that these IgSF proteins might function in GCN fusion and/or in formation of this structure (Zhuang, 2009).
This study, along with that of Weavers (2009) demonstrates that Sns and Kirre are present in, and crucial for, the nephrocyte diaphragm (ND). Knockdown of Kirre or Sns results in a severely diminished number of NDs and smoothening of ND-associated furrows on the GCN surface, implicating Sns and Kirre in their formation. Mutations in the extracellular domain of Sns cause major perturbations in the ND, establishing that Sns also dictates fundamental aspects of its structure. Similar smoothening of the GCN surface occurs upon knockdown of Polychaetoid (Pyd), the Drosophila ortholog of the zonula occludens (ZO-1) tight junction protein that interacts with Neph1, providing strong support for functional conservation of these molecules. The ND controls access of molecules to the labyrinthine channels for uptake by endocytosis, and can discriminate between molecules of different sizes in a rate-dependent manner. Finally, in contrast to that reported by Weavers (2009) and reminiscent of their action in the embryonic musculature, Sns and Kirre contribute to the adhesion of the GCNs into an organized garland and their fusion into binucleate cells (Zhuang, 2009).
These data those of Weavers (2009) demonstrate that the GCNs have significant structural and functional similarities to podocytes in the mammalian kidney. Sns and Kirre are instrumental in directing and/or stabilizing interactions at sites of membrane invagination that become the NDs. These proteins parallel the role of their mammalian orthologs Nephrin and Neph1 in the SD that forms between podocyte foot processes in the kidney glomerulus. In addition, Sns and Kirre mediate tight adhesion between GCNs in the embryo, and, in contrast to Weavers this study notes that these proteins also direct GCN fusion. Both proteins are expressed during larval life and significant cell death occurs in their absence. Sns clearly plays a specific structural role in the ND that is perturbed by mutations in its extracellular domain. Finally, the SD and ND both mediate the flow of molecules between the circulatory system and the excretory system, and appear to discriminate between molecules on the basis of size and rate of passage (Zhuang, 2009).
The GCNs are thought to process waste material and detoxify the insect hemolymph, its open circulatory system, through a process of endocytosis and degradation. Endocytosis occurs from sites deep within labyrinthine channels that form by invagination of the plasma membrane, and proteins associated with endocytosis localize to the cortical region of the cytoplasm in membranes associated within these channels. The channels and associated membranes expand in mutants that block endocytosis, and compounds such as horseradish peroxidase, dye-conjugated BSA or avidin, and various dextrans, readily pass through the plasma membrane into these channels. Access appears to occur through a structure that is reminiscent of the SD in vertebrates. This study has shown that this nephrocyte diaphragm is dependent on the presence of Sns and Kirre, and that perturbation of the Sns extracellular domain causes obvious defects in the ND. Thus, IgSF homologs appear to be a structural component of this access point in both insects and vertebrates (Zhuang, 2009).
The number of NDs decreases significantly upon knockdown of Sns or Kirre, but a small number still remain. The uptake of large molecular tracers is severely diminished under these conditions, suggesting that the NDs are a major route of access to the endocytic machinery within the labryinthine channels. Perhaps more revealing relative to the initial findings of Weavers, it was found that the uptake of small molecules is slower under conditions of Sns or Kirre knockdown but ultimately achieves normal levels. Thus, like the SD, the ND appears to be more permeable to small molecules. Interestingly, studies in vertebrates have addressed the relative contributions of the podocyte basement membrane and the slit diaphragm to glomerular permeability, and Nephrin and Neph1 were found to be crucial. Moreover, electron tomography has identified Nephrin as a decisive determinant for filtration of molecules larger than BSA (Zhuang, 2009).
Nephrin and Neph1 are capable of forming both homodimers and heterodimers, and these abilities could reflect interactions that occur in vivo in cis and/or in trans. The diameter of the vertebrate SD is consistent with a model in which this distance could be spanned by homophilic interaction of Nephrin or heterophilic interaction between Neph1 and Nephrin in trans. The similar diameter of the Drosophila ND therefore supports a model in which interactions between the Kirre and Sns ectodomains determine this distance. The exact molecular interactions remain to be determined, however, and may differ in vertebrates and Drosophila. For example, Nephrin is capable of homophilic interactions in trans, a property that Sns does not appear to have. Thus, it seems unlikely that Sns spans this distance, as suggested for Nephrin. Homophilic interactions of Kirre, which can occur, could serve this purpose. One might then predict the spacing to be decreased from the observed 30-35 nm due to the shorter extracellular domain of Kirre. Of note, kinetic studies in Drosophila S2 cells indicate a strong preference for interaction with Sns. Moreover significant levels of Sns or Kirre remain in GCNs from second instar larvae upon knockdown of the corresponding partner, yet the number of NDs is diminished. Localization of each protein by immunoEM analysis under these conditions may prove to be illustrative in this regard. Given the above interaction studies and fact that both proteins are continuously present in the GCN, a model is favoed in which heterotypic interactions are preferred as in the embryonic musculature. One fundamental difference between Sns and Kirre in the embryonic musculature and the GCNs is that they are expressed in different myoblast cell types but co-expressed within individual garland cells. However, their co-expression in GCNs is another feature in common with Nephrin and Neph1 in vertebrate podocytes (Zhuang, 2009).
It is unclear whether Sns and Kirre function through interactions with signal transduction components that parallel those of Nephrin and Neph1 in the GCNs. Signaling molecules thought to be downstream of Sns and/or Kirre in the musculature, and known to be downstream of Nephrin, include N-WASp and components of the Arp2/3 pathway. One other functional parallel between the SD and ND is that of the tight junction protein Pyd, which contributes to formation of ND-associated furrows on the surface of the GCN. Although Pyd interacts biochemically with two different forms of Kirre, it remains to be shown whether this interaction occurs through postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding sites in Kirre, as observed for binding of its vertebrate counterpart ZO-1 to Neph1 (Zhuang, 2009).
GCNs become binucleate before or immediately after their assimilation into the garland of cells that surrounds the esophagus at its junction with the proventriculus. This binucleate nature seems almost invariant, with cells rarely remaining mononucleate or having more than two nuclei. Although an explanation for this invariance is not apparent, the cell appears to accommodate multiple processes to ensure it. Quantitation of cells and nuclei over time, the absence of dying GCNs, and time-lapse imaging suggest that cell fusion is the primary mechanism utilized by wild-type GCNs, and that the IgSF proteins contribute to this process. Some mutant cells are still binucleate, but the possibility cannot be eliminate that other molecules contribute to GCN fusion or that these IgSF proteins function in yet more redundant ways to drive this fusion. Perhaps a drive to become binucleate has forced the cell to compensate for defects in fusion in other ways, such as cell division without cytokinesis. Although all efforts to address such a mechanism have yielded negative results, behavior of this type may be another common property between insect garland cell nephrocytes and mammalian podocytes (Zhuang, 2009).
To assay the effects of loss of hbs function, both new deficiencies were generated around region 51D7 and EMS-induced mutations were generated. For two of the EMS-induced mutations recovered, 2593 and 459 (hereafter hbs2593 and hbs459), nucleotide changes were identified in the hbs transcription unit, therefore establishing that they were hbs alleles. hbs2593 mutation is a T to A transversion in the second nucleotide of the intron between exons 9 and 10. The mutation leads to an aberrantly spliced transcript that retains the whole intron and results in a prematurely truncated protein. hbs459 is an A to T transversion in exon 7 that generates a stop codon (Artero, 2001).
Immunohistochemistry provided further confirmation that these mutations led to loss of hbs function. In these experiments, hbs2593 embryos showed a dramatic decrease in Hbs protein, while hbs459 homozygous embryos show no detectable protein. Allele hbs361 shows protein expression at levels indistinguishable from wild type but behaves at least as strongly as hbs459 in phenotypic assays (Artero, 2001).
In order to determine the function of Hbs during embryonic development, a series of deletions were generated by irradiation of nearby P-elements. For EP(2)2590, over 50 w- flies were isolated from the 71,700 progeny screened and 13 stable lines were successfully established, while for l(2)k04218, 55,600 progeny were screened and 29 lines established. Deletion 12 removes hbs expression as assayed by in situ hybridization with cDNA4, and is lethal over l(2)k06403 but not l(2)k04218. As the smallest deletion removing hbs, this line has a phenotype where the ventral muscle pattern is abnormal in two to three hemisegments per mutant embryo. The abnormality consists of a loss of some muscle fibers from the ventral muscle group. In hemisegments where the muscle patterning is normal, motor innervation is also normal. Deletion 11 does not remove or disrupt hbs expression, yet also shows the ventral muscle defect that was seen in Deletion 12 (as does Deletion 6). Furthermore, in deletions 11 and 12 transheterozygotes, the muscle phenotype is present. As such, the ventral muscle phenotype maps to a gene(s) in the region other than hbs. It appears that hbs mutant embryos do not have an overt muscle phenotype because: (1) the muscle phenotype in deletion 12 is no different from that in the transheterozygote deletions 11 and 12 embryos; (2) muscle number, insertion sites and innervation are normal in the unaffected hemisegments; and (3) unfused myoblasts are barely evident in late stage 16. The nervous system was also examined across stages 12-17 with mAbs BP102, 1D4, 22C10, anti-Wrapper and anti-Repo, and no defects were found. As such, Hbs appears functionally redundant in the development of the embryonic somatic mesoderm and central nervous system. Deletion 12 in trans with sns, irreC-rst or the duf/irreC-rst deletion does not produce phenotypes in the embryonic muscles or the adult (Dworak, 2001).
Overexpression of hbs in the mesoderm in homozygous UAS-hbs;twi-GAL4 embryos partially disrupts myoblast fusion, but not muscle fiber number or sites of muscle attachment. This phenotype is evident in all hemisegments of all embryos. When hbs is misexpressed using the da-GAL4 driver, unfused myoblasts are again present, and in all hemisegments, some muscle fibers are inserted at inappropriate attachment locations. twi-GAL4 expression is restricted to mesoderm, while da-GAL4 is expressed in all tissues, suggesting that aberrant muscle fiber attachments may be due to hbs misexpression in the epidermis. To further test this idea, hbs was misexpressed in the epidermis with additional GAL4 drivers. Driving in the Engrailed pattern in the epidermis with en-GAL4 disrupts attachments made by several lateral muscle fibers in most hemisegments of all embryos. This is strongly exacerbated, occurring in all hemisegments of all embryos, by overexpressing more broadly and strongly in the lateral epidermis with sca-GAL4, a GAL4 line that drives in the sca pattern in the epidermis and CNS. Driving with pnr-GAL4 in the dorsal ectoderm, dorsal muscle attachments sites are radically disrupted, with the muscle fibers often failing to cross the segment and instead aligning with the segment boundary. Unfused myoblasts are also seen in the epidermal gain-of-function embryos (Dworak, 2001).
The effects of increased hbs expression in imaginal discs were assessed using drivers giving general or specific domains of expression. The distal wing margin was abnormal in omb-GAL4/+;UAS-hbs/+ and UAS-hbs/+;da-GAL4/+ flies. There was a blistered appearance at the distal wing edge in all omb-GAL4/+;UAS-hbs/+ flies, and blistered or notched distal wing margins in the UAS-hbs/+;da-GAL4/+ flies. On the dorsal thorax (notum) the large anterior section (scutum) has two mechanosensory bristle populations: macrochaetes (large bristles) and microchaetes (small bristles). Microchaetes on the central scutum are in 10 longitudinal rows and fairly constant numbers. Misexpression of hbs in the wing discs with pnr-GAL4, sca-GAL4 or da-GAL4 perturbs the linear arrangement of the microchaetes, but their number is unaltered (Dworak, 2001).
Driving UAS-hbs with GMR-GAL4 or sca-GAL4 in the eye-antennal disc gave a rough eye phenotype with disorganization of the ommatidia and bristles. Driving with the da-GAL4 driver gave the strongest rough eye phenotype with occasional fusion of ommatidia. Examination with anti-Elav antibody shows that the photoreceptor clusters are irregularly placed, and pigment cells are absent at sites of ommatidia fusions. Larval photoreceptor pathfinding and targeting appears normal when examined with mAb 24B10 (Dworak, 2001).
Overexpressing a secreted form of Hbs with all of the aforementioned GAL4 drivers did not generate gain-of-function phenotypes. In addition, the adult hbs gain-of-function phenotypes were not suppressed as transheterozygotes with sns, irreC-rst or the duf/irreC-rst deletion (Dworak, 2001).
Thus misexpression of hbs in the wing disc yields abnormal distal wing margins and disorganized microchaetes on the notum. At first glance, these phenotypes look like mild loss-of-function Notch defect; however, microchaete numbers do not deviate from normal. Hence, the microchaete phenotype appears to be due to displacement of cells, rather than changes in cell number caused by disturbance of lateral inhibition. This could arise if: (1) the cell-cell associations within proneural clusters are slightly perturbed, resulting in subtle changes in the location of the founder cells; or (2) founder cells are normally specified but subsequent alterations in cell-cell associations lead to their being slightly displaced (Dworak, 2001).
Misexpression of hbs in the eye disc results in a rough eye phenotype, which is reminiscent of that seen when irreC-rst is absent or misexpressed in the eye disc. Yet neither gain-of-function eye phenotype is suppressed by a 50% decrease in the expression of the other gene, and no rough eye phenotype, is observed in the hbs and irreC-rst transheterozygotes. Since Hbs and IrreC-Rst did not give a positive result in the S2 cell aggregation assay, and the overexpression of irreC-rst in the wing imaginal disc causes a notal microchaete phenotype that differs from that for hbs, there is still no direct support that Hbs and IrreC-Rst interact directly with one another in a simple one-to-one trans binding relationship. More suitable interaction analyses await elucidation of whether Kirre and Sns have roles in eye (Dworak, 2001).
The body wall muscle of a Drosophila larva is generated by fusion between founder cells and fusion-competent myoblasts (FCMs). Initially, a founder cell recognizes and fuses with one or two FCMs to form a muscle precursor, then the developing syncitia fuses with additional FCMs to form a muscle fiber. These interactions require members of the immunoglobulin superfamily (IgSF), with Kin-of-IrreC (Kirre) and Roughest (Rst) functioning redundantly in the founder cell and Sticks-and-stones (Sns) serving as their ligand in the FCMs. Previous studies have not resolved the role of Hibris (Hbs), a paralog of Sns, suggesting that it functions as a positive regulator of myoblast fusion and as a negative regulator that antagonizes the activity of Sns. The results reported in this study resolve this issue, demonstrating that sns and hbs function redundantly in the formation of several muscle precursors, and that loss of one copy of sns enhances the myoblast fusion phenotype of hbs mutants. It was further shown that excess Hbs rescues some fusion in sns mutant embryos beyond precursor formation, consistent with its ability to drive myoblast fusion, but show using chimeric molecules that Hbs functions less efficiently than Sns. In conjunction with a physical association between Hbs and SNS in cis, these data account for the previously observed UAS-hbs overexpression phenotypes. Lastly, it was demonstrated that either an Hbs or Sns cytodomain is essential for muscle precursor formation, and signaling from IgSF members found exclusively in the founder cells is not sufficient to direct precursor formation (Shelton, 2009).
Sns and Hbs function redundantly in the initial fusion event between founder cells and FCMs. As observed in other mutants, precursor formation in sns mutant embryos is delayed over that occurring in wild-type embryos, but is readily observed in stage 13 embryos in at least some segments. By contrast, no fusion was observed by late stage 15 in sns, hbs double mutant embryos. Although the possibility of a temporal delay of fusion in sns, hbs double mutants cannot be eliminated because reporter expression declines after this stage, a model is favored in which a crucial first step is not occurring in the absence of both Sns and Hbs. Using new FCM reporters that facilitate quantitation of unfused myoblasts, re-examination of the hbs loss-of-function phenotype reveals that the loss of one copy of sns actually worsens the hbs mutant phenotype, as expected if these proteins have some functional redundancy. Finally, both snsGal4 and mef2Gal4 directed Hbs can drive a significant amount of fusion in sns mutants, arguing that Hbs is capable of directing fusion beyond precursor formation (Shelton, 2009).
Although Hbs can rescue the sns mutant phenotype beyond precursor formation, replacing any domain of Hbs with the comparable domain of Sns improves the ability of the chimeric protein to rescue fusion over that achieved by Hbs alone. The activity of the Hbs cytodomain is most dramatically different from that of Sns, providing an explanation for the observation that intact Hbs or a membrane-anchored Hbs cytoplasmic domain both interfere with myoblast fusion in wild-type embryos. Rather than acting as an antagonist of Sns, these high levels of Hbs probably interfere competitively with endogenous Sns. First, an excess of Hbs may drive its interaction with a limiting component that is normally used more efficiently by Sns. Alternatively, given their ability to form hetero- and homodimers in vivo, excess Hbs may sequester Sns in a less functional form. Although the data do not fully resolve this issue, the co-localization of Hbs and Sns is consistent with the latter model. Of note, dimer formation between the related IgSF proteins Boc and Cdo can be directed by sequences in both the extracellular and intracellular domains (Kang, 2002), and both the extracellular and intracellular domains of Sns are capable of mediating its interaction with Hbs, raising the possibility that either full-length Hbs or a membrane-anchored cytodomain may sequester Sns under conditions of overexpression (Shelton, 2009).
The finding that Hbs functions positively but much less efficiently than Sns in directing later rounds of myoblast fusion provides an explanation for the previously observed behavior of Hbs in overexpression assays (Artero, 2001; Dworak, 2001). Additionally, the data appear to be inconsistent with a model in which excess Sns is deleterious, as inferred if a decrease in sns copy number compensates for the loss of hbs (Artero, 2001). The possibility that Sns activity is negatively regulated cannot be excluded. Possible mechanisms could include limitations in the machinery for tyrosine phosphorylation, such that unphosphorylated Sns even in excessive amounts would be unable to transduce a signal to downstream events. Downstream targets of Sns may also be limiting, such that no further activation of the pathway can be accomplished by Sns. It is also noted that Sns protein is transient, appearing just before fusion and being eliminated shortly thereafter. Despite the issue of whether Sns activity is regulated in some fashion, the data are not consistent with a model in which its activity is negatively regulated by endogenous Hbs (Shelton, 2009).
Current models for myoblast fusion suggest that it occurs in two steps that differ genetically and/or temporally. Consistent with the two genetically distinct steps, fusion does not occur in embryos mutant for genes encoding the guanine nucleotide exchange factors Schizo, Mbc or Duf and Rst. By contrast, precursor formation is observed in embryos lacking the Hem-2/Nap1 homolog Kette, the Kirre-associated protein Rols, the Arp14D/66B regulators WASp and Vrp1 or Sns. These data support a model in which the molecular requirements for precursor formation differ from those for subsequent myotube formation. An alternative model, using three dimensional analyses and quantitating fusing myoblasts over time, revealed that fusion occurs in two temporal phases, comprising an initial phase of limited fusion between cells that are in close proximity and a second phase when most myoblast fusion occurs. Moreover, precursor formation is temporally delayed in embryos lacking molecules such as Rols and Kette, suggesting that these molecules do influence the first step in fusion (Shelton, 2009).
The present study does not address whether the genetic requirements for precursor formation differ from those for subsequent rounds of fusion, or whether these steps utilize the same set of proteins. The data do not eliminate the possibility of two distinct genetic steps, with Sns and Hbs acting redundantly in precursor formation but not in later events. Hbs is capable of directing precursor formation in the absence of Sns. However, the ability of Hbs to drive fusion beyond precursor formation when in excess, and the observation that removal of one copy of sns enhances fusion defects in hbs mutants, suggests that Hbs can assist in later rounds of myoblast fusion. These data are consistent with models in which molecular interactions in precursor formation and subsequent fusion differ kinetically but not genetically. One possibility, independent of the process of fusion itself, is that Sns and Hbs differ in their ability to drive FCM cell migration. Although the role of cell migratory behavior in myoblast fusion is unclear, the ability to migrate may contribute to the rate of fusion. While these questions remain to be addressed, the present study advances the understanding of fusion by resolving the interaction of two proteins that function early in the process, thereby providing additional perspectives for sorting out the different mechanisms of myoblast fusion (Shelton, 2009).
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date revised: 10 February 2010
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