hibris : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - hibris
Cytological map position - 51D11--E1
Function - receptor
Keywords - muscle fusion
Symbol - hbs
FlyBase ID: FBgn0029082
Genetic map position -
Classification - nine Ig-C2 type repeats and a Fibronectin type III domain
Cellular location - surface transmembrane
Hibris (Hbs) is a transmembrane immunoglobulin-like protein that shows extensive homology to Drosophila Sticks and stones (Sns) and the human kidney protein Nephrin. Hbs is expressed in embryonic visceral, somatic and pharyngeal mesoderm among other tissues. In the somatic mesoderm, Hbs is restricted to fusion competent myoblasts (the cells that fuse to founder cells) and is regulated by Notch and Ras signaling pathways. Embryos that lack or overexpress hbs show a partial block of myoblast fusion, followed by abnormal muscle morphogenesis. Abnormalities in visceral mesoderm are also observed. In vivo mapping of functional domains suggests that the intracellular domain mediates Hbs activity. Hbs and its paralog, Sns, co-localize at the cell membrane of fusion-competent myoblasts. The two proteins act antagonistically: loss of sns dominantly suppresses the hbs myoblast fusion and visceral mesoderm phenotypes, and enhances Hbs overexpression phenotypes. hbs is not continuously expressed in all fusion-competent myoblasts during the fusion process. S2 cell aggregation assays have revealed a heterotypic interaction between Hibris and Kin-of-irre (Kirre, formerly Dumbfounded), but not between Hibris and Irregular Chiasm-Roughest (Dworak, 2001). It is proposed that Hibris is an extracellular partner for Dumbfounded and potentially mediates the response of myoblasts to this attractant. The temporal pattern of hbs expression within fusion-competent myoblasts may reflect previously undescribed functional differences within this myoblast population (Artero, 2001; Dworak, 2001).
The general framework for understanding muscle cell fusion was provided by Doberstein (1997), who subdivided the process into steps: cell-cell recognition, adhesion, alignment of membranes and membrane fusion. Genetic studies in Drosophila have also provided fusion mutants (Paululat, 1999). Essential loci for myoblast fusion include the transcriptional regulator Mef2, the membrane bound protein Rolling stone (Paululat, 1997), cytoplasmic proteins such as Blown fuse (Doberstein, 1997), and components of the Rac1 signaling pathway such as Myoblast city and Drac1. Interestingly, overexpression of weak gain-of-function Notch constructs throughout the mesoderm can completely block fusion without interfering with earlier roles for Notch, suggesting that Notch is also involved in muscle morphogenesis. Another family of proteins that plays crucial roles in myoblast fusion in Drosophila is the immunoglobulin (Ig) superfamily. These Ig-containing proteins include Kirre (Ruiz-Gómez, 2000) and Sns (Bour, 2000). Although the described mutant phenotypes for kirre and sns are the same -- a complete fusion block -- their expression patterns in the somatic mesoderm are strikingly different. kirre is expressed exclusively in founder cells, while Sns is expressed exclusively in fusion-competent cells. Moreover, kirre is expressed during the entire fusion process and acts as an attractant for myoblasts, indicating that founder cells actively recruit fusion-competent cells until the final muscle size is achieved (Ruiz-Gómez, 2000). Sns, however, is a general marker for fusion-competent cells and, consistent with the segregation of these myoblasts, its expression depends on Notch signaling (Bour, 2000). Both Kirre and Sns are involved in the initial steps of myoblast cell-cell recognition, since free myoblasts in embryos mutant for either gene do not cluster around founder cells, suggesting that there is no recognition between founder cells and fusion-competent cells (Artero, 2001 and references therein).
To better understand muscle diversity and morphogenesis, a differential display screen was devised to identify genes specifically expressed in founder versus fusion-competent myoblasts. In short, use was made of the observation that muscle progenitor specification depends on Notch and Ras signaling to either repress or enhance muscle progenitor fate, respectively. Activated forms of Notch and Ras were overexpressed in Toll10b mutant embryos to reduce tissue complexity. Toll10b mutant embryos differentiate almost exclusively as somatic mesoderm (Artero, 2001).
A single differentially displayed band was chosen for further study because it was strongly upregulated under the activated Notch conditions. Corresponding cDNAs were isolated and the gene was named hibris. hbs is indeed dependent on Notch signaling. Northern blot signals suggested that hbs expression is upregulated at least twofold upon Notch activation, and repressed at least fivefold upon Ras activation (Artero, 2001).
Although hbs mutant flies survive to adult stages in normal numbers and show visible phenotypes such as rough eyes, reduced viability and semisterility, clear abnormalities are found in muscle development in mutant embryos. Embryos heterozygous for hbs459/Df(2R)X28, and different hbs transallelic combinations, were probed with anti Myosin antibody and compared with their wild-type counterparts. In embryos lacking hbs function, it was found that the muscle pattern is specified, although some muscles are occasionally missing or show abnormal morphologies (e.g. look thinner than normal or have fewer nuclei than normal). Immunocytochemistry with antibodies directed against founder cell markers such as Krüppel, Even-skipped and Runt reveals a normal pattern. These embryos, however, reproducibly showed a partial fusion block: an increased number of free myoblasts are present when compared with wild-type embryos. Unfused myoblasts are detected scattered around the muscles, surrounding the gut, heart and CNS. These free myoblasts show extended processes, indicative of their competence to scan for founder cells. In these experiments, stage 16 embryos were studied to ensure that free myoblasts have had the chance to fuse and that in this mutant background they fail to do so. Note, however, that by this developmental stage many unfused myoblasts might have undergone apoptosis, been phagocytosed and lost Myosin expression, decreasing the apparent number of unfused myoblasts. Loss of hbs function also interferes with the normal gut development as shown both by an enlargement of the first chamber as well as by loss of visceral muscle progenitors at stage 12 (Artero, 2001).
The GAL4/UAS system was used to assess the effect of hbs overexpression. When UAS-hbsFL was expressed throughout the embryonic mesoderm driven by twist-GAL4;DMef2-GAL4, defects were detected both in the somatic and visceral mesoderm similar to loss of hbs function. In the somatic mesoderm, a partial fusion block with free myoblasts scattered at several locations was found. In addition, these embryos normally show a greater degree of muscle loss than hbs homozygous mutant embryos. Defects in the visceral mesoderm, as revealed by anti-Fasciclin III staining, were also found. Because delivering extra Hbs to both founder and fusion-competent myoblasts leads to muscle losses, tests were performed to see whether all founder cells were correctly specified in these embryos. Antibody staining with the founder cell markers Krüppel, Even-skipped and Runt indicated that founder cells are correctly specified in embryos overexpressing Hbs. However, at later stages, aberrant morphologies were detected in the growing muscles (i.e. odd shapes), suggesting that prolonged Hbs expression interferes with muscle morphogenesis rather than the initial specification (Artero, 2001).
To dissect which parts of the protein are important for Hbs function, two additional constructs were tested. The constructs either deleted the extracellular (UAS-hbsDeltaECD) or the intracellular (UAS-hbsDeltaICD) region of the protein, but maintained the transmembrane domain such that the truncated proteins remain membrane anchored. Overexpression of UAS-hbsDeltaECD in the mesoderm results in detectable phenotypes, indicating that the intracellular domain of Hbs alone can interfere with muscle development. Specifically, a greater degree of fusion block and muscle loss as well as defects in the late pattern of founder cell markers were detected compared with overexpression of the full-length Hbs. This construct also causes greater defects in visceral mesoderm, leading to a greater reduction of visceral muscle progenitors. Altogether, these results suggest that the cytoplasmic domain of Hbs is required for hbs function in the mesoderm. By contrast, overexpression of UAS-hbsDeltaICD does not affect somatic muscle development appreciably but blocks visceral mesoderm development (Artero, 2001).
Since Sns, Kirre and Roughest are all Ig-C2-containing proteins implicated in myoblast fusion, their possible functional relationship with hbs was examined by testing for genetic interactions. For this analysis Df(1)w67k30 was tested: this deficiency removes both kirre and roughest, both of which are suggested to be partially redundant in the fusion process. hbs and Df(1)w67k30 do not interact genetically, either in loss- or gain-of-function hbs backgrounds. In addition, overexpression of hbs does not rescue any aspect of the Df(1)w67k30 phenotype (Artero, 2001).
Hbs and Sns co-localize at the cell membrane at discrete points, which would be consistent with both proteins working together in the fusion process. The doses of sns and hbs were manipulated. Stage 16 hbs homozygous mutant embryos characteristically show groups of free myoblasts throughout the embryo, and a gut phenotype was revealed early by anti-Fasciclin III or late by anti-Myosin staining. hbs-null embryos in which the sns dose is halved show dominant suppression of both the partial fusion block and gut phenotype. When hbs and sns double mutant embryos were analyzed, no noticeable change in the sns phenotype was detected. Thus, sns and hbs appeared to act antagonistically (Artero, 2001).
Consistent with the hypothesis that hbs and sns act antagonistically rather than cooperatively in myoblast fusion, sns mutations dominantly enhance hbs overexpression phenotypes. In the somatic muscles, there is an increase in the number of free myoblasts. This interaction also leads to a greater loss in visceral muscle progenitors, as measured by Fasciclin III expression. To quantify these observations, the number of gaps in Fasciclin III expression in stage 12 embryos was used as a measurement of genetic interaction between hbs and sns. The result of this quantification is consistent with the qualitative analysis. The dominant increase in Fasciclin III expression gaps when sns dose is lowered is statistically significant at the P<0.0001 level, indicating that a synergistic, rather than an additive, effect between hbs overexpression and heterozygosity for sns accounts for the increase in Fasciclin III expression gaps observed. As an additional test to determine the relationship between hbs and sns, attempts were made to rescue the sns loss-of-function phenotype by overexpression of hbs. In this experiment, Hbs does not rescue the sns loss-of-function phenotype in the somatic muscles in agreement with the genetic data. Altogether, the results of these experiments suggest that hbs antagonizes sns function during mesoderm development, but cannot differentiate whether sns is downstream of hbs or both genes are working in parallel pathways (Artero, 2001).
These data show that hbs overexpression gives a similar phenotype to hbs loss and suggest that either Hbs transduces a signal or these constructs behave as dominant negatives. In support of Hbs acting as a signal transducer, it was found that overexpression of UAS-hbsDeltaECD results in a partial block in somatic muscle development, indicating that the cytoplasmic domain of Hbs mediates the activity of the protein. A similar dependence on the cytoplasmic domain of Notch in the embryonic nervous system or EGF receptor signaling pathway antagonist Echinoid in the eye imaginal discs (Bai, 2001) has been used as support for these proteins acting as signal transducers. Alternatively, the overexpression data with both the full-length and intracellular domain constructs could be interpreted as Hbs exerting a dominant negative effect through the titration of another crucial component, such as another membrane protein or cytoskeletal component. This key protein could be a target of Hbs without Hbs actually transducing a signal in the classical sense. However, since the behavior of mutant combinations of sns and hbs is different (Hbs loss of function is suppressed by sns mutations whereas hbs overexpression is enhanced by sns mutations) the possibility is favored that Hbs transduces a signal rather than behaves as a dominant negative in the overexpression studies. It would be expected that sns heterozygosity would suppress both the hbs loss and overexpression phenotypes if hbs overexpression were acting as a dominant negative. These data also indicate that the extracellular domain of hbs does not behave as a dominant negative construct either, at least not in the somatic mesoderm, because overexpression of this construct does not cause a detectable phenotype in the somatic musculature (Artero, 2001).
Given the lack of enzymatic features in the cytoplasmic domain, it is proposed that Hbs associates with adapter proteins to connect to the cytoskeleton, cytoplasmic kinases or other transmembrane receptors. Interestingly, the intracellular domain contains motifs that are conserved in Sns. These motifs could potentially serve as interfaces for adaptor proteins. Work with Nephrin provides a putative candidate, the mouse CD2-associated protein (CD2AP). CD2AP, an SH3-containing cytoplasmic protein, has been shown to interact with the intracellular domain of Nephrin and has been proposed to anchor Nephrin to the actin cytoskeleton at the slit diaphragm in the kidney (Shih, 1999) and to participate in setting up the immunological synapse in T cells (Dustin, 1998). The Drosophila genome contains a CD2AP homolog, which is currently being analyzed (Artero, 2001).
Overexpression of UAS-hbsDeltaECD or UAS-hbsFL throughout the mesoderm results in a partial fusion block, but with greater muscle loss and morphological defects when compared with the loss of function phenotype. This increased muscle loss could reflect that, in overexpression experiments, Hbs is expressed in both fusion-competent as well as founder cells, thereby breaking the expression asymmetry that is found under normal situations. However, it could equally be supposed that prolonged and higher levels of Hbs block muscle fusion and disrupt later events in muscle morphogenesis. It is noted that both loss and gain of hbs result in a fusion phenotype. Although it is obvious why gain of a negative regulator could result in a fusion block, it is less clear why loss results in a similar phenotype. It is speculated that hbs highlights a novel aspect of the regulated events in the fusion process (Artero, 2001).
Cell recognition and adhesion between myoblasts is the first step of the fusion process (Doberstein, 1997). Kirre has been shown to be expressed in founder cells and behave as a myoblast attractant. By contrast, Sns is expressed in fusion competent myoblasts exclusively. A simple model can be proposed with these data: Kirre interacts at the cell surface with Sns to allow recognition between founder and fusion-competent cells (Taylor, 2000; Frasch, 2000). Subsequently, this recognition would signal the start of an orderly fusion process that requires such proteins as Rac, Blown fuse and Rolling stone. Later, myotubes continue to 'grow' in a directed manner by this process of attraction, recognition and fusion, sending growth cone-like structures toward specific attachment sites. This analysis suggests that Hbs acts at the first step of this process, since free myoblasts in hbs mutant embryos do not cluster nearby muscles (like sns) (Bour, 2000). In addition, Hbs serves as a negative regulator of Sns activity. This negative regulation is necessary to complete the fusion process since unfused myoblasts are found in hbs mutant embryos (Artero, 2001).
There are several ways in which Hbs could antagonize Sns function at the molecular level. (1) Hbs could compete for a putative Sns 'ligand'. Given the similarity (69%) of the extracellular domains of Hbs and Sns, this appears reasonable. However, overexpression of the extracellular domain of Hbs alone can not block myoblast fusion, while such fusion is blocked by the full-length construct. Moreover, the data support a requirement for activity of the intracellular domain of Hbs. (2) Hbs and Sns could form a receptor complex that switches the positive Sns signal to a negative signal. (3) Another possibility would have Hbs and Sns function independently of one another but converge intracellularly on downstream proteins involved in fusion. For example, Echinoid, an Ig domain protein resembling Hbs in overall structure, has been shown to antagonize the Ras pathway in the eye, not at the cell membrane but intracellularly at the level of transcription of a target gene, tramtrack (Bai, 2001). Hbs, Sns and Kirre may also be functioning together similarly in the generation of the visceral muscles, since recent data indicate that visceral muscles are not mononucleated, as previously reported, but syncytial (Artero, 2001 and references therein).
Hbs and Sns co-localize at the cell membrane, which could correspond to focal adhesion points between founder cell and fusion-competent cells or among fusion competent myoblasts. However, Hbs precedes Sns temporally in the somatic and visceral mesoderm at stage 11, prior to the initiation of fusion. The two expression patterns largely overlap during stage 12-13 as fusion begins, with some Sns and Hbs-specific foci of expression, and by late stage 14, unfused myoblasts are basically devoid of Hbs but maintain Sns as fusion continues towards completion. These differences in the pattern of expression of Sns and Hbs are meaningful since when hbs is genetically removed such that fusion competent cells express Sns exclusively, a muscle fusion phenotype is detected. Hbs could, therefore, be revealing different potentials for the fusion-competent cells during muscle formation. Initially, cells that fail to become muscle progenitors during the first round of segregation may be included in a new equipotential cluster. These clusters appear largely to overlap and form in rapid sequence. Early Hbs expression could participate in maintaining myoblasts that fail to become progenitors in the first round of selection in an uncommitted state, ready for successive rounds of progenitor segregation. This provides a reasonable explanation for occasional muscle losses in hbs mutant embryos. Subsequently, upon adoption of fusion-competent cell fate and expression of Sns, Hbs could regulate early events in fusion, maintaining an orderly process. During later events in the fusion process (i.e. while myotubes are already growing towards their specific attachment sites), when there are fewer free fusion-competent cells, Hbs may no longer be necessary to regulate or orient fusion. Thus, three 'fusion-competent cell' stages are proposed (Artero, 2001):
Since founder cell markers such as Krüppel and Even-skipped define subsets of founders, it is envisaged that additional markers will be found that serve to characterize these different fusion-competent cell stages. Moreover, the hbs enhancer trap provides a novel tool to recognize subsets of fusion-competent cells without the technical difficulties associated with a punctate, membrane-bound signal (Artero, 2001).
Clearly, Sns is not the only potential partner for Hbs. Although Sns is expressed in the visceral mesoderm and muscle attachments, it is not expressed in heart, midline, hindgut, Malpighian tubules, imaginal discs or adult tissues where hbs expression and phenotypes are found. Either Hbs is acting alone in these tissues or it is interacting with other Ig domain proteins. Similarly, Sns does not always act with Hbs. Like Kirre (and unlike Hbs) Sns is expressed in garland cells (Artero, 2001).
Although Hbs operates in multiple places, one unifying theme to hbs pleiotrophy is found -- an association with Notch signaling. The evidence demonstrates that hbs is a novel target of Notch activity in the somatic musculature. There are also several compelling similarities between the hbs and Notch activities over the course of development. Weak gain-of-function Notch constructs block myoblast fusion, one of the aspects of hbs phenotype. Notch is involved in planar polarity in the eye and both hbs loss- and gain-of-function lead to polarity defects. Preliminary results suggest that the hbs semisterility phenotype is due to failure to specify stalk cells in the ovarioles, a phenotype described for Notch loss-of-function mutations. hbs overexpression in adults results in misplacement of bristles and hbs and Notch interact weakly in the wings, resulting in a measurable increase in wing nicks. Perhaps like the Enhancer of split complex, hbs could mediate part of Notch signaling. Future work will be directed to uncover the interaction between these two genes throughout development (Artero, 2001 and references therein).
With the identification of Sns and Hbs on fusion-competent myoblasts, the question arises as to what the corresponding extracellular partners may be on the founder cells/forming myotubes. IrreC-rst and Kirre are the Drosophila members of the DM-GRASP/BEN/SC1 subfamily of the IgSF. kirre is expressed by the founder cells but not the fusion-competent myoblasts (Ruiz-Gómez, 2000), and irreC-rst is expressed in the embryonic mesoderm but the identity of the cells was not specified. The return of kirre expression to the mesoderm can rescue the phenotype; however, rescue with irreC-rst was not attempted. So the respective contribution of the two proteins to the fusion phenotype is uncertain. However, since Kirre misexpression can guide myoblasts to novel locations, it is considered a founder cell-derived attractant (Dworak, 2001 and references therein).
The similar fusion phenotype for the sns mutant and the kirre/irreC-rst deletion and the attractive properties of Kirre suggest Sns and Kirre underscore a fusion-competent myoblast-founder cell attraction mechanism (Frasch, 2000). Meanwhile, hbs overexpression in the somatic mesoderm partially phenocopies the sns loss-of-function mutant and the irreC-rst/duf deletion, suggesting reduced attraction of myoblasts to the myotubes. However myoblast fusion is also partially blocked when IrreC-Rst is overexpressed in the mesoderm, and myoblasts go to ectopic locations when Kirre is presented in the epidermis. So the Hbs gain-of-function phenotype could also be interpreted as the response of myoblasts to an imbalance of attractive forces (Dworak, 2001).
Support for Hbs mediating an attractive function comes from the S2 cell assays. Under the given assaying conditions, neither Sns nor Hbs interacts homotypically, and Hbs does not bind to Sns in trans. These observations contradict a model where Hbs might block in trans an Sns-mediated attraction between fusion competent myoblasts and bias the interaction towards the Kirre-expressing founder cells. In the S2 cell aggregation assay, both Hbs and Sns show an interaction with Kirre, mediating heterophilic adhesion between S2 cells. But neither Sns nor Hbs induce aggregates in combination with IrreC-Rst or Side. These results support a model where both Hbs and Sns facilitate the Kirre-induced attraction of fusion competent myoblasts to founder cells. But the results do not rule out other interaction combinations between these different proteins. Further experiments are required to determine if they act in cis or in complexes, or whether they require different conditions for binding to one another in trans in the S2 cell assay (Dworak, 2001).
When hbs is globally expressed with the da-GAL4 driver, the myoblast fusion defect is enhanced, and muscle fiber insertions are also misplaced. To determine whether the latter is due to hbs misexpression in the musculature or the epidermis, hbs was misexpressed in the epidermis with several GAL4 drivers. When misexpressed in the epidermis within a hemisegment, subsets of muscles fail to traverse the hemisegment and either bunch ventrally (en-GAL4 and sca-GAL4 drivers) or align with the segment boundary (pnr-GAL4 drivers). As such, the misplaced muscle attachment phenotype observed in the da-GAL4 gain-of-function condition is attributed to epidermally rather than mesodermally misexpressed hbs (Dworak, 2001).
hbs is broadly expressed in the epidermis around and at the sites where muscles will ultimately attach, then becomes confined to the muscle attachment sites themselves. During normal development, Hbs may assist in slowing and constraining myotube exploration in the region where attachments must ultimately form. The data on myoblast fusion links Hbs to an attraction/adhesion mechanism. Furthermore, Kirre is present in developing mytotubes (Ruiz-Gómez, 2000) and Sns is also present at the muscle attachment sites (Bour, 2000). Given these expression patterns and the heterotypic interaction of Kirre with Hbs and Sns, it is possible that these proteins also interact during myotube guidance, serving to direct myotubes to their expidermal attachment sites (Dworak, 2001).
This is the first evidence of a direct physical interaction between extracellular molecules that are expressed on either fusion-competent myoblasts and at muscle attachment sites (Hbs and Sns), or on muscle founder cells and developing myotubes (Kirre). These observations suggest that adhesion between these molecules may aid recognition between fusion competent myoblasts and founder cells, and between myotubes and epidermal attachment sites. Whether the large cytoplasmic domains on these proteins have signaling abilities must now be determined (Dworak, 2001).
hibris is regulated by Notch and Ras in a Toll10b mutant background. This regulation was confirmed in vivo in wild-type embryos. hbs expression was examined in Notch and Ras loss-of-function embryos and embryos overexpressing activated forms of Notch and Ras in the mesoderm. A dominant negative Ras construct activates hbs expression in the somatic mesoderm. Zygotic null Notch embryos show lower hbs transcription. Conversely, an activated form of Notch upregulates hbs in the mesoderm, while an activated form of Ras almost completely inhibits hbs expression. These results argue that, upon stimulation, Notch activates hbs, while Ras acts as a negative signal, and predicts that hbs expression in the somatic mesoderm would be restricted to fusion-competent cells (Notch dependent) and excluded from founder cells (Ras dependent). It is not known whether this regulation is direct, that is, Notch or Ras effectors act directly on the hbs promoter, or indirect, that is, Notch/Ras converts cell fate, which in turn would lead to hbs upregulation/downregulation by some other effector (Artero, 2001).
Sporadic evidence suggests Notch is involved in cell adhesion. However, the underlying mechanism is unknown. This study has investigated an epithelial remodeling process in the Drosophila eye in which two primary pigment cells (PPCs) with a characteristic 'kidney' shape enwrap and eventually isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that in the developing Drosophila eye the ligand Delta is transcribed in cone cells and Notch is activated in the adjacent PPC precursors. In the absence of Notch, emerging PPCs fail to enwrap cone cells, and hibris (hbs) and sns, two genes coding for adhesion molecules of the Nephrin group that mediate preferential adhesion, are not transcribed in PPC precursors. Conversely, activation of Notch in single IOCs leads to ectopic expression of hbs and sns. By contrast, in a single IOC that normally transcribes rst, a gene coding for an adhesion molecule of the Neph1 group that binds Hbs and Sns, activation of Notch leads to a loss of rst transcription. In addition, in a Notch mutant where two emerging PPCs fail to enwrap cone cells, expression of hbs in PPC precursors restores the ability of these cells to surround cone cells. Further, expression of hbs or rst in a single rst- or hbs-expressing cell, respectively, leads to removal of the counterpart from the membrane within the same cell through cis-interaction and forced expression of Rst in all hbs-expressing PPCs strongly disrupts the remodeling process. Finally, a loss of both hbs and sns in single PPC precursors leads to constriction of the apical surface that compromises the 'kidney' shape of PPCs. Taken together, these results indicate that cone cells utilize Notch signaling to instruct neighboring PPC precursors to surround them and Notch controls the remodeling process by differentially regulating four adhesion genes (Bao, 2014).
hbs expression in the mesectoderm and developing CNS midline partially overlaps with the expression of the transcription factor single minded (sim). In sim embryos, the mesectodermal progeny survive but fail to differentiate or migrate to appropriate locations. In sim mutant embryos, hbs expression is abolished at the CNS midline. When sim was misexpressed in all neuroblasts with the sca-GAL4 driver, the domain of hbs expression at the CNS midline was expanded. Yet when sim was misexpressed in all post-mitotic neurons using elav-GAL4 drivers hbs expression was unaltered (Dworak, 2001).
Notch is pivotal in the development of multiple tissue types. At the developing ventral midline, Notch activity is essential for establishing sim expression. Consequently, in NXK11 mutant embryos hbs expression is also lost at the CNS midline at stage 12 and onwards, after depletion of the Notch maternal contribution. In addition, Notch is crucial for the development of fusion competent myoblasts. In NXK11 mutant embryos, where myoblasts are transformed to founder cells, hbs expression is absent in the mesoderm. An examination was made to determine whether hbs is downstream of two mesoderm-specific transcription factors, bap and mef2. hbs expression in visceral mesoderm is greatly decreased in bap mutant embryos, but unaffected in the mef2 mutant embryos (Dworak, 2001).
Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).
The expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).
Proteins belonging to the Ig superfamily are frequently implicated in cell-cell adhesion. The ability of Hbs, Sns, Kirre, IrreC-Rst and Sidestep (Side) to bind homotypically was tested with the S2 cell aggregation assay. As a negative control, S2 cells were transfected with RmHa3 vector, and as a positive control, S2 cells were transfected with Fasciclin II-RmHa3. Homotypic aggregation was observed for Fasciclin II and Kirre. To test for heterotypic interactions, the S2 cells were labeled with either DiI (red) or DiO (green), and the aggregates were examined using confocal microscopy. When Fasciclin II-transfected cells (red) were mixed with RmHa3-transfected cells (green), all aggregates formed contained only red cells. Similarly, when Kirre-transfected cells (red) were mixed with RmHa3-transfected cells (green), aggregates were again all comprised of only red cells. When Kirre-transfected cells (red) were mixed with Hbs- or Sns-transfected cells (green), the resultant aggregates all had both red and green cells, but when Kirre-transfected cells (red) were mixed with RmaHa3- or Irrec-transfected (green) cells, all the resultant aggregates contained only red fluorescent cells. This is the first evidence suggesting that Nephrin proteins interact heterophilically in trans with other potentially non-Nephrin extracellular partners (Dworak, 2001).
Cell adhesion is essential for morphogenesis; however, the mechanisms by which cell adhesion coordinates precisely regulate morphogenesis are poorly understood. This study analyzes the morphogenetic processes that organize the interommatidial precursor cells (IPCs) of the Drosophila pupal eye. The Drosophila immunoglobulin superfamily members Hibris and Roughest are essential for IPC morphogenesis in the eye. The two loci are expressed in complementary cell types, and Hibris and Roughest proteins bind directly in vivo. Primary pigment cells employ Hibris to function as organizers in this process; IPCs minimize contacts with neighboring IPCs and utilize Roughest to maximize contacts with primaries. In addition, evidence is provided that interactions between Hibris and Roughest promote junction formation and that levels of Roughest in individual cells determine their capacity for competition. These results demonstrate that preferential adhesion mediated by heterophilic interacting cell-adhesion molecules can create a precise pattern by minimizing surface free energy (Bao, 2005).
To properly organize the ommatidia into a precise pattern, the interommatidial precursor cells (IPCs) undergo dynamic cell rearrangements between 18 and 42 hr after puparium formation (APF). These cells will eventually differentiate as secondary and tertiary pigment cells (2ºs, 3ºs) and mechanosensory bristles. Emergence of the interommatidial lattice was further analyzed with an antibody to the β-catenin ortholog Armadillo (Arm), a core component of the adherens junction. Based on this work, IPC and ommatidial patterning was classified into four stages (hours are based on the approximate center of the eye field), which are briefly described (Bao, 2005).
(1) Initial cell sorting (18-24 hr APF). Initially, IPCs are scattered between ommatidia with a relaxed apical profile. As development progresses, two cells emerge from the IPC pool to enwrap the cone cells and become 1ºs; the remaining IPCs simultaneously line up in single file to contact 1ºs from adjacent ommatidia. Concurrently, some cells are removed by apoptosis (Bao, 2005).
(2) Emergence of 3ºs (24-27 hr APF). Typically, three cells are initially positioned equally at a vertex. One cell reaches past the other two to contact a third 1º ; this cell will then physically “invade” the vertex and mature as a 3º (Bao, 2005).
(3) Selection of 2º s (27-36 hr APF). Cells that fail to become 3ºs either become 2ºs or are removed by programmed cell death. During this final cell-fate decision, cell-cell adhesion becomes visibly polarized as IPCs form detectable junctional contacts with 1º s but not with other IPCs. In addition, a 'scalloping' of membrane profiles is observed as 1ºs push between IPCs, further confirming that the adhesion between 1ºs and IPCs is greater than between neighboring IPCs. By 36 hr APF, the hexagonal pattern is essentially complete: it is composed of a single 2º at each side and a 3º or bristle organule at each vertex (Bao, 2005).
(4) Maturation (36-42 hr APF). Visible adherens junctions return to the interfaces between IPCs (now 2ºs and 3ºs). Contacts are now smoothed as the scalloping caused by invasive 1º contacts is now relaxed (Bao, 2005).
One particularly striking feature of this morphogenetic process is the dynamic nature of the cell junctions, which were visualized with the junctional protein Arm. For example, the level of Arm in the cone cells was constant but the levels of Arm in the IPCs decreased: this was seen by comparing the levels of Arm in the two cell groups. This drop in Arm levels is followed by its complete loss between IPCs after 3ºs emerge and eventual reemergence at the final maturation stage to levels similar to cone cells. Thus, junctions appear to be diminished during the period of maximal cell rearrangement, suggesting that IPCs are free to move during these stages (Bao, 2005).
Using laser ablation studies, it has been demonstrated that 1ºs are centrally important for the process of organizing IPCs into a correctly patterned interommatidial lattice. However, the mechanism by which one cell can provide such remarkably precise patterning information to a larger collection of uncommitted cells has not been not clear. The dynamic interactions between Hibris and Roughest provide such a mechanism (Bao, 2005).
The 'differential adhesion hypothesis' (DAH) proposes that sorting-out and segregation of cell populations are driven by differences in the intensities of cell adhesions. Given motile and cohesive cell populations, DAH predicts that weakly cohesive cells will tend to be displaced by more strongly cohesive ones; this process can direct cells to segregate away from unlike cell populations, and it can control tissue spreading during, for example, germ layer maturation in the embryo. DAH has been supported by several observations. For example, quantitative differences in the level of cadherin expression can lead two cell populations to be mutually immiscible: less cohesive cells will envelope more cohesive ones, creating a 'sphere within a sphere' configuration. Recently, the importance of differential adhesion for patterning developing tissue has been demonstrated in the pupal retina. Cone cells segregate from other cells and assemble into a simple pattern by minimizing surface area, as do soap bubbles. This assembly is mediated at least in part by E- and N-cadherins, and manipulating cadherin levels within the cone cells or their neighbors can alter the final cone cell pattern. These experiments illustrate that differential adhesion caused by differences in cadherin expression can mediate morphogenesis and pattern formation (Bao, 2005).
The current data indicate that IPC patterning follows a mechanism that shows unique aspects when compared with these classical DAH experiments. First, manipulating E-cadherin levels does not alter the morphogenesis or arrangement of IPCs. Even when two neighboring IPCs have higher levels of E-cadherin, adhesion between these two IPCs or their final patterning is not affected. More critically, IPCs do not aggregate together or segregate away from their neighbors. Rather, they separate away from each other to minimize IPC:IPC contacts, and aggregate with ommatidial cores to maximize 1º :IPC contacts. That is, the data indicate that IPCs have a preference for adherence to 1ºs. This preference can be seen most clearly at 27 hr APF: the junctions between IPCs and 1ºs are strong and elaborate; the junctions between IPCs are indistinct, and 1ºs are seen to push between IPCs to maximize contact and create a scalloping effect. The result is the precise aggregation of two different cell populations (Bao, 2005).
Why do IPCs sort away from other IPCs and preferentially adhere to 1ºs? The data indicate that interactions between Hibris and Roughest provide the mechanism. The immunoglobulin-class proteins Roughest and Hibris are utilized by IPCs and 1ºs, respectively, to form heterophilic interactions. Several lines of evidence support this view: (1) both Hibris and Roughest are required for proper interommatidial lattice assembly; (2) hibris is expressed in 1ºs as well as in cone cells and roughest is expressed in IPCs at the time of IPC rearrangement in the eye; (3) expression of ectopic Hibris in either the 1º or IPC is sufficient to relocalize Roughest protein -- conversely, downregulation of Hibris in 1ºs leads to decreased levels of Roughest protein at the 1º :IPC interface; (4) Hibris and Roughest are capable of directly binding each other when isolated in tissue culture experiments (Bao, 2005).
After 1ºs are specified and start to express Hibris, levels of Roughest protein decrease between IPCs and increase in the borders between IPCs and 1ºs; for example, at 30 hr APF, Roughest protein is undetectable between IPCs. Furthermore, ectopic Hibris in 1ºs is sufficient to attract still more Roughest protein toward the 1º :IPC border; by contrast, ectopic Roughest in 1ºs does not attract additional Roughest. It is concluded that although Roughest can show homophilic interactions in S2 cells, it strongly prefers heterophilic interactions with Hibris in situ (Bao, 2005).
Ubiquitous Hibris expression greatly increases the levels of cell-junction proteins between IPCs. Similarly, individual IPCs that received ectopic Hibris form E-cadherin-rich borders with neighboring IPCs that are sharp, straight, and significantly enlarged (Bao, 2005).
The evidence indicates that 1ºs and IPCs prefer to adhere to each other based on their expression of Hibris and Roughest, respectively. One principle of thermodynamics states that the binding of two adherent molecules will lead to a reduction of free energy within the system, provided the equilibrium constant of association (ka) is greater than the equilibrium constant of dissociation (kd). The essential role of Hibris and Roughest in IPC morphogenesis prompts making an assumption: among the various molecules being displayed in the surfaces of 1ºs and IPCs, Hibris and Roughest play a major role in determining the flow of free energy. Roughest has a higher affinity for Hibris than itself, and therefore heterophilic binding between Roughest and Hibris leads to a greater reduction in free energy. As a result, contacts between IPCs and 1ºs contribute to a reduction of free energy and are favored, while contacts between IPCs and IPCs do not contribute to reduction of free energy and are disfavored (Bao, 2005).
Other features of the developing pupal eye provide important components to this patterning process. After 1ºs are specified, they establish cell junctions with each other and with cone cells. These cone cell/1º units are not free to move within the epithelial plane and form a functional patterning unit. Therefore, 1ºs function as the organizers in this context. In contrast, IPCs have reduced levels of junctional proteins and are free to move within the epithelium. Numerous filopodia from IPCs observed by SEM studies also point to their potential for high motility. Taken together, these data suggest that IPC morphogenesis follows a preferential adhesion model: IPCs exhibit preferential adhesion to 1ºs; 1ºs function as organizers for IPC morphogenesis, and IPC:1º contacts are free energy favored while IPC:IPC contacts are disfavored (Bao, 2005).
The ommatidial clusters are poorly organized until 18 hr APF, when the morphogenetic movements of the IPCs begin to organize clusters into a hexagonal array. Preferential adhesion of IPCs to 1ºs yields two major outcomes. (1) IPCs compete to adhere directly to the limited, Hibris-rich surface presented by the 1ºs. High motility of IPCs permits this competition to proceed and achieve a favored configuration. (2) Preferential adhesion can also lead to the removal of cells that fail to contact a 1º . Specifically, IPCs that adhere to 1ºs have an increased chance to survive since the Hibris:Roughest interactions provide a greater opportunity to establish a stable junction. By the same token, those cells that do not have access to 1ºs are disadvantaged and are commonly dropped from the apical surface; these cells are likely to be eventually removed by programmed cell death. As a result, each stage proceeds with a progressive reduction of the IPC:IPC contacting surfaces and an increase in IPC:1º contacting surfaces (Bao, 2005).
At the onset of IPC morphogenesis (18 hr APF), the average size of IPC:IPC contacts is not significantly different from the size of IPC:1º contacts. During the time cells in multiple layers are sorted into single file after the initial cell-sorting stage (24 hr APF), IPC:IPC contacts are significantly reduced. After emergence of 3ºs, this reduction in IPC:IPC contacts is particularly dramatic. The IPC:1º contacts are increased by a scalloped profile, a further demonstration that IPC:IPC contacts are disfavored. To complete this pattern, therefore, IPC:IPC contacts are further minimized by reducing the number of candidate 2ºs to one cell between each 3º and bristle. Thus, IPC morphogenesis reveals a mechanism by which pattern is determined through minimizing disfavored cell-cell contacts and maximizing preferred cell-cell contacts (Bao, 2005).
Finally, it is interesting to note how 2ºs are selected. After emergence of 3ºs, two IPCs are commonly found between a 3º and bristle. In many ways, these two IPCs are equal: each contacts two 1ºs and each establishes equally strong cell junctions; each forms a scalloped contour with two neighboring 1ºs, and each is exposed to the same molecular cues. However, evidence is provided that these two cells have a low affinity for each other, a situation that is not favored by minimum free energy principles. One cell will be removed. How is this cell chosen? Clues came from manipulating levels of Roughest, which altered each cell's capacity for competition. Artificially high levels of Roughest rendered a cell a supercompetitor: the targeted cell even replaced two cells to become both a 2º and a 3º. Presumably, high levels of Roughest promote a higher level of cell junctions, which makes a cell more competitive and determines the survivor. Conversely, low levels of Roughest put the targeted cell at a disadvantage during this competition. Therefore, during the selection of a 2º , differing levels of Roughest expressed by each cell may determine its fate: survival or death (Bao, 2005).
Neph1/Nephrin family members are required for the development of a wide array of tissues including axonal pathfinding and myoblast fusion in Drosophila and formation of the slit diaphragm in the developing mammalian kidney. The role observed for preferential adhesion in IPC morphogenesis and patterning in the Drosophila eye leads to the interesting possibility that similar mechanisms are utilized broadly in pattern formation (Bao, 2005).
Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found in many parts of the nervous system. This study focused on the communication between retinal axons and their postsynaptic partners, lamina neurons, in the first ganglion of the Drosophila visual system, as a model for the formation of topographic maps. Post-mitotic lamina precursor cells differentiate upon receiving Hedgehog signals delivered through newly arriving retinal axons and, before maturing to extend neurites, extend short processes toward retinal axons to create the lamina column. The lamina column provides the cellular basis for establishing stereotypic synapses between retinal axons and lamina neurons. This study identified two cell-adhesion molecules: Hibris, which is expressed in post-mitotic lamina precursor cells; and Roughest, which is expressed on retinal axons. Both proteins belong to the nephrin/NEPH1 family. Evidence is provided that recognition between post-mitotic lamina precursor cells and retinal axons is mediated by interactions between Hibris and Roughest. These findings revealed mechanisms by which axons of presynaptic neurons deliver signals to induce the development of postsynaptic partners at the target area. Postsynaptic partners then recognize the presynaptic axons to make ensembles, thus establishing a topographic map along the anterior/posterior axis (Sugie, 2010).
This study shows that cell recognition between pre- and postsynaptic neurons via the Hbs-Rst interaction is required for the establishment of precise retinotopic mapping. During the development of the Drosophila visual center, presynaptic photoreceptors extend their axons to the lamina layer. Postsynaptic lamina precursor cells (pLPCs) start to differentiate in response to Hh delivered through newly arriving R axons. They then express Hbs, which interacts with Rst on R axons (see Model for the specific interaction between R axons and pLPCs mediated by an interaction between Hbs and Rst). This Hbs-Rst interaction is required for lamina column assembly, which underlies the topographic connections of the synapses along the anteroposterior axis (Sugie, 2010).
The process of lamina column assembly is unique in that presynaptic neurons regulate the development of postsynaptic partners in the target area, and the somata of postsynaptic neurons recognize the presynaptic axons at the developing stage well before neurite formation. This mechanism appears to be an efficient and accurate way to make a topographic map along the anterior/posterior axis. In addition, unlike the well-known axon guidance process, in which growth cones search for their targets, postsynaptic cells actively contribute to the pre- and postsynaptic interactions via direct communication. The changes in the Hbs localization that are associated with rst mutation were not only observed in pLPCs adjacent to R axons, but also in pLPCs far from R axons. This finding could be ascribed to the fact that pLPCs that are distant from R axons can contact R axons through their protrusions. Hbs might be preferentially localized at the protrusions of pLPCs that interact with R axons. The behavior of pLPCs is analogous to that of developing muscle cells, which extend filopodia to the axonal targeting of innervating motoneurons (Sugie, 2010).
Tests were performed to see whether the cell-adhesion mechanism mediated by Hbs and Rst was sufficient to rescue the sim phenotypes. Induction of exogenous hbs in pLPCs did not rescue sim loss-of-function mutants. Consistent with this finding, overexpression of sim using the NP6099-Gal4 driver caused the premature incorporation of pLPCs into the assembling domain, but overexpression of hbs did not. These results suggest that other molecules under the control of sim must be required for lamina column assembly (Sugie, 2010).
hbs expressed in photoreceptor cells does not play an essential role in lamina column assembly. The reason that Hbs originating in R axons does not interfere with the Hbs-Rst association remains unknown. The intracellular interaction of the two proteins might be blocked in R axons as a result of alternative subcellular localization and/or steric hindrance, or additional intermediates might be required for Hbs function in pLPCs, but not in R axons (Sugie, 2010).
Nephrin and NEPH1 homolog proteins tend to be located on opposing cell membranes so that they are brought into close apposition. This arrangement underlies the amazingly similar patterns of immunoreactivity in the eye disc, wing disc and somatic muscle as well as in the pupal optic lobe. These proteins are located in opposing cell membranes in the lamina. Consistent with previous studies, Hbs and Sns proteins were expressed in pLPCs, whereas Rst and Kirre were expressed in R axons; however, Hbs was also expressed in R axons. Recent studies have demonstrated that proteins of the nephrin and NEPH subfamilies are also expressed in neighboring cell types in vertebrate nervous systems. These observations reveal the conservation of nephrin/NEPH1 expression patterns across tissues and species (Sugie, 2010).
Previous work has identified SYG-1, a homolog of Rst, Kirre and NEPH1, as well as SYG-2, a homolog of Hbs, Sns and nephrin, which are necessary for synaptic specificity in Caenorhabditis elegans. The first Ig domain of SYG-1 and the first five Ig domains of SYG-2 are necessary and sufficient for binding and synapse formation in vivo. Similarly, it was found that the extracellular domain of Hbs and the first Ig domain of Rst are important for the association of pLPCs with R axons. These observations show remarkable functional conservation of the restricted domains of Drosophila and C. elegans nephrin/NEPH1 homologs (Sugie, 2010).
Further study of the preferential cell adhesion between nephrin/NEPH1 homolog proteins may reveal a common mechanism underlying the interaction between pre- and postsynaptic neurons in both Drosophila and vertebrate brains (Sugie, 2010).
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).
Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which is specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier (Kestilä, 1998).
Mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement and nephrotic range proteinuria; the recognition of these results emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. A cDNA including the full-length mouse nephrin open reading frame has been cloned and sequenced and immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin has been developed. Nephrin identified in mouse glomerular extract is a glycoprotein with an apparent molecular mass of 185 kDa. Nephrin is located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalizes to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. Nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm (Holzman, 1999).
The size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm, is described in this study. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognize a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein reveal reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remain negative. In immunogold-stained thin sections, nephrin label is found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggest an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data (Ruotsalainen, 1999).
The developmental expression of nephrin, ZO-1 and P-cadherin was examined in normal fetal kidneys and in NPHS1 kidneys. Nephrin and zonula occludens-1 (ZO-1) are first expressed in late S-shaped bodies. During capillary loop stage, nephrin and ZO-1 localize at the basal margin and in the cell-cell adhesion sites between developing podocytes, especially in junctions with ladder-like structures. In mature glomeruli, nephrin and ZO-1 concentrate at the slit diaphragm area. P-cadherin is first detected in ureteric buds, tubules, and vesicle stage glomeruli. Later, P-cadherin is seen at the basal margin of developing podocytes. Fetal NPHS1 kidneys with Fin-major/Fin-major genotype do not express nephrin, whereas the expression of ZO-1 and P-cadherin is comparable to that of control kidneys. Although early junctional complexes proved structurally normal, junctions with ladder-like structures and slit diaphragms were completely missing. The results indicate that nephrin is dispensable for early development of podocyte junctional complexes. However, nephrin appears to be essential for formation of junctions with ladder-like structures and slit diaphragms (Ruotsalainen, 2000).
Nephrin is a central component of the glomerular podocyte slit diaphragm and is essential for the normal renal filtration process. This study describes the complete structure of the mouse nephrin gene, which was shown to be homologous to the human gene, the major difference being 30 exons in the mouse gene as opposed to 29 in human. The complete primary structure of mouse and rat nephrins was also determined. The sequence identity between the mouse and rat proteins is 93%, while both rodent proteins have only about 83% sequence identity with human nephrin. The availability of the three mammalian sequences is significant for the interpretation of sequence variants and mutations in the nephrin gene in patients with congenital nephrotic syndrome. In situ hybridization analyses of whole mouse embryos and tissues has revealed high expression of nephrin in kidney glomeruli and, surprisingly, an intense and highly restricted expression in a set of cells in hindbrain and spinal cord. No expression is observed elsewhere. This expression pattern may explain occasionally occurring neural symptoms caused by inactivating mutations in the nephrin gene in patients with congenital nephrotic syndrome (Putaala, 2000).
CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associates with nephrin, the primary component of the slit diaphragm (Shih, 1999).
CD2-associated protein (CD2AP) is an adapter molecule that can bind to the cytoplasmic domain of nephrin, a component of the glomerular slit diaphragm. Mice lacking CD2AP exhibit a congenital nephrotic syndrome characterized by extensive foot process effacement, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function. The patterns of expression of both CD2AP and nephrin have been examined in developing mouse and human kidney. Both proteins are first detected in developing podocytes at the capillary loop stage of glomerulogenesis and eventually became concentrated near the glomerular basement membrane. CD2AP is observed diffusely in collecting duct and apically in many cells of proximal and distal tubule. Kidneys from Cd2ap -/- mice initially exhibit normal nephrin localization, but as the mice age and foot processes become effaced, nephrin disappears. In laminin-beta(2) mutant mice exhibiting nephrotic syndrome, CD2AP in glomeruli is aberrantly localized in a primarily punctate pattern. Extensive extrarenal expression of CD2AP is observed in endothelial and epithelial cells, in many cases with a specific subcellular localization. Together, these results suggest that CD2AP is not only involved in maintaining the slit diaphragm but may also have a general role in maintaining specialized subcellular architecture. The severity of kidney disease in Cd2ap mutant mice may have eclipsed manifestation of defects in other tissues (Li, 2000).
CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. CD2AP has been demonstrated to localize to the slit diaphragm in podocytes using immunoelectron microscopy, and nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm (Shih, 2001).
Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Src-family kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was co-transfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin (Li, 2004).
Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Using a surface plasmon resonance biosensor this study shows that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+) concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin forms cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that can promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction (Khoshnoodi, 2003).
Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin (Lenkkeri, 1999).
A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis has revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice shows that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy has demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space (Donoviel, 2001).
A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene is expressed in the brain and pancreas of (+/-) and (-/-) mice. In the brain, the gene is expressed in the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, expression is seen in radial glial cells. Neither anatomical nor morphological abnormalities are observed in the brains of null mice (Putaala, 2001).
Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. This study demonstrates that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria (Huber, 2001).
The recently identified gene NPHS1 with its mutations causing congenital nephrotic syndrome of the Finnish type (CNF) is highly promising in providing new understanding of pathophysiology of proteinuria. Changes in the expression levels of nephrin-specific mRNA in commonly used experimental models of proteinuria were examined using semiquantitative reverse transcription-polymerase chain reaction, immunofluorescence, and immunoelectron microscopy (IEM) of nephrin. Notably, a 40% down-regulation of the nephrin-specific mRNA of cortical kidney was seen already at day 3 after induction of the puromycin aminonucleoside nephrosis (PAN), while no major elevation of urinary protein secretion was seen at this stage. A further decrease of 80% of nephrin message was seen at the peak of proteinuria at day 10. A similar decrease of up to 70% from the basal levels was seen in mercuric chloride-treated rats. Changes in the protein expression paralleled those of the mRNA in indirect immunofluorescence. Interestingly, a remarkable plasmalemmal dislocation from the normal expression site at the interpodocyte filtration slits could be observed in IEM. It is concluded that Nephrin appears to be an important causative molecule of proteinuria and shows a remarkable redistribution from the filtration slits to the podocyte plasma membrane, especially in PAN (Ahola, 1999).
Since the discovery of the nephrin gene, many mutations have been reported in the NPHS1 gene in patients from diverse ethnic backgrounds. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, the fate of 21 such mutations identified in NPHS1 patients has been examined. Immunostaining of stable transfected cells expressing the nephrin mutants has demonstrated that most of the mutants show only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly shows an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, the data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1 (Liu, 2001).
The distribution of nephrin was examined by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. An extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern was found in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin co-localizes with IgG immune deposits. In the attempt to explain these results, an in vitro investigation was carried out to see whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induces the shedding of nephrin with a loss of surface expression. This phenomenon is abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site (Doublier, 2001).
Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. This study demonstrates that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, this study demonstrates that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. These findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes (Huber, 2003a).
The PDZ domain-containing protein zonula occludens-1 (ZO-1) selectively localizes to the cytoplasmic basis of the slit diaphragm, a specialized cell-cell contact in between glomerular podocytes necessary to prevent the loss of protein in the urine. However, the function of ZO-1 at the slit diaphragm has remained elusive. Deletion of Neph1, a slit diaphragm protein of the immunoglobulin superfamily with a cytoplasmic PDZ binding site, causes proteinuria in mice. This study demonstrates now that Neph1 binds ZO-1. This interaction was mediated by the first PDZ domain of ZO-1 and involved the conserved PDZ domain binding motif present in the carboxyl terminus of the three known Neph family members. Furthermore, Neph1 co-immunoprecipitates with ZO-1 from lysates of mouse kidneys, demonstrating that this interaction occurs in vivo. Both deletion of the PDZ binding motif of Neph1 as well as threonine-to-glutamate mutation of the threonine within the binding motif abrogated binding of ZO-1, suggesting that phosphorylation may regulate this interaction. ZO-1 binding was associated with a strong increase in tyrosine phosphorylation of the cytoplasmic tail of Neph1 and dramatically accelerated the ability of Neph1 to induce signal transduction. Thus, these data suggest that ZO-1 may organize Neph proteins and recruit signal transduction components to the slit diaphragm of podocytes (Huber, 2003b).
The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. This study shows that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. This Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo (Jones, 2006).
Search PubMed for articles about Drosophila hibris
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Doublier, S., et al. (2001). Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome. Am. J. Pathol. 158(5): 1723-31. 11337370
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date revised: 23 July 2014
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