polychaetoid

DEVELOPMENTAL BIOLOGY

Polychaetoid protein is localized at the cell-cell junction. Observation of the presumptive wing blade region of the wing imaginal disc reveals that Pyd has a more basal distribution compared with Shotgun (Takahisa, 1996). Pyd colocalizes with the scaffolding protein Canoe. In the cellular blastoderm (stage 5), the Cno protein is distributed diffusely in the cytoplasm, with significant accumulation at the apical surface. The cytoplasmic staining decreases before gastrulation. In stage 13 embryos undergoing germ-band retraction, marked accumulation of Cno is observed in the amnioserosa, with persistent expression of Cno in the lateral epidermis. The intense staining of the amnioserosa and the apposed edges of the lateral epidermis continues during dorsal closure. At this stage of embryogenesis, the trachea in each segment begins to elongate laterally to form the tracheal system across the segments. In addition, Cno is localized in Malpighian tubules, hindgut and the central nervous system. The tissue localization of Pyd is remarkably similar to that of Cno. It is present in the cytoplasm in the blastoderm stage embryo. In the later stages, Pyd is exclusively localized to cell boundaries. The epidermis, amnioserosa, the margin of the closing epidermis, the tracheal system, the Malpighian tubes, the hindgut and the CNS all express Pyd at high levels (Takahashi, 1998).

Ectodermal epithelium was examined for Pyd and Cno colocalization. The two proteins partially colocalize: Pyd expression is more widespread than Cno expression. The domain of Cno expression and that of Fas III expression are mutally exclusive, whereas the distributions of Arm and Drosophila alpha-catenin coincide with that of Cno. In contrast, Pyd is expressed in areas at which Fas III is localized. Fas III distribution is known to be restricted to septate junctions, and Drosophila alpha-catenin and Armadillo are confined to adherens junctions. Cno colocalizes with Arm but not with Fas III in the embryonic epidermis. Thus the results indicate that Pyd is present at both the septate and adherens junctions while Cno exists predominantly at adherens junctions (Takahashi, 1998).

To examine the Cortactin cellular localization, Cortactin was immunostained in epithelial cells of imaginal discs. The typical honeycomb-like images indicate that the protein distributes in a cell-cell contact-associated manner. To clarify the subcellular localization, the double stainings of Cortactin with Pyd, F-actin, and DE-cadherin (Shotgun) were conducted using a laser-scanning confocal microscope. DE-cadherin is a component of the adherens junction and localizes at the apicolateral region of epithelial cell junctions. The distribution of Pyd partially overlaps with that of DE-cadherin and extends to the slightly basal region, corresponding to the site of the septate junction. Colocalization of Cortactin, Pyd, and DE-cadherin is evident, while the staining area of Cortactin in the periplasm seemed slightly broader than that of either Pyd or DE-cadherin. Colocalization of Cortactin and F-actin in a periplasmic region is also observed. Regarding the apical-basal axis, the distribution of Cortactin extends from the basal half side of the adherens junction to the more baso-lateral region (Katsube, 1998).

Polychaetoid is required for dorsal closure of the embryo, sensory organ patterning, and cell fate specification in the developing eye. pyd is alternatively spliced resulting in two isoforms that differ by the presence or absence of exon 6. To determine the role of alternative splicing in Pyd function, antibodies specific for each isoform were generated. The exon 6⁺ form of Pyd is localized at adherens junctions of embryonic and imaginal epithelia, while the exon 6^- form is distributed broadly along the lateral membrane. These results suggest that localization of Pyd is controlled by alternative splicing and raises the possibility that exon 6 represents a distinct protein-protein interaction domain (Wei, 2001).

The temporal expression pattern of the two alternatively spliced pyd transcripts was determined by performing stage-specific RT-PCR, with the primers ex5-5' and ex7-3', using samples from 0-2, 2-4, 4-6, 6-8, and 8-24 h embryos, L3 larvae, early pupae and adults. The pyd6¹ form is present at all stages examined. However, transcripts at 0-2 h of embryogenesis represent both maternal and zygotic gene expression. RT-PCR with RNA isolated from unfertilized eggs demonstrates that the pyd6⁺ transcript is expressed maternally. The pyd6^- form is detected in 8-24 h embryos, L3 larvae, pupae and adults but the amount of PCR product is always less than that of the pyd6⁺ form. To determine more precisely when the pyd6^- transcript can first be detected, RT-PCR experiments with 8-12, 12-16, and 16-24 h embryos were conducted. The transcript is present at 8-12 h of embryogenesis, but the amount of PCR product is extremely low compared to the level of pyd6⁺ at the same stage. The level of the pyd6^- product increases at later developmental stages (late embryonic, L3, pupae, and adult) and the relative expression level of the two isoforms remains quite constant at these stages. Given that the same primers are used to detect both the 6⁺ and 6^- forms, and that the 6^- product is smaller than the 6⁺ product, it is thought that the consistently lower levels of the 6^- product are likely to reflect lower levels of expression of the 6^- isoform of the pyd transcript (Wei, 2001).

Cells are connected to neighboring cells and to the extracellular matrix by specialized junctions. In invertebrates, cell-to-cell junctions include adherens, septate and gap junctions. Note that the septate junction, which is thought to be functionally similar to the vertebrate tight junction, is basal to the adherens junctions. To determine the cell junction localization of Pyd proteins a series of immunofluorescent labeling experiments was performed in conjunction with specific cell junction markers (Wei, 2001).

The Drosophila wing disc is a monolayer of epithelial cells connected by adherens and septate junctions. Double labeling of wing discs with anti-Pyd (6⁺ and 6^- forms, respectively) and antibodies against septate junction proteins, Dlg or Coracle (Cor) shows that the 6⁺ form is apical to Dlg and Cor, suggesting that it is localized apically to septate junctions. The 6^- form is distributed more broadly since anti- Pyd 6^- staining is detected both apically and basally to Dlg and Cor (Wei, 2001).

Armadillo and E-cadherin are located at adherens junctions. Double labeling of wing discs was performed with antibody against E-cadherin (Shotgun) or Armadillo, and antibodies against the Pyd 6⁺ or Pyd 6^- isoforms. The Pyd 6⁺ isoform demonstrates co-localization with Shotgun and Arm, suggesting that Pyd 6⁺ is localized at adherens junctions. However, antibody staining of the Pyd 6^- isoform and Arm shows slight displacement of Pyd 6^- from the adherens junctions and its extension to a more basal region. These results suggest that the Pyd 6^- form is localized broadly around the cell membrane in imaginal discs. Results from double staining of the ectodermal epithelia of stage 11-14 embryos with antibodies against Pyd 6⁺ and Shotgun or antibodies against Pyd 6^- and Arm also support the above observations (Wei, 2001).

Expression of pyd6⁺ cDNA from a heat shock promoter can rescue the lethality of a pyd null mutation (pyd^C5) and also greatly suppresses the extra bristle phenotype of pyd^C5/pyd^J14 flies. The following experiments were carried out to study the role pyd6^- plays in SOP specification. The pyd6^- cDNA was inserted in the transformation vector CaSpeR under the control of the hsp70 promoter, and transgenic lines were generated. Flies were subjected to a heat shock regime. The experimental conditions were the same as those used for rescue of pyd^C5 lethality by the pyd6⁺ cDNA. Progeny were collected in vials and exposed repeatedly to a 37°C water bath for 1 h every 12 h from early embryogenesis (about 4-8 h after the eggs were laid) to the mid-pupal stage. Parents were heat-shocked at 37°C for 1 h and allowed to recover at 25°C for 1 h before collecting eggs, or remained continuously at 25°C (no heat shock). pyd 6^- does not rescue the lethal phenotype of pyd^C5. However, overexpression of pyd 6^- cDNA does significantly reduce the extra bristle phenotype of pyd^J14/pyd^C5 flies. Overexpression of Pyd 6^- in a second independent hs-pyd6^- transgenic line also fails to rescue the lethal phenotype of pyd^C5, but does suppress the extra bristle phenotype of pyd^J14/pyd^C5 flies. A similar suppression of the extra bristle phenotype is observed with overexpression of the pyd6⁺ cDNA, suggesting that both isoforms can function in SOP patterning. Overexpression of both isoforms simultaneously produces a more complete suppression of the extra bristle phenotype than either isoform alone, suggesting that both isoforms are involved in patterning of SOPs (Wei, 2001).

The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm

The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation1 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. This study shows that the insect nephrocyte has remarkable anatomical, molecular and functional similarity with the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialised filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. Fly orthologues of the major constituents of the slit diaphragm, including nephrin, neph1, CD2AP, ZO-1 and podocin are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, the nephrocyte diaphragm is completely lost in flies mutant for nephrin or neph1 orthologues, a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in the human kidney disease NPHS1) or neph1. These changes drastically impair filtration function in the nephrocyte. The similarities described between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting the two cell types are evolutionarily related and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated disease (Weavers, 2009).

Filtration of blood in the vertebrate kidney occurs within the glomerulus of the nephron (see The glomerular and nephrocyte filtration barriers are anatomically similar). The filtration barrier is formed by podocytes, specialised epithelial cells, which send out interdigitating foot processes to enwrap the glomerular capillaries. These processes are separated by 30-50nm wide slit pores spanned by the slit diaphragm, which together with the glomerular basement membrane (GBM), form a size- and charge-selective filtration barrier. Disruption to this barrier in disease leads to leakage of blood proteins into the urinary space and to kidney failure (Weavers, 2009).

Although invertebrate excretory systems are considered to lack nephrons, 'nephron-like' components, such as filtration cells and ducts in which the filtrate is modified, are widespread (see The glomerular and nephrocyte filtration barriers are anatomically similar). Insect nephrocytes regulate haemolymph composition by filtration, followed by endocytosis and processing to sequester and/or secondarily metabolise toxic materials. Drosophila has two types - garland and pericardial nephrocytes. They are tethered to the oesophagus, and are bathed in haemolymph. Extensive infolding of the plasma membrane generates a network of labyrinthine channels or lacunae flanked by nephrocyte foot processes. The channel entrances are narrow slits 30nm in width, spanned by a single or double filament forming a specialised filtration junction; the nephrocyte diaphragm. Each nephrocyte is enveloped by basement membrane. The nephrocyte diaphragm and basement membrane behave as a size and charge-selective barrier and filtrate is endocytosed from the sides of the lacunae. Thus the anatomy of the nephrocyte and podocyte filtration barriers are remarkably similar (Weavers, 2009).

In view of this similarity, whether the nephrocyte diaphragm is molecularly related to the slit diaphragm was investigated. The major slit diaphragm components, the transmembrane Ig-domain superfamily proteins nephrin and neph1 are co-expressed in the podocyte and interact across the slit pore by homo- and hetero-typic binding to form the diaphragm. Mutations in nephrin, as in human congenital nephrotic syndrome of the Finnish type (NPHS1), cause slit diaphragm loss and foot process effacement, resulting in breakdown of the filtration barrier and proteinuria (Weavers, 2009).

Drosophila has two nephrin orthologues - sticks and stones (sns) and hibris (hbs) - and two neph1 orthologues - dumbfounded (duf) and roughest (rst). Since hbs and rst are expressed in only a subset of nephrocytes, focus was placed on sns and duf. Sns and Duf are expressed throughout life in both nephrocyte types, from midembryogenesis for garland cells and from the first larval instar for pericardial cells. Interestingly, the onset of Sns and Duf expression correlates in time with the appearance of the nephrocyte diaphragm at the ultrastructural level and double labelling reveals precise co-localisation. This finding is initially surprising because in most contexts Sns and Duf are expressed in complementary patterns and mediate interaction between cells of different type. The only other situation where the two types of Ig-domain proteins are co-expressed in the same cell is the vertebrate podocyte. Sns and Duf are dependent on one another for stabilization at the plasma membrane. Loss or knockdown of either protein in embryonic or larval nephrocytes leads to a loss, severe reduction or mislocalisation of the other. These data demonstrate an essential interaction between the two proteins in the same cell, similar to those between nephrin and neph1 in the podocyte. The precise subcellular location of the proteins was revealed by immuno-electron microscopy. Both Sns and Duf specifically localise to the nephrocyte diaphragm and double labelling reveals close colocalisation between the two proteins (Weavers, 2009).

Garland and pericardial nephrocytes are correctly specified in sns and duf mutants. However, given the importance of the Ig-domain proteins in slit diaphragm formation, the ultrastructure of the diaphragm was examined in sns and duf mutants. In wild-type garland cells, nephrocyte diaphragms and associated lacunae appear during mid-embryogenesis, progressively increasing in number. Diaphragms densely populate the cell periphery in third instar larvae. Strikingly, sns or duf mutant garland cells completely lack nephrocyte diaphragms at every stage and lacunae are rarely detected. Occasional infoldings do form, but are never bridged by diaphragms. Instead, the nephrocyte surface contains frequent, small patches of electron-dense subcortical material, possible remnants of undercoat normally associated with the wild-type diaphragm. These observations suggest that in the absence of the diaphragm, foot processes are unstable and undergo effacement. Scanning electron microscopy reveals the surface smoothening in mutant garland cells. These phenotypes are remarkably similar to those of podocytes lacking nephrin or neph1. Thus, by analogy with nephrin and neph1 in the slit diaphragm, it is suggested that Sns and Duf interact through their extracellular domains to form the nephrocyte diaphragm itself (Weavers, 2009).

It is noted that the basement membrane in sns knockdown and duf larval nephrocytes was irregular and dramatically expanded. The basement membrane in duf nephrocytes has an average depth of 202nm compared with 57nm for wild-type. This results from an increase in deposition of the Drosophila collagen IV (Viking). However this is unlikely to account for the four-fold thickening observed, and it is suggested that a further contributing factor is accumulation of haemolymph proteins that clog the basement membrane due to inefficient filtration (Weavers, 2009).

Given the similarities between the morphology and molecular requirements for podocyte and nephrocyte diaphragms, the ability of human nephrin to rescue the sns mutant phenotype was tested. However nephrocytes are sensitive to absolute levels of sns, so that even moderate overexpression produced abnormal phenotypes. Therefore the effects were compared of overexpressing Drosophila sns with human nephrin. Resulting phenotypes are strikingly similar, including abnormal nephrocyte foot process morphology and marked thickening of diaphragm filaments. These data indicate that precise levels of Sns are critical for diaphragm formation and more importantly that human nephrin and Drosophila Sns function in equivalent ways (Weavers, 2009).

Vertebrate nephrin and neph1 form a multi-protein complex at the slit diaphragm with zonula occludens-1 (ZO-1). Mutations in these genes result in kidney disease. It was asked whether the fly orthologues contribute to the nephrocyte diaphragm. in situ hybridisation reveals that pyd (ZO-1), CG31012 (CD2AP) and Mec2 (NPHS2/podicin) are expressed in nephrocytes. Furthermore, Pyd-GFP precisely co-localises with Duf to the membrane, mirroring co-localisation of ZO-1 and neph1 in the podocyte (Weavers, 2009).

Molecular interactions between these vertebrate slit diaphragm-associated proteins have been established. To test whether fly orthologues form a similar complex, a yeast two-hybrid analysis was performed with Sns and Duf intracellular domains. Sns interacts with Mec-2 (podocin) and Duf interacts with Pyd (ZO-1). Interaction between Duf and Pyd was independently confirmed by co-immunoprecipitation. A previous report established direct association between Sns and Duf. These interactions between the fly proteins closely resemble those described for slit diaphragm-associated proteins. These data, taken together with those described above, provide strong evidence that the nephrocyte diaphragm slit diaphragm are molecularly homologous structures (Weavers, 2009).

Insect nephrocytes are size and charge-selective in their sequestration of materials from the haemolymph. Selectivity is based on the characteristics of the diaphragm and basement membrane, which act together as a filtration barrier. To test filtration capacity of the Drosophila nephrocyte diaphragm the passage of fluorescently-labelled dextrans of different sizes was assayed. If the nephrocyte diaphragm acts as a size-selective filter it was reasoned that, like the vertebrate slit diaphragm, it would allow free passage of small (10,000mw) but exclude large (500,000mw) dextrans. In agreement with these expectations, uptake of the 500,000mw dextran in wild-type nephrocytes is significantly lower than the 10,000mw dextran. These data strongly suggest that the nephrocyte diaphragm functions as a size-based filtration diaphragm (endocytosis from foot process tips could account for low levels of large dextran uptake). Higher uptake of the large dextran is anticipated in Ig-domain mutant nephrocytes because they lack diaphragms. However, while the level of uptake of the small dextran in duf or sns nephrocytes is unaltered compared to wild-type, a dramatic reduction is found in large dextran uptake; large to small ratio is 1:22.5 for duf and 1:15.3 for sns. Instead, the large dextran appears as a halo surrounding the cell. The thickening of basement membrane observed in duf nephrocytes could explain the exclusion of large dextran. This highlights a further parallel between nephrocytes and podocytes. An endocytosis-based clearance mechanism in podocytes prevents clogging of the GBM with blood plasma proteins; the slit-diaphragm associated protein CD2AP has been implicated in this process. It is suggested that an equivalent clearance mechanism exists in nephrocytes and that this mechanism requires Sns and Duf functions (Weavers, 2009).

Whatever the causes of reduction in filtration capability, the animal's haemolymph physiology will be disturbed. This hypothesis was tested by feeding larvae silver nitrate, a toxin endocytosed and concentrated in nephrocytes. At low concentrations of silver nitrate, viability of control larvae is not compromised but duf larvae show a greatly reduced viability. A previous study showed a requirement for nephrocytes in the face of toxic stress (Das, 2008). The current data show that Ig-domain proteins are essential for this functions (Weavers, 2009).

This study has highlighted similarities between podocytes and nephrocytes but podocytes are an integral part of the nephron, whereas the nephrocyte is spatially separated from its renal (Malpighian) tubule. Such differences have contributed to the traditional view that vertebrate and invertebrate excretory systems are unrelated. Nevertheless, nephron-like features are present in the excretory systems of a wide variety of invertebrates and in the protochordate Amphioxus, suggesting a common origin. The molecular parallels between nephrocytes and podocytes described in this study support this hypothesis, and it will be of interest to determine whether nephrin/neph-like protein complexes are found in other invertebrate filtration diaphragms (Weavers, 2009).

Defects in the slit diaphragm complex underlie human diseases whose unifying feature is proteinurea and kidney failure. These symptoms result from defective filtration, but in addition the nephrin/neph1 complex regulates podocyte behaviours such as cell survival, polarity, actin dynamics and endocytosis. How these functions of the slit diaphragm relate to disease pathologies is presently unclear. The fly nephrocyte also depends on the activity of a nephrin/neph1 complex for survival, shape and selective endocytosis and thus provides a simple and genetically tractable model in which the multiple roles of the slit diaphragm complex can be addressed (Weavers, 2009).

Effects of Mutation or Deletion

In Drosophila sensory organ development, the balance of activities between proneural genes and repressor genes defines a proneural cluster as a population of competent cells for neural development. The tamou gene (Tamou means 'hairy' in Japanese), now termed polychaetoid, encodes a cell-cell junction-associated protein, which is homologous to mammalian ZO-1, a member of the membrane-associated guanylate kinase homolog family. The polychaetoid mutation reduces the transcription of a repressor gene, extramacrochaetae, and causes enlargement of a proneural cluster where supernumerary precursor cells emerge, resulting in extra mechanosensory organs in the fly. Homozygotes of tam1, a mutation caused by a P-element insertion, are fully viable and fertile but ectopic macrochaetae are conspicuous on the dorsal side of the head and thorax of adults. The P-element insertion is 85 bases upstream of the 5'-terminal of the cDNA. The ectopic macrochaetae are not uniformly distributed on the notum, since almost all of them emerge near the positions of the dorsocentral (DC), postalar (PA) and/or scutellar (SCu) bristles, and rarely form near the postions of the supraalar or presutural bristles. The microchaete distribution pattern is slightly affected by the ectopic microchaetae formed between the rows of normal microchaete; however, the spacing of the microchaetae within each row seem normal. These phenotypes resemble those of some hypomorphic alleles of extramacrochaetae. Daughter cells of single sensory organ precursor (SOP) cells are known to be closely associated with one another during sensory organ development. In the tam1 mutant, epithelial cells are always observed between the bristles of the notum. Therefore, the ectopic bristle (shaft and socket) do not seem to be generated by the transformation of daughter cells, which normally give rise to a sheath cell and a neuron, leading to the prediction that the additional bristle in the tam1 mutant appear through the formation of additional SOPs (Takahisa, 1996).

In the most severe pyd genotypes, nearly all macrochaetae positions on the head and notum are affected. However, individual macrochaete positions display differences in sensitivity to reduction of pyd function: in general, as the level of pyd function decreases, more positions are affected and the number of extra bristles at the most sensitive positions increases. However, it is not possible to define an allelic series that is consistent for all macrochaete positions. A slight increase in macrochaete density is noted in pyd mutants. A reduction in pyd function results in the formation of additional SOPs in proneural clusters. In pyd mutant discs, the proneural cluster pattern of Achaete expression appears unchanged from the wild-type pattern: ectopic expression of Achaete is not observed. However, the subsequent refinement of Achaete expression is altered as additional cells express the high levels of Achaete characteristic of SOPs. In many pyd mutants, SOPs within a single cluster appear to be at different stages, as reflected in the normal transition from Achaete to Asense expression (Chen, 1996).

polychaetoid is required for cell fate specification in the eye. In pyd mutants a slight roughness of the eye is detected. This roughness is due to the presence of mispositioned and duplicated mechanosensory bristles and occasional enlarged, irregularly shaped facets. In addition, the rows of ommatidia are misaligned. In the midpupal stage eyes of pyd mutants, a small fraction of ommatidia are observed to have extra cone cells and/or primary pigment cells. Some ommatidia have too few cone cells. Ommatidia with extra cone cells also have extra photoreceptors. Reduction of Delta or Notch function in pyd mutants strongly enhances both the pyd extra bristle and rough eye phenotypes (Chen, 1996).

Polychaetoid is required for cell specification and rearrangement during Drosophila tracheal morphogenesis

The development of the complex network of epithelial tubes that ultimately forms the Drosophila tracheal system relies on cell migration, cell shape changes, cell rearrangements, cell differentiation, and branch fusion. Most of these events are controlled by a combination of distinct transcription factors and cell-cell signaling molecules, but few proteins that do not fall within these two functional classes have been associated with tracheal development. This study shows that the MAGUK protein Polychaetoid (Pyd/ZO-1), the Drosophila homolog of the junctional protein ZO-1, plays a dual role in the formation of tracheal tubes. pyd/ZO-1 mutant embryos display branch fusion defects due to the lack of reliable determination of the fusion cell fate. In addition, pyd/ZO-1 mutant embryos show impaired cell intercalation in thin tracheal branches. Pyd/ZO-1 localizes to the adherens junctions (AJs) in tracheal cells and might thus play a direct role in the regulation of the dynamic state of the AJ during epithelial remodeling. This study suggests that MAGUK proteins might play important roles during AJ remodeling in epithelial morphogenesis (Jung, 2006).

Cell fate determination in the Drosophila tracheal system is regulated by the Trachealess (Trh) transcription factor. In order to identify novel Trh target genes, a comparison of the transcriptional profiles of wild-type and loss-of-function trh mutant Drosophila stage 11 embryos was compared using the Affymetrix GeneChip technology. It was found the expression of the polychaetoid (pyd) gene is downregulated in a trh mutant background. pyd/ZO-1 is expressed in the tracheal placodes of stage 11 embryos. Expression was also observed in the foregut and hindgut and in the epidermis. In trh mutant embryos, pyd/ZO-1 transcription is still observed in the epidermis, the foregut, and the hindgut but was absent from the tracheal placodes. pyd/ZO-1 encodes the Drosophila homolog of the vertebrate cell-cell junction-associated protein zonula occludens 1 (ZO-1) and is a member of the MAGUK family of proteins (Jung, 2006).

MAGUK proteins are known to be incorporated in macromolecular protein complexes located at the tight junctions (TJs) in vertebrates. In Drosophila, Pyd/ZO-1 localizes to the adherens junctions (AJs) of the embryonic epidermis and the larval imaginal disc epithelium. An N-terminal eGFP-tagged full-length Pyd/ZO-1 protein was expressed in tracheal cells under the control of the btl-Gal4 driver and a GFP signal was observed at the level of intercellular AJs of the dorsal trunk (DT) in a mesh-like pattern similar to the D-α-catenin-GFP and Shotgun/E-cadherin-GFP (Shg/E-Cad-GFP) fusion constructs. GFP fluorescence was also seen in single lines corresponding to the autocellular AJs of the dorsal branches (DBs), the ganglionic branches (GBs), and the lateral trunk (LT). Double-labeling experiments showed a colocalization of the GFP-Pyd fusion construct and Shg/E-Cad at the AJs in the tracheal system. These results demonstrate that Pyd/ZO-1 is enriched with Shg/E-Cad and D-α-catenin-GFP at the AJs of tracheal cells (Jung, 2006).

Since the DB fusion-defect phenotype is not fully penetrant, occurring phenotypes were grouped into three categories: only one metamere affected (weak phenotype), two to four metameres affected (intermediate phenotype), and five or more metameres affected (severe phenotype). The results suggest that pyd/ZO-1 is required for proper fusion of the DBs and, to a lesser extent, the LT, as well as for proper outgrowth of the GBs. These phenotypes are due to specific lesions in the pyd/ZO-1 locus, as shown by the rescue experiments (Jung, 2006).

Terminal- and fusion-cell fate determination was examined by analyzing both blistered/Drosophila serum reponse factor (bs/Dsrf) and escargot (esg) expression patterns in pyd/ZO-1 loss-of-function mutant embryos. It was found that, instead of a single Bs/Dsrf-expressing cell at the tip of DBs, some DBs were characterized by the presence of an ectopic terminal cell in pyd/ZO-1 mutants. This situation is similar to the previously reported ectopic expression of acheate and asense in Drosophila wing imaginal discs of pyd/ZO-1 mutants, with consequent formation of extra macrochaetes on the adult notum. Using an esg-lacZ line, it was observed that in wild-type embryos or pyd^C5 heterozygous embryos, one single lacZ-positive fusion cell is found at the tip of each DB, whereas in pyd^C5 homozygous individuals, anti-β-galactosidase staining was absent in some DBs that had failed to undergo branch fusion. It was confirmed that branches that had not fused showed an excess of terminal cells and a loss of fusion cells by using the esg-lacZ; pyd^C5 strain to perform a 2A12/Dsrf/β-galactosidase triple immunostaining. These results suggest that pyd/ZO-1 is involved in fusion- versus terminal-cell specification via the regulation of bs/Dsrf or esg gene expression (Jung, 2006).

Interestingly, DBs with a wild-type Dsrf and Esg pattern (one terminal cell and one fusion cell) were identified, that did not undergo branch fusion. Therefore, it appears that the cell fate shift observed in pyd/ZO-1 mutants does not alone explain all fusion defects, and xa mechanical role of Pyd/ZO-1 in the branch fusion process itself cannot be excluded (Jung, 2006).

To determine whether pyd/ZO-1 function is required in the terminal or in the fusion cell, clonal analysis was performed in mosaic third-instar larvae using the MARCM system. pyd/ZO-1 mutant clones affecting the terminal cells did not affect branch fusion to a significant extent. However, most pyd/ZO-1 mutant clones including the fusion cells were associated with branch fusion defects, whereas only a few DBs displaying pyd/ZO-1 mutant fusion cells showed a wild-type branch fusion pattern. In some cases, it was observed that pyd/ZO-1 homozygous mutant cells in the position of the fusion cell displayed ectopic terminal branches, an observation that is reminiscent of the esg loss-of-function phenotype. It was therefore concluded that pyd/ZO-1 acts in the fusion cell to specify fusion- versus terminal-cell fate (Jung, 2006).

It was observed that overexpression of pyd/ZO-1 in tracheal cells of wild-type embryos mimics the pyd/ZO-1 DB fusion-defect phenotype. Taken together, these results suggest a model in which Pyd/ZO-1 regulates the subcellular localization and/or activation of a factor of unknown nature involved in the regulation of gene expression by either inducing the fusion-cell fate or repressing the terminal-cell fate in the fusion cell (Jung, 2006).

Several MAGUK proteins are known to be used as scaffold proteins organizing signaling pathways . pyd/ZO-1 itself regulates the expression of the acheate-scute gene complex. Further insights into the molecular aspects of this regulation will provide a clearer idea about how Pyd/ZO-1 fulfills its function (Jung, 2006).

Sincc these results indicated that Pyd/ZO-1 localizes to the AJs of the tracheal system, the dynamics of tracheal cell rearrangements were analyzed by examining the D-α-catenin-GFP pattern in pyd/ZO-1 mutant embryos using confocal live imaging techniques. In stage 16 wild-type embryos, the cell rearrangement process is completed in unicellular branches. Cells are in an end-to-end configuration, with long lines of autocellular AJs and very tiny rings of intercellular AJs. In contrast to these control embryos, large loops of intercellular AJs remained in fine branches of pyd homozygous embryos, indicating that cell intercalation was not completed. These cell intercalation defects were not fully penetrant. In the case of the strongest allele with regard to this intercalation phenotype, 26% of the DBs were affected; this phenotype was never observed in wild-type embryos (Jung, 2006).

Strikingly, in most cases, DBs with a cell intercalation phenotype were still able to stretch and eventually reached the dorsal midline. Some were even able to undergo branch fusion. These results suggest that branch fusion and branch stretching are not required for DB cell intercalation and vice versa. Similar observations were made in the case of the LT, where loops of D-α-catenin-GFP were still present between LT cells although adjacent metameres had undergone fusion (Jung, 2006).

It was observed that pyd/ZO-1 mutant embryos showed GB outgrowth defects. Defective cell intercalation was also found in GBs, and affected branches were shorter than neighboring GBs. Therefore, it is proposed that, in contrast to DBs, proper cell intercalation is necessary for GBs to adopt an elongated tube shape (Jung, 2006).

Expression of the GFP-pyd construct in the tracheal system of BG02748 homozygous embryos allowed a full rescue of the intercalation phenotype, even though DB fusion defects were not fully rescued. A live confocal time-lapse analysis was performed and it was observed that, in contrast to stage 16 heterozygous pyd^C5 embryos, in which DB cells were intercalated and remained so until the end of the analysis, homozygous pyd^C5 embryos of the same age showed cell intercalation defects where loops of D-α-catenin-GFP were interspersed with autocellular AJs. It is concluded that the zipping process seems to be stopped or frozen, leaving pairs of cells in a partially intercalated state. It was not observe that autocellular AJs converted back into intercellular AJs, which could also have been a possible explanation for the phenotype. It is very likely that Pyd/ZO-1 is part of the adhesion E-cadherin/α-/β-catenin protein complex that governs cell-cell adhesion (Jung, 2006).

Altogether, these results suggest a role for pyd/ZO-1 in facilitating the cell rearrangement process that underlies the formation of fine, elongated tubes with autocellular AJs in the tracheal system. This phenotype appears to be functionally independent from the branch-fusion-defect phenotype (Jung, 2006).

This study has shown that Pyd/ZO-1 plays a dual role during the morphogenesis of the Drosophila tracheal system and it is proposed that the transcriptional and mechanistic roles of pyd/ZO-1 are independent. This suggests that Pyd/ZO-1 uses distinct partners to achieve its two functions in the tracheal system. This is the first report of an in vivo analysis of pyd/ZO-1 in a cell biological process, and Pyd/ZO-1 is the first junction-associated protein described to have an effect on cell rearrangements during tracheal branching morphogenesis (Jung, 2006).

Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina

Correct cellular patterning is central to tissue morphogenesis, but the role of epithelial junctions in this process is not well-understood. The Drosophila pupal eye provides a sensitive and accessible model for testing the role of junction-associated proteins in cells that undergo dynamic and coordinated movements during development. Mutations in polychaetoid (pyd), the Drosophila homologue of Zonula Occludens-1, are characterized by two phenotypes visible in the adult fly: increased sensory bristle number and the formation of a rough eye produced by poorly arranged ommatidia. It was found that Pyd is localized to the adherens junction in cells of the developing pupal retina. Reducing Pyd function in the pupal eye results in mis-patterning of the interommatidial cells and a failure to consistently switch cone cell contacts from an anterior-posterior to an equatorial-polar orientation. Levels of Roughest, DE-Cadherin and several other adherens junction-associated proteins are increased at the membrane when Pyd protein is reduced. Further, both over-expression and mutations in several junction-associated proteins greatly enhances the patterning defects caused by reduction of Pyd. These results suggest that Pyd modulates adherens junction strength and Roughest-mediated preferential cell adhesion (Seppa, 2008).

The data demonstrate that Pyd is an AJ-associated protein that is required for patterning of the pupal lattice cells. Live imaging of the developing eye indicates that Pyd is necessary for the directed movements of interommatidial precursor cells (IPCs) that allow cell sorting into defined niches. Membrane contacts are dynamically exchanged in the pupal eye: each shift in the position of a cell requires the removal of previous contacts and the establishment of new ones. Pyd regulates patterning at least in part through modulating levels of the AJ-associated proteins DE-Cadherin, β-Catenin, and α-Catenin. Other studies have suggested that cell adhesion is necessary both to facilitate and restrict cell movement within the eye epithelium; the interplay between these two processes requires tight regulation of the levels of both cell adhesion molecules and junctional proteins. The data indicate that removal of Pyd from the AJ compromises this tightly-regulated system and biases the cells toward poorly-directed movements, perhaps because of dysregulation of the timing or function of the mechanisms that control the stability of AJ proteins. This failure in precise regulation of adhesion was also highlighted in the inability of cone cells to exchange their membrane contacts: the apical interfaces of pyd-RNAi expressing cone cells were locked in place. Ectopic DE-Cadherin further increased the percentage of ommatidia affected, again emphasizing the link between pyd activity and the AJ (Seppa, 2008).

The localization of Pyd to the AJ in the pupal eye is dependent on both DE-Cadherin and α-Catenin. However, it was found that ectopic expression of either junctional protein is not sufficient to alter the localization of Pyd. Taken together, these data indicate that DE-Cadherin and α-Catenin are necessary to build or maintain the AJ and to localize Pyd but that, in excess, they are not sufficient to attract ectopic Pyd. This suggests that either Pyd protein levels are not easily altered or that Pyd may be binding to proteins other than the core AJ constituents. Recent work demonstrated that E-Cadherin was necessary for the initial steps of AJ formation while α-Catenin was essential for both the establishment and maintenance of the junction; only when α-Catenin was reduced was ZO-1 lost from established junctions. The results suggest that in dynamically restructured tissues such as the eye, both E-Cadherin and α-Catenin are necessary for the localization of AJ-associated proteins (Seppa, 2008).

The immunoglobulin superfamily member Roughest is necessary for appropriate sorting of IPCs during pupal eye development. Reducing Pyd increased Roughest protein levels specifically at the AJ. Roughest is the Drosophila orthologue of Neph1, a cell adhesion molecule necessary for the structure and function of the glomerular slit diaphragm in the mammalian kidney. The slit diaphragm is the main size-selective barrier in the filtration apparatus of the kidney and retains many characteristics of both the tight and AJ complexes from which it was derived. The Hibris orthologue Nephrin also forms part of the physical structure of the slit diaphragm and both cell adhesion molecules have been reported to bind to each other as well as to ZO-1. Perhaps ZO-1, as with Pyd, has a role in regulating the localization or levels of cell adhesion molecules such as Neph1 and Nephrin (Seppa, 2008).

The Dpp pathway has emerged as a major contributor to patterning of the Drosophila pupal eye. Its role requires functional connections to both DE-Cadherin and Roughest. For example, mutations in shotgun (the locus that encodes DE-Cadherin) suppresses the roughest eye phenotype but enhances Dpp pathway-dependent phenotypes in the eye and wing. Together, these data suggest a model in which (1) Roughest acts to promote the stability of membrane contacts to drive directed cell movements and (2) the Dpp pathway and Pyd act to destabilize the adherens junction complex and local cell contacts to allow for proper IPC sorting. Consistent with this view, it was observed that reducing pyd enhances the effects of reduced Dpp pathway activity in the eye and wing. Thus, Pyd appears to act in concert with the Dpp pathway to regulate select core components of the AJ during development (Seppa, 2008).

This study has shown that Pyd is required specifically for patterning the interommatidial cells of the Drosophila pupal eye. Pyd appears to regulate both cell shape and cell positioning by controlling the levels of AJ proteins such as DE-Cadherin and adhesion proteins such as Roughest. Thus, Pyd provides a link between adhesion and junction formation; a further understanding of its role in the pupal eye will shed light on how these processes are coordinated to generate precise cellular movements during epithelial patterning (Seppa, 2008).

The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. This study describes the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. A novel role was uncovered for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1 (Goossens, 2011).

This study demonstrates a requirement for Neuroglian signaling in different steps of mushroom body (MB) axonogenesis, namely (1) axonal projection into the peduncle, and (2) branching, outgrowth and guidance of axonal sister branches. The two steps in mushroom body axonogenesis are genetically separable and seem to involve distinct NRG signaling complexes (Goossens, 2011).

In peduncle formation, NRG signaling does not rely on the NRG-180-specific intracellular domain, but on the extracellular domain and the part of the cytoplasmic domain common to both NRG isoforms. The extracellular domain contributes intercellular adhesive properties, necessary for axon fasciculation into a peduncle. This conclusion is supported by the defective adhesive properties of the NRG⁸⁴⁹ mutant protein in cell aggregation assays, and by the fact that Nrg⁸⁴⁹ hemizygotes frequently lack the peduncle. Interaxonal fasciculation in the peduncle probably involves binding to and stabilization by the actin cytoskeleton network via the ankyrin-binding domain shared by the two NRG isoforms. This conclusion is supported by previous aggregation experiments in Drosophila S2 cells in which it was shown that homophilic binding of NRG leads to recruitment of ankyrin to the contact sites and by the observation that RNAi-mediated knockdown of neuron-specific ank2-RNA results in MB phenotypes similar to those seen in Nrg mutants (Goossens, 2011).

MB lobe development, on the other hand, requires the NRG-180-specific intracellular fragment. This study showed that PYD acts downstream of NRG-180 during the formation of α and γ lobes. Consistent with this, axon stalling defects (i.e. lack of peduncle formation) were never observed in pyd mutants, whereas defects were observed in lobe outgrowth, branching and guidance. Furthermore, the neuron-specific NRG-180 isoform can bind directly to the first PDZ-domain of this MAGUK protein. The observation that NRG and PYD interact to mediate proper sister neurite projections defines a novel role for the ZO-1 homolog PYD in axonogenesis. Thus far, the best-known role of MAGUKs in the nervous system has been in synapse development and function, as is the case for one of the prototypic MAGUKs, Drosophila Discs Large 1 (Dlg1), whereas PYD is known as a component of adherens junctions (Goossens, 2011).

Sema-1a, trio and Rac1 were also found to be a part of the genetic network that interacts with Nrg. The observation that heterozygosity for mutations in Sema-1a and trio both suppress NRG-180 overexpression induced MB phenotypes indicates that Sema-1a and trio are genetically downstream of Nrg and possibly in the same pathway. By contrast, the introduction of a Rac1 mutation in a Nrg gain- or loss-of-function background results in both cases in enhancement of MB phenotypes. This argues against a one-to-one signaling model between NRG and RAC1, in which RAC1 acts only downstream of NRG-180. Consistent with the genetic data, no direct physical interaction could be detected between NRG-180 and RAC1, but preliminary co-immunoprecipitation data suggest that NRG-180 can bind to TRIO. Further experiments will be necessary to assess whether this binding also occurs in vivo, and whether it is instrumental for NRG-180-dependent modulation of RAC1 signaling (Goossens, 2011).

Contrary to the observed genetic interaction between Nrg and Sema-1a, this study found no evidence for interaction between Nrg and two genes that code for well-characterized Semaphorin receptors, plexin A and plexin B. This is an unexpected observation in light of the fact that Sema-1a and plexin A and plexin B interact during mushroom body development. Therefore, this suggests that during mushroom body development Sema-1a acts both in a plexin-dependent and a plexin-independent way. A plexin-independent role in axon outgrowth has previously been described for vertebrate Sema7a (Goossens, 2011).

The distinct requirement for NRG in peduncle and lobe formation is reminiscent of what has been shown for DSCAM. This protein has an early and essential role for selective fasciculation of young axons in the peduncle and is subsequently required for bifurcation and branch segregation. In light of this, it is interesting to note that the different cell-adhesion molecules that have been implicated in MB development have a different MB expression pattern or temporal requirement for MB development. NRG is expressed in the MBs throughout its entire development, but no essential function was found for Neuroglian in larval MB development. By contrast, DSCAM expression disappears with fiber maturation and mutants have larval MB phenotypes. Likewise, Fas2 mutants display larval lobe defects, whereas no lobe defects were found in adult mutants. Taken together, these observations suggest that different steps in MB axonogenesis depend on combinations not only of isoforms of the same cell-adhesion molecule (e.g. DSCAM) but also of different cell surface molecules (e.g. NRG, FAS2 and SEMA-1A). Jointly, the cell-surface molecule complement of any given axon combined with guidance signals will then control the signaling required for proper neural circuit formation in the MBs (Goossens, 2011).

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date revised: 23 December 2019

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