InteractiveFly: GeneBrief

Notopleural: Biological Overview | References

Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. This study shows by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. These data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade (Drees, 2019).

This paper reports that the vertebrate matriptase-prostasin proteolytic cascade, which is crucial for extracellular matrix differentiation and tissue homeostasis, is conserved in Drosophila. Np acts as a functional Drosophila homologue of matriptase, and tpr mediates prostasin function in the Drosophila tracheal system. Cleavage targets of these conserved extracellular proteolytic pathways are the ZP domains, present in many extracellular proteins of both vertebrates and invertebrates. The Np-mediated protease cascade controls at least three distinct cellular processes during tracheal development, i.e. morphogenesis of the taenidial folds, degradation of the tracheal Dpy cable in the tracheal lumen, and maintenance of the transepithelial barrier function (Drees, 2019).

In vertebrates, proteolysis by matriptase plays a key role in regulating epithelial differentiation. Ectopic expression of human matriptase in the developing tracheal system of Np mutant embryos rescues all aspects of the Np mutant phenotype, i.e. degradation of the Dpy luminal cable, taenidial folds formation, and gas filling of the tubes. The fact that matriptase can functionally substitute for the lack of Np activity indicates that the two proteins share similar functions. Furthermore, similar to matriptase, Np is differentially localized in tissue and stage-dependent patterns at the apical plasma membrane and the apical extracellular space. These findings suggest processing of Np by ectodomain shedding similar to what has been described for matriptase. However, the processing of human matriptase involves not only ectodomain shedding but also the transient interaction of the stem region with its cognate inhibitor HAI-2. Absence of HAI-2 prevents cell surface translocation of matriptase and causes its accumulation in the Golgi compartment (Lai, 2015). Ectopic HAI-2 expression in combination with matriptase facilitates secretion of matriptase in the Drosophila tracheal system. This finding and the lack of a Drosophila HAI-2 homologue indicate different regulatory mechanisms for the translocation and ectodomain shedding of Np and matriptase. This assumption is also consistent with the lack of conserved regulatory stem regions of Np and matriptase. Therefore, different protein processing mechanisms of the two otherwise functionally equivalent proteins are proposed. However, the apparent diverse regulation of both proteins provides the possibility to establish an in vivo experimental system to analyse aspects of matriptase regulation and processing in Drosophila (Drees, 2019).

The matriptase-prostasin proteolytic cascade is initiated by rapid matriptase autoactivation as shown by an in vitro cell-free system as was observed with Np. Thus, the proteolytic cascade involved in aECM formation and maturation in Drosophila is likely to be initiated by Np autoactivation. Once activated, it acts on Tpr, a direct downstream target of Np in the developing tracheal system. This conclusion is based on the observation that in vitro-purified Np is able to cleave Tpr at the zymogen activation site, implying a direct activation of Tpr zymogen by Np in vivo. Furthermore, the tracheal aECM phenotype of tpr mutant embryos is less pronounced than the Np phenotype, since taenidial folds are wild-type like in tpr mutant embryos, while Np mutant embryos lack taenidial folds. The observation that human matriptase also cleaves Tpr at the same zymogen activation site provides additional support for the functional identity of matriptase and Np (Drees, 2019).

Ectopic tracheal expression of human prostasin, together with human HAI-2 in tpr mutant embryos, rescues the defects of the aECM and the LC phenotype of tpr mutants. Thus, Tpr is a functional homologue of human prostasin in the developing trachea. Also, human HAI-2 is required for prostasin secretion into the tracheal lumen, as has been reported for prostasin in vertebrate tissues [43]. LC defects, as observed in tpr mutants, are often caused by an impaired transepithelial barrier function. However, tpr mutant embryos develop a normal barrier and, thus, it is supposed that the LC defects are likely caused by hampered degradation of luminal material and/or Tpr might affect epithelial sodium channels (ENaCs). ENaCs are located in the apical membrane of tracheal cells and are critical for tracheal gas fillin. In vertebrates, prostasin activates ENaCs by inducing proteolytic cleavage of the gamma subunit. It will be interesting to see whether Tpr plays a similar role for ENaC activation in Drosophila (Drees, 2019).

Vertebrate prostasin is widely expressed in ectodermal tissue and most functional aspects of human matriptase are mediated via prostasin in the various tissues. However, the functional relationship of matriptase and prostasin remains to be clarified since matriptase activation and shedding is prostasin-dependent in specific tissues. In contrast to vertebrate prostasin, Drosophila Tpr expression is confined to the tracheal system and not detectable in other ectodermal tissues. Thus, while vertebrate matriptase and prostasin are co-expressed in most tissues, a different scenario is proposed in Drosophila. Tpr belongs to a small group of seven very similar proteases in Drosophila. The corresponding genes are differentially expressed in specific spatial patterns in various ectodermal tissues.It is proposed that such protease zymogens represent putative Np cleavage targets that mediate Tpr-like functions in the different ectodermal tissues that express Np including salivary glands, hindgut, and epidermis (Drees, 2019).

Drosophila tracheal development is a paradigm for the generation of branched tubular systems. Early steps of tracheal maturation, notably tube formation and tubular network assembly, develop independently of Np, while tracheal aECM formation and the transepithelial barrier function during late embryonic tracheogenesis depend on Np. Main differentiation events of the aECM, such as taenidial folds morphogenesis and degradation of luminal protein matrix, are controlled by Np and Tpr (Drees, 2019).

The taenidial folds of the tracheal aECM mainly consist of chitin running perpendicular to the tracheal tube length along the lumen. Their main function is to provide stiffness combined with concurrent flexibility of the tube. Taenidial folds formation is severely affected in Np mutant embryos. The outmost taenidial structure, the hydrophobic envelope, is not detectable and the chitin strands of taenidial folds are highly disorganized. Chitin-interacting proteins, involved in chitin organization, may represent putative targets of Np activity. The aECM phenotype of tpr mutant embryos suggests that tpr function is more specific and confined to establish a proper adhesion between the apical side of tracheal cells and the overlaying taenidial folds of the aECM (Drees, 2019).

Both Np and Tpr are also involved in the degradation of tracheal luminal Dpy, a large ZP domain-containing protein. Luminal Dpy is part of a chitin-proteinous matrix within the tracheal lumen and is essential for normal tracheal network and tube formation. The luminal matrix is degraded and removed from the tracheal lumen during formation of the tracheal taenidial folds and the subsequent gas filling of the tracheal system. Np appears to be the key factor in Dpy cable degradation since Np-deficient embryos completely lack Dpy degradation. Some degradation, however, is mediated via Tpr proteolysis because tpr mutants display remaining Dpy material in the tracheal lumen during late embryogenesis. Thus, the combination of Np and Tpr accomplish luminal Dpy degradation. Alternatively or in addition, Np activates unknown proteases that mediate complete Dpy degradation prior to the gas filling of the tracheal tubes (Drees, 2019).

The transepithelial barrier function is established by the septate junction (SJ) protein complexes, localized at the apico-lateral membrane of epithelial cells [50]. The lack of bona fide SJ proteins like the Drosophila claudin Mega causes a disruption of the ladder-like SJ ultrastructure and a barrier function defect [32]. In Np mutants, the ladder-like ultrastructure of SJs and the barrier function appear to be properly established, but the barrier function collapses during the end of embryogenesis. Thus, Np is essential for the maintenance of the transepithelial barrier function mediated by SJs. This function of Np is reminiscent of matriptase function in mammals. Tracer injection experiments into the dermis of matriptase-deficient mice indicate impaired epidermal tight junction function in such animals [18]. In intestinal epithelial model cell layers and hypomorphic matriptase mice, the essential tight junction component Claudin-2 is deregulated. This observation suggests that reduced barrier integrity was caused, at least in part, by an impaired claudin-2 protein turnover [51]. Furthermore, matriptase cleaves EpCAM, which in turn decreases EpCAM ability to associate with claudin-7 followed by lysosomal degradation of claudin-7 [52]. Thus, it is speculated that Np may also control the maintenance of the epithelial barrier in Drosophila by regulating the function or turnover of claudins in SJs, the invertebrate analogue of the vertebrate tight junction (Drees, 2019).

The aECM protein Dpy is an in vivo downstream target of both Np and human matriptase proteolytic activity. This observation was puzzling since Dpy is not conserved in vertebrates. However, Dpy contains a conserved region, the ZP domain. The ZP domain defines a conserved family of aECM proteins, originally identified in the zona pellucida coat surrounding the mammalian oocyte. The 260 amino acids long ZP domain is proposed to act as a module promoting polymerization of proteins into threads and matrices essential for the organization of highly specialized apical extracellular structures. In fact, the ZP domain was confirmed as a target of Np and matriptase by data showing that Pio, an aECM protein containing a ZP domain, is cleaved by Np and matriptase. Both cleave the ZP domain of Pio within the short linker region, which separates ZP-N and ZP-C, the two subdomains of the ZP domain. Pio is secreted apically in the tracheal lumen and establishes together with Dpy, possibly via ZP-domain polymerization, a structural matrix in the tracheal lumen that is essential for the formation of an interconnected branched network. Cleavage of ZP domains within a meshwork of Dpy and Pio filaments may facilitate rapid degradation of the luminal extracellular matrix, the prerequisite for normal gas filling of the tracheal system. This conclusion is supported by phenotypic analysis of Np mutant embryos, which exhibit a stable, undegraded luminal Dpy cable and lack tracheal gas filling. ZP-domain proteins also play crucial roles in development of embryonic epidermal cuticle, an aECM that protects the animal against the external milieu. Eight ZP-domain proteins are required for the localized reorganization of epidermal cells and to sculpture the actin-rich apical extensions, the denticles. The observation that Np mutants exhibit reduced and rudimentary denticles in the epidermis underlines the possibility that the epidermal ZP-proteins are also targets of Np protease activity (Drees, 2019).

The results showing that human matriptase cleaves the ZP domain of Pio open future directions to explore novel targets of the matriptase-prostasin catalytic pathway. In vertebrates, ZP-domain proteins are involved in remodelling apical extracellular structures, such as ZP1-ZP3, important in the mammalian ovary for fertilization and uromodulin, which is released into the tubular kidney lumen where it polymerizes in a gel-like matrix that controls salt transport and urine concentration. Also, mutations in genes encoding ZP-domain proteins cause human diseases such as deafness, triggered by mutations in alpha- and beta-tectorin. The tectorins are components of the tectorial membrane, an aECM necessary for sound transmission to neural cells in the cochlea. These examples already demonstrate the importance of ZP-domain proteins for mammalian physiology. Based on the results reported in this study, it is proposed that ZP-domain cleavage by the matriptase-prostasin proteolytic cascade may represent a conserved process to control ZP-domain protein functions, which are crucial for apical matrix remodelling during development, wound repair, and differentiation (Drees, 2019).

The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila

Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. This study identified a mechanism of sensing and responding to the membrane-driven expansion of tracheal tubes. The apical membrane is anchored to the apical extracellular matrix (aECM) and causes expansion forces that elongate the tracheal tubes. The aECM provides a mechanical tension that balances the resulting expansion forces, with Dumpy being an elastic molecule that modulates the mechanical stress on the matrix during tracheal tube expansion. The zona pellucida (ZP) domain protein Piopio was shown to interact and cooperates with the ZP protein Dumpy at tracheal cells. To resist shear stresses which arise during tube expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-Dumpy-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane, tears the apical matrix, and impairs tubular network function. Conserved ectodomain shedding was shown of the human TGFβ type III receptor by Notopleural and the human Matriptase, providing novel findings for in-depth analysis of diseases caused by cell and tube shape changes (Drees, 2023).

Tracheal tube lumen expansion requires mechanical stress regulation at apical cell membranes and attached aECM. This involves the proteolytic processing of proteins that set local membrane-matrix linkages. Thus, the membrane microenvironment exhibits critical roles in regulating tube and network functionality (Drees, 2023).

ZP domain proteins organize protective aECM in the kidney, tectorial inner ear, and ZP, as well as in Drosophila epidermis, tendon cells, and appendages. Drosophila ZP domain proteins link the aECM to actin and polarity complexes in epithelial cells. Dumpy establishes force-resistant filaments for anchoring tendon cells to the pupal cuticle (Drees, 2023).

This study found that ZP protein-mediated microenvironmental changes increase the flexibility of membrane-matrix association, resulting from the activity of ZP domain proteins. Shear stress stimulates the activity of membrane-anchored proteases and potentially also apical transmembrane protease Np since the misdistribution of the tracheal cytoskeleton was not observed when blisters arise. The dynamic membrane-matrix association control is based on findings that loss of Np prevents Pio ectodomain shedding at the apical cell membrane resulting in immobile localization of Pio at the membrane and Dpy localization within the matrix. Direct interaction and overlapping subcellular localization at the cell surface showed that both proteins form a ZP matrix that potentially attaches membrane and ECM. Deregulation of Pio shedding blocks ZP matrix rearrangement and release of membrane-matrix linkages under tube expansion and subsequent shear stress. This destabilizes the microenvironment of membranes, causing blister formation at the membrane due to ongoing membrane expansion. Additionally, Pio could be part of a force-sensing signal transduction system destabilizing the membrane and matrix. The observation that the membrane deformations are maintained in Np mutant embryos supports the postulated Np function to redistribute and deregulate membrane-matrix associations in stage 16 embryos when tracheal tube length expands. In contrast, Np overexpression potentially uncouples the Pio-Dpy ZP matrix membrane linkages resulting very likely in unbalanced forces causing sinusoidal tubes (Drees, 2023).

The membrane defects observed in both Pio and Np mutants indicate errors in the coupling of the membrane matrix due to the involvement of Pio. In pio mutants, gaps appear between the deformed membrane and the apical matrix. These changes in apical cell membrane shape are consistent with increased cell and tube elongation in pio mutant embryos because the matrix is uncoupled from the membrane in such mutants. In contrast to pio mutants, the large membrane bulges in Np mutants affect the membrane and the apical matrix. Since apical Pio is not cleaved in Np mutants, the matrix is not uncoupled from the membrane as in pio mutant embryos but is likely more intensely coupled, which leads to tearing of the matrix axially along the membrane bulges, when the tube expands in length. If apical Pio detachment reduces coupling between the matrix and apical membrane, then it is likely that Np mutant embryos may exhibit a reduced tube length phenotype. In Np mutant embryos, average tracheal dorsal trunk length tends to be reduced compared to wt embryos, suggesting that Pio shedding is critical in controlling tracheal tube lumen length (Drees, 2023).

Is Pio ectodomain shedding in response to tension? Tension was not measured directly. However, the developmental profile of mechanical tension during tracheal tube length elongation in stage 16 embryos is consistent with the profile of Pio shedding. Np cleaves apical Pio during stage 16 when tube length expands. In contrast, Pio shedding decreases sharply at early stage 17 when tube elongation is completed. The model, therefore, predicts that loss of Pio or increased Pio secretion at stage 16 may reduce the coupling of the membrane matrix so that increased tracheal tube elongation is maintained until the end of stage 16, which is found in pio mutants and upon Np overexpression. Unknown proteases may likely be involved in Pio processing since cleaved mCherry::Pio is also detectable in inactive NpS990A cells. Previously a mutation at the Pio ZP domain (R196A) resistant to NP cleavage in cell culture experiments (Drees, 2019). Establishing a corresponding mutant fly line would be essential in determining whether the observed phenotype resembles the phenotype of the Np mutant embryos. In addition, unknown mechanisms, such as distinct membrane connections during development and emerging links to the developing cuticle, may also influence tension at the apical membrane during tube length control (Drees, 2023).

Indeed, the anti-Pio antibody, which detects all different Pio variants, showed a punctuate Pio pattern overlapping with the apical cell membrane markers Crb and Uninflatable (Uif) at the dorsal trunk cells of stage 16 embryos. Additionally, Pio antibody also revealed early tracheal expression from embryonic stage 11 onward, and due to Pio function in narrow dorsal and ventral branches, strong luminal Pio antibody staining is detectable from early stage 14 until stage 17, when airway protein clearance removes luminal contents. In the pio^5m and pio^17c mutants, Pio stainings were strongly reduced although some puncta were still detectable in the trachea. Similarly, Pio antibody staining is intracellular in the trachea of stage 11 pio^2R-16 point mutation embryos. Interestingly, also dpy mutants showed strongly reduced and intracellular Pio antibody staining (Drees, 2023).

mCherry::Pio was generated as a tool for in vivo Pio expression and localization pattern analysis during tube lumen length expansion. The mCherry::Pio resembled the Pio antibody expression pattern from early tracheal development onward. However, luminal mCherry::Pio enrichment occurs specifically during stage 16, when tubes expand. The stage 16 embryos showed mCherry::Pio puncta accumulating apically in dorsal trunk cells. Moreover, mCherry::Pio puncta partially overlapped with Dpy::YFP and chitin at the taenidial folds, forming at apical cell membranes. Supported by several observations, such as antibody staining, video monitoring, FRAP experiments, and western blot studies, these findings indicate that Pio may play a significant role at the apical cell membrane and matrix in dorsal trunk cells of stage 16 embryo (Drees, 2023).

Furthermore, it was shown that Np mediates Pio ZP domain cleavage for luminal release of the short Pio variant during ongoing tube length expansion. The luminal cleaved mCherry::Pio is enriched at the end of stage 16 and finally internalized by the subsequent airway clearance process during stage 17 after tube length expansion. Such rapid luminal Pio internalization is consistent with a sharp pulse of endocytosis rapidly internalizing the luminal contents during stage 17. Wurst is required to mediate the internalization of proteins in the airways. Consistencly, during stage 17, luminal Pio antibody staining fades in control embryos but not in Wurst deficient embryos (Drees, 2023).

Nevertheless, Pio and its endocytosis depend on its interaction with the chitin matrix and the Np-mediated cleavage. In stage 16 wurst and mega mutant embryos, Pio antibody staining at the chitin cable, suggesting that Pio is cleaved and released into the dorsal trunk tube lumen. Also, the Cht2 overexpression did not prevent the luminal release of Pio. However, reduced wurst, mega function, and Cht2 overexpression caused an enrichment of punctuate Pio staining at the apical cell membrane and matrix . Although the three proteins are involved in different subcellular requirements, they all contribute to the determination of tube size by affecting either the apical cell membrane or the formation of a well-structured apical extracellular chitin matrix, indicating that changes at the apical cell membrane and matrix in stage 16 embryos affect the Pio pattern at the membrane. It also shows that local Pio linkages at the cell membrane and matrix are still cleaved by the Np function for luminal Pio release, which explains why those mutant embryos do not show pio mutant-like membrane deformations and Np-mutant-like bulges. This is in line with observations that tracheal Pio overexpression cannot cause tube size defects as the Np function is sufficient to organize local Pio linkages at the membrane and matrix. Therefore, it is unlikely that tracheal tube length defects in wurst and mega mutants as well as in Cht2 misexpression embryos are caused by the apical Pio density enrichment (Drees, 2023).

Nevertheless, oversized tube length due to the misregulation of the apical cell membrane and adjacent chitin matrix may cause changes to local Pio set linkages and the need for Np-mediated cleavage. Strikingly, a lack of Pio release was observed in Np mutants. This shows that Pio density at the membrane versus lumen depends predominantly on Np function. The molecular mechanisms that coordinate the Np-mediated Pio cleavage are unknown and will be necessary for understanding how tubes resist forces that impact cell membranes and matrices. On the other hand, Pio is required for the extracellular secretion of its interaction partner Dpy. At the same time, Dpy is needed for Pio localization at the cell membrane and its distribution into the tube lumen. Consistently, in vivo, mCherry::Pio, and Dpy::eYFP localization patterns overlap at the apical cell surface and within the tube lumen. These observations support the model that Pio and Dpy interact at the cell surface where Np mediates Pio cleavage to support luminal Pio release by the large and stretchable matrix protein Dpy (Drees, 2023).

Taenidial organization prevents the collapse of the tracheal tube. Therefore, cortical (apical) actin organizes into parallel-running bundles that proceed to the onset of cuticle secretion and correspond precisely to the cuticle’s taenidial folds. Mutant larvae of the F-actin nucleator formin DAAM show mosaic taenidial fold patterns, indicating a failure of alignment with each other and along the tracheal tubes. In contrast, pio mutant dorsal tracheal trunks contained increased ring spacing. Fusion cells are narrow doughnut-shaped cells where actin accumulates into a spotted pattern. Formins, such as Diaphanous, are essential in organizing the actin cytoskeleton. However, dorsal trunk tube fusion defects were not found as found in the presence of the activated Diaphanous (Drees, 2023).

On the other hand, ectopic expression of DAAM in fusion cells induces changes in apical actin organization but does not cause any phenotypic effects. DAAM is associated with the tyrosine kinase Src42A, which orients membrane growth in the axial tube dimension. Src42 overexpression elongates tracheal tubes due to flattened axially elongated dorsal trunk cells and AJ remodeling. Although flattened cells and tube overexpansion are similar in pio mutant embryos, mislocalization of AJ components was not observed, as found upon constitutive Src42 activation. Instead, an unusual stretched appearance of AJs was detected at the fusion cells of pio mutant dorsal trunks, which has not been observed before and may play a role in regulating axial taenidial fold spacing and tube elongation (Drees, 2023).

Self-organizing physical principles govern the regular spacing pattern of the tracheal taenidial folds. The actomyosin cortex and increased actin activity before and turnover at stage 16 drive the regular pattern formation. However, the cell cortex and actomyosin are in frictional contact with a rigid apical ECM. The Src42A mutant embryos contain shortened tube length but increased taenidial fold period pattern due to decreased friction. In contrast, the chitinase synthase mutant kkv1 has tube dilation defects and no regular but an aberrant pearling pattern caused by zero fiction (Drees, 2023).

In contrast, pio mutant embryos do not contain tube dilation defects or shortened tubes but increased tube length. Furthermore, chitin-binding probe (cbp) and antibody stainings reveal the presence of a luminal chitin cable and a solid aECM structure in pio mutant stage 16 embryos. In addition, apical actin enrichment in tracheal cells of pio mutant embryos appeared wt-like. Nonetheless, pio mutant embryos show an increased taenidial fold period compared with wt, indicating a decreased friction. Thus, it is proposed that the lack of Pio reduces friction. Reasons might be subtle defects of actomyosin constriction or chitin matrix, which was not detected in the pio mutant tracheal cells. Further reasons for lower friction might also be the loss of Pio set local linkages between apical cortex and aECM in stage 16 embryos, which are modified by Np, as proposed in in a model (see Model of apical Pio and Dpy matrix at the apical cell surface and Pio proteolysis and release. (Drees, 2023).

Heterozygous and homozygous pio mutant embryos generally do not show tubal collapse. However, the loss of Pio and accompanying lack of Dpy secretion in stage 17 pio mutant embryos led to the loss of a Pio/Dpy matrix, impacting the late embryonic maturation and differentiation of a normal chitin matrix at the apical cell surface. TEM images reveal reduced dense chitin matrix material at taenidial folds and misarranged taenidial fold pattern, suggesting impaired taenidial function prevents tube lumen from collapsing after tube protein clearance. wurst knockdown and mutant embryos do not show general tube collapse, but luminal chitin fiber organization is disturbed in stage 17 embryos. Therefore, transheterozygous wurst;pio mutant embryos may combine both defects and suffer from maturation deficits of the chitin/ZP matrix at the apical cell surface and within the tube lumen, which finally causes a high number of embryos with incomplete gas-filling due to tube collapse. These maturation deficits are even more dramatic in the wurst;pio double mutants, which show no gas-filling (Drees, 2023).

These studies on human Matriptase provide evidence for a mechanistic conservation of ZP domain protein as a substrate for ectodomain shedding. The upregulation of Matriptase activity and increased TGFβ receptor density affect human and mouse model idiopathic pulmonary fibrosis cells on pulmonary fibrogenesis. Furthermore, the human Matriptase induces the release of proinflammatory cytokines in endothelial cells, which contribute to atherosclerosis and probably also to abdominal aortic aneurysms. The membrane bulges arising in the Drosophila model during tracheal tube elongation upon Np loss of function showed analogy to the appearance of artery aneurysms. Bulges with varying phenotypic expression in different organs can lead to aortic rupture due to fragile artery walls or degeneration of layers in responses to stimuli, such as shear stresses. Indeed, aneurysm development is forced by alterations in the ECM and is characterized by extensive ECM fragmentation caused by shedding of membrane-bound proteins (Drees, 2023).

This study identified a dynamic control of matrix proteolysis, very likely enabling fast and site-specific uncoupling of membrane-matrix linkages when tubes expand. Such a scenario has not yet been studied in angiogenesis. It may represent a new starting point for genetic studies to decipher the putative roles of ZP domain proteins and Matriptase in clinically relevant syndromes, including the formation of aneurysms caused by membrane deformation and defects in size determination of airways and vessels (Drees, 2023).

Control of tissue morphogenesis by the HOX gene Ultrabithorax

Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. This study shows that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. These results provide a long-awaited explanation for how Ubx controls morphogenetic transformation (Diaz-de-la-Loza, 2020).

The results reveal how Ubx – a homeotic gene that encodes the founding member of the HOX-family of transcription factors – regulates apical and basal matrix remodelling to control epithelial morphogenesis (see Ubx controls apical and basal ECM degradation to regulate morphogenesis). Ubx strongly represses two genes encoding apical matrix proteases (Np and Sb), as well as partially repressing two genes encoding basal matrix metalloproteases (Mmp1 and Mmp2), while inducing an inhibitor of Mmp1/2 (Timp) in the haltere. In this way, Ubx prevents both apical and basal matrix remodelling in the haltere, a key event in the homeotic wing-to-haltere transformation. In addition to regulating morphogenesis, Ubx controls many other genes affecting wing growth and pattern. Together, the combined repression of morphogenesis, growth and patterning by Ubx is responsible for the full transformation of wing to haltere (Diaz-de-la-Loza, 2020).

Ubx controls apical and basal ECM degradation to regulate morphogenesis. Schematic of Ubx expression and function in Drosophila and a hypothetical four-winged ancestor. Ubx controls organ shape via regulation of aECM and bECM proteases, in addition to its known functions in regulating organ growth and patterning. These target genes have presumably evolved to be specifically regulated in the Drosophila wing and/or haltere, and must be insensitive to Ubx in four-winged ancestors (Diaz-de-la-Loza, 2020).

These findings also support the general view that transcriptional control of matrix synthesis and degradation is a conserved mechanism by which information encoded in the genome is deployed to govern the shape of tissues and organs in animals. Although this concept is broadly appreciated for the regulation of the bECM, the notion that the aECM is also developmentally regulated during tissue morphogenesis needs further investigation, particularly in mammals. Beyond animals, morphogenesis of plants, fungi and bacteria is also known to be fundamentally dependent on patterned synthesis and degradation of the cell wall, a type of ECM. Thus, genetic control of the matrix appears to be a general principle that shapes all life forms (Diaz-de-la-Loza, 2020).

Dual function for Tango1 in secretion of bulky cargo and in ER-Golgi morphology

Tango1 enables ER-to-Golgi trafficking of large proteins. Loss of Tango1, in addition to disrupting protein secretion and ER/Golgi morphology, causes ER stress and defects in cell shape. The previously observed dependence of smaller cargos on Tango1 is a secondary effect. If large cargos like Dumpy, which this study identifies as a Tango1 cargo, are removed from the cell, nonbulky proteins reenter the secretory pathway. Removal of blocking cargo also restores cell morphology and attenuates the ER-stress response. Thus, failures in the secretion of nonbulky proteins, ER stress, and defective cell morphology are secondary consequences of bulky cargo retention. By contrast, ER/Golgi defects in Tango1-depleted cells persist in the absence of bulky cargo, showing that they are due to a secretion-independent function of Tango1. Therefore, maintenance of ER/Golgi architecture and bulky cargo transport are the primary functions for Tango1 (Rios-Barrera, 2017).

The endoplasmic reticulum (ER) serves as a major factory for protein and lipid synthesis. Proteins and lipoproteins produced in the ER are packed into COPII-coated vesicles, which bud off at ER exit sites (ERES) and then move toward the Golgi complex where they are sorted to their final destinations. Regular COPII vesicles are 60-90 nm in size, which is sufficient to contain most membrane and secreted molecules. The loading of larger cargo requires specialized machinery that allows the formation of bigger vesicles to accommodate these bulky molecules. Tango1 (Transport and Golgi organization 1), a member of the MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family, is a key component in the loading of such large molecules into COPII-coated vesicles. Molecules like collagens and ApoB (apolipoprotein B)-containing chylomicrons are 250-450 nm long and rely on Tango1 for their transport out of the ER, by physically interacting with Tango1 or Tango1 mediators at the ERES (Rios-Barrera, 2017).

Tango1 is an ER transmembrane protein that orchestrates the loading of its cargo into vesicles by interacting with it in the ER lumen. The interaction of Tango1 with its cargo then promotes the recruitment of Sec23 and Sec24 coatomers on the cytoplasmic side, while it slows the binding of the outer layer coat proteins Sec13 and Sec31 to the budding vesicle. This delays the budding of the COPII carrier. Tango1 also recruits additional membrane material to the ERES from the Golgi intermediate compartment (ERGIC) pool, thereby allowing vesicles to grow larger. It also interacts directly with Sec16, which is proposed to enhance cargo secretion. A shorter isoform of mammalian Tango1 lacks the cargo recognition domain but nevertheless facilitates the formation of megacarrier vesicles (Rios-Barrera, 2017).

Apart from bulky proteins, some heterologous, smaller proteins like secreted horseradish peroxidase (ssHRP, 44 kDa) and secreted GFP (27 kDa) also depend on Tango1 for their secretion . Unlike for collagen or ApoB, there is no evidence for a direct interaction between Tango1 and ssHRP or secreted GFP. It is not clear why Tango1 would regulate the secretion of these molecules, but it has been proposed that in the absence of Tango1, the accumulation of nonbulky proteins at the ER might be due to abnormally accumulated Tango1 cargo blocking the ER; however, this has not been tested experimentally (Rios-Barrera, 2017).

Drosophila Tango1 is the only member of the MIA/cTAGE family found in the fruit fly, which simplifies functional studies. Like vertebrate Tango1, the Drosophila protein participates in the secretion of collagen. And as in vertebrates, ssHRP, secreted GFP, and other nonbulky molecules like Hedgehog-GFP also accumulate in the absence of Tango1. These results have led to the proposal that Tango1 participates in general secretion. However, most of the evidence for these conclusions comes from overexpression and heterologous systems that might not reflect the physiological situation (Rios-Barrera, 2017).

This study describes a tango1 mutant allele that was identified in a mutagenesis screen for genes affecting the structure and shape of terminal cells of the Drosophila tracheal system. Tracheal terminal cells form highly ramified structures with branches of more than 100 μm in length that transport oxygen through subcellular tubes formed by the apical plasma membrane. Their growth relies heavily on membrane and protein trafficking, making them a very suitable model to study subcellular transport. Terminal cells were used to study the function of Tango1, and loss of Tango1 was found to affect general protein secretion indirectly, and it also leads to defects in cell morphology and in the structure of the ER and Golgi. The defects in ER and Golgi organization of cells lacking Tango1 persist even in the absence of Tango1 cargo (Rios-Barrera, 2017).

These studies led to an explanation of why, in the absence of Tango1, nonbulky proteins accumulate in the ER despite not being direct Tango1 cargos. These cargos are retained in the ER as a consequence of nonsecreted bulky proteins interfering with their transport. However, the effect of loss of Tango1 on ER/Golgi morphology can be uncoupled from its role in bulky cargo secretion (Rios-Barrera, 2017).

This study has described a role of Tango1, which was initially identified through its function in tracheal terminal cells and other tissues in Drosophila embryos, larvae, and pupae. Due to their complex shapes and great size, terminal cells are a well-suited system to study polarized membrane and protein trafficking, with the easily scorable changes in branch number and maturation status providing a useful quantitative readout that serves as a proxy for functional membrane and protein trafficking machinery. Moreover, this analyses are conducted in the physiological context of different tissues in the intact organism (Rios-Barrera, 2017).

The loss-of-function allele tango1^2L3443 has a stop codon eight amino acids downstream of the PRD domain and eliminates the 89 C-terminal amino acids of the full-length protein. It is unlikely that the mutation leads to a complete loss of function. First, terminal cells expressing an RNAi construct against tango1 show stronger defects, with fewer branches per cell than homozygous tango1^2L3443 cells. Second, the mutant protein appears not to be destabilized nor degraded, but instead is present at apparently normal levels, albeit at inappropriate sites. Predictions of the deleted fragment of the protein suggest it is disorganized and that it contains an arginine-rich domain that has no known interaction partners and that is not present in human Tango1. In homozygous mutant terminal cells, the mutant tango1^2L3443 protein fails to localize at ERES. In mammalian Tango1, the Sec16-interacting region within the PRD domain is necessary for the localization of Tango1 to the ERES and for its interaction with Sec23 and Sec16, but since this domain is fully present in tango1^2L3443, the results mean that either the missing 89 C-terminal amino acids contain additional essential localization signals, or that the PRD domain is structurally affected by the truncation of the protein. The latter is considered less likely, as a truncation of eight amino acids downstream of the PRD domain is unlikely to destabilize the polyproline motifs, especially as the overall stability of the protein does not seem to be affected. Furthermore, this region shows a high density of phosphoserines (Ser-1345, Ser-1348, Ser-1390, and Ser-1392), suggesting it might serve as a docking site for adapter proteins or other interactors (Rios-Barrera, 2017).

Terminal cells lacking Tango1 have fewer branches than control cells and are often not properly filled with air. This loss-of-function phenotype is not due to a direct requirement for Tango1, as it is suppressed by the simultaneous removal of Dumpy (Dpy), an extracellular protein involved in epidermal-cuticle attachment, aposition of wing surfaces and trachea development. It also cannot be explained by the individual loss of crb, Piopio (Pio) a zona pellucida (ZP) domain protein that mediates the adhesion of the apical epithelial surface and the overlying apical extracellular matrix, or dpy, since knocking down any of these genes has no effect on cell morphology. Instead, it is proposed that the cell morphological defect is due at least in part to the activation of the ER stress response, since expression of Xbp1 is sufficient to recapitulate the phenotype. Xbp1 regulates the expression of genes involved in protein folding, glycosylation, trafficking, and lipid metabolism. It is possible that one or a small number of specific genes downstream of Xbp1 are responsible for defective branch formation or stability, but the phenotype could also be a secondary consequence of the physiological effects of the ER stress response itself, for example, a failure to deliver sufficient lipids and membrane from the ER to the apical plasma membrane (Rios-Barrera, 2017).

Collagen, with a length of 300 nm, and ApoB chylomicrons with a diameter of > 250 nm, have both been biochemically validated as Tango1 cargos. These molecules are not expressed in terminal cells, and therefore it was clear that Tango1 must have a different substrate in these cells. Given that Tango1 is known for the transport of bulky cargo, that Dpy is the largest Drosophila protein at 800 nm length, and that Dpy vesicles are associated with Tango1 rings in tracheal cells, it is proposed that Dpy is a further direct target of Tango1. Colocalization of Tango1 with its cargo has also been observed in other tissues: with collagen in Drosophila follicle cells and with ApoB in mammalian cell lines (Rios-Barrera, 2017).

No regions of sequence similarity that could represent Tango1-binding sites have been found in Tango1 cargos. There are several possible explanations for this. First, these proteins may contain binding motifs, but the motifs are purely conformational and not represented in a linear amino acid sequence. There is no evidence for or against this hypothesis, but it would be highly unusual, and there is support for alternative explanations. Thus, as a second possibility, all three proteins may require Tango1 for their secretion, but variable adapters could mediate the interactions. In vertebrates, Tango1 can indeed interact with its cargo through other molecules; for instance, its interaction with collagen is mediated by Hsp47. However, in Drosophila, there is no Hsp47 homolog. In the case of ApoB, it has been suggested that microsomal triglyceride transfer protein (MTP) and its binding partner, protein disulphide isomerase (PDI), might associate with Tango1 and TALI to promote ApoB chylomicrons loading into COPII vesicles. Evidence supporting this is that the lack of MTP leads to ApoB accumulation at the ER. It is not known if secretion of other Tango1 cargos like collagen or Dpy also depends on MTP and PDI, but PDI is known also to form a complex with the collagen-modifying enzyme prolyl 4-hydroxylase. Previous work has shown that terminal cells lacking MTP show air-filling defects and fail to secrete Pio and Uninflatable to the apical membrane, and that loss of MTP in fat body cells also affects lipoprotein secretion, as it does in vertebrates. Since cells lacking MTP or Tango1 have similar phenotypes, it is plausible that the MTP function might be connected to the activity of Tango1 (Rios-Barrera, 2017).

The data is interpreted to mean that in the absence of Tango1, primary cargo accumulates in the ER, and in addition, there are secondary, indirect effects that can be suppressed by reducing the Tango1 cargo that overloads the ER. The secondary effects include activation of the ER stress response and intracellular accumulation of other trafficked proteins like Crb, laminins, and overexpressed proteins and probably also the accumulation of heterologous proteins like secreted HRP or GFP in other systems (Rios-Barrera, 2017).

The data suggest that primary and secondary cargo reach the ERES but fail to be trafficked further along the secretory pathway. In this model, primary cargo, probably recruited by adaptors, would be competing with other secondary cargo for ERES/COPII availability, creating a bottleneck at the ERES. This is consistent with recent experiments that show that in tango1-knockdown HeLa cells, VSVG-GFP trafficking does not stop completely, but is delayed. Furthermore, in these experiments, VSVG-GFP is mostly seen in association with Sec16 and Sec31, supporting the clogging model (Rios-Barrera, 2017).

It is not immediately clear why cargo accumulation in terminal cells lacking Tango1 affects the secretion of Crb but not of βPS integrin. While steady states are looked at in this analyses, Maeda (2016) measured the dynamics of secretion and found that loss of Tango1 leads to a reduced rate of secretion of VSVG-GFP, an effect that would have been missed for any proteins the current study classified as not affected by loss of Tango1. Irrespective, a range of mechanisms can be thought of that might be responsible for this difference, including alternative secretion pathways and differences in protein recycling. Alternative independent secretory pathways have been reported in different contexts. For instance, while both αPS1 and βPS integrin chains depend on Sec16 for their transport, the αPS1 chain can bypass the Golgi apparatus and can instead use the dGRASP-dependent pathway for its transport. It would be possible then that in terminal cells, βPS integrin is also trafficked through an alternative pathway that is not affected by loss of Tango1. Similarly, tracheal cells lacking Sec24-CD accumulate Gasp, Vermiform, and Fasciclin III, but not Crb, supporting a role for alternative secretion pathways for different proteins, as has been already proposed. Following this logic, overexpressed βPS integrin would then also be trafficked through a different route from that of the endogenous βPS integrin, possibly because of higher expression levels or because of the presence of the Venus fused to the normal protein (Rios-Barrera, 2017).

Drosophila Tango1 was initially found to facilitate collagen secretion in the fat body. More recently, the accumulation of other nonbulky proteins at the ER in the absence of Tango1 has led to the proposal of two models to explain these results: one in which Tango1 regulates general secretion, and the second one where Tango1 is specialized on the secretion of ECM components, since loss of Tango1 leads to the accumulation of the ECM molecules SPARC and collagen. The current results suggest a third explanation, where cargo accumulation in the ER might not necessarily be a direct consequence of only the loss of Tango1. Instead, in addition to depending on Tango1, some proteins of the ECM appear also to depend on each other for their efficient secretion. This is the case for laminins LanB1 and LanB2, which require trimerization before exiting the ER, while LanA can be secreted as a monomer. Loss of collagen itself leads to the intracellular accumulation of ECM components in fat body cells, such as the laminins and SPARC. Conversely, SPARC is required for proper collagen and laminin secretion and assembly in the ECM. Furthermore, intricate biochemical interactions take place between ECM components. Hence, due to the complex genetic and biochemical interactions between ECM components, the dependence of any one of them on Tango1 is difficult to determine without further biochemical evidence. The concept of interdependent protein transport from the ER as such is not new, as it has also been observed in other systems, for instance in immune complexes. During the assembly of T-cell receptor complexes and IgM antibodies, subunits that are not assembled are retained in the ER and degraded (Rios-Barrera, 2017).

Nevertheless, these observations in glial cells, which express laminins but not collagen, allow at least these requirements to be partly separate. This study found that laminins are accumulated due to general ER clogging and not because they rely on Tango1 for their export. This is based on observations that once the protein causing the ER block is removed, laminin secretion can continue in the absence of Tango1. It is still unclear why glial cells can secrete laminins in the absence of collagen whereas fat body cells cannot, but presumably laminin secretion can be mediated by different, unidentified cargo receptors expressed in glial cells (Rios-Barrera, 2017).

This study found that Sec16 forms aberrant aggregates in cells lacking Tango1, as in mammalian cell lines, and that the number of Sec16 particles is reduced. Other studies have shown that Tango1 overexpression produces larger ERES, and that Tango1 and Sec16 depend on each other for localization to ERES. In addition, lack of Tango1 also affects the distribution of Golgi markers. Thus, Tango1 influences not only the trafficking of cargos, but also the morphology of the secretory system (Rios-Barrera, 2017).

It had been suggested that the disorganization of ER and Golgi apparatus in cells lacking Tango1 might be an indirect consequence of the accumulation of Tango1 cargo. The work of Maeda (2016) has provided a possible explanation for the molecular basis, and proposed that Tango1 makes general secretion more efficient, but it has not formally excluded the possibility that the primary cause for the observed defects is secretory protein overload. This study has now shown that this is not the case: In the absence of Tango1, an aberrant ER and Golgi morphology is still observed even after the main primary substrates of Tango1 were removed and, thereby, secretion of other molecules was restored and the ER stress response was prevented (Rios-Barrera, 2017).

ERGIC53 accumulates at the ERES in the absence of Tango1, and this can be partly reversed by removing dpy. This is in apparent contradiction to findings in mammalian cells where Tango1 was necessary for the recruitment of membranes containing ERGIC53 to the budding collagen megacarrier vesicle. However, ERGIC53 also has Tango1-independent means of reaching the ER. The current results indicate that its retrieval from the ER to the ERGIC compartment depends directly or indirectly partly on Tango1. As a cargo receptor for glycoproteins, ERGIC53 may be retained at the ERES as a consequence of the accumulation of its own cargo at these sites. This would mean that it cannot be delivered back to the ERGIC or the cis-Golgi apparatus for further rounds of retrograde transport, which may, in turn, be an explanation for the enlargement of the Golgi matrix protein 130 kD (GM130) compartment seen after Tango1 knockdown (Rios-Barrera, 2017).

The finding that Tango1-depleted cells have a functional secretory pathway despite the ER-Golgi disorganization was unexpected. Stress stimuli like amino acid starvation (but not ER stress response itself) lead to Sec16 translocation into Sec bodies and inhibition of protein secretion. However, uncoupling of ER-Golgi organization from functional secretion has also been observed in other contexts. Loss of Sec23 or Sec24-CD leads to a target peptide sequence KDEL appearing in aggregates of varying sizes and intensities similar to those observed for Sec16 and for KDEL-RFP in cells lacking tango1. Also, GM130 is reduced in Sec23 mutant embryos. Despite these structural problems, these embryos do not show generalized secretion defects and also do not affect the functionality of the Golgi apparatus, as determined by glycosylation status of membrane proteins (Rios-Barrera, 2017).

Thus, Tango1 appears to have an important structural function in coordinating the organization of the ER and the Golgi apparatus, and this, in turn, may enhance vesicle trafficking. This fits with the role of Tango1 in recruiting ERGIC membranes to the ERES, and also with the effects of loss of Tango1 in the distribution of ER and Golgi markers. It has been proposed that the ER and Golgi apparatus in insects, which unlike in mammalian cells is not centralized but spread throughout the cytoplasm, is less efficient for secretion of bulky cargo than mammalian cells that can accommodate and transport it more efficiently through the Golgi ribbon. This difference could explain why tango1 knockout mice seem to have only collagen secretion defects and die only as neonates. However, a complete blockage of the ER might also be prevented by the activity of other MIA3/cTAGE5 family homologs. In mammalian cell culture experiments, even if loss of tango1 affects secretion of HRP, the secretion of other overexpressed molecules like alkaline phosphatase is not affected. This could also be because of the presence of other MIA3/cTAGE5 family homologs. By contrast, because there are no other MIA3/cTAGE5 family proteins in Drosophila, loss of tango1 may lead to the accumulation of a wider range of overexpressed proteins and more overt mutant phenotypes than in mammals (Rios-Barrera, 2017).

Patterned anchorage to the apical extracellular matrix defines tissue shape in the developing appendages of Drosophila

How tissues acquire their characteristic shape is a fundamental unresolved question in biology. While genes have been characterized that control local mechanical forces to elongate epithelial tissues, genes controlling global forces in epithelia have yet to be identified. This study describes a genetic pathway that shapes appendages in Drosophila by defining the pattern of global tensile forces in the tissue. In the appendages, shape arises from tension generated by cell constriction and localized anchorage of the epithelium to the cuticle via the apical extracellular-matrix protein Dumpy (Dp). Altering Dp expression in the developing wing results in predictable changes in wing shape that can be simulated by a computational model that incorporates only tissue contraction and localized anchorage. Three other wing shape genes, narrow, tapered, and lanceolate, encode components of a pathway that modulates Dp distribution in the wing to refine the global force pattern and thus wing shape (Ray, 2015).

This study has identified a group of genes that define the global force patterns that shape the appendages in Drosophila. During pupal development, shape is determined by a general contraction of the tissue in combination with localized anchorage to the pupal cuticle, which is mediated by the apical extracellular matrix (aECM) protein Dp. In the developing wing, Dp is localized to the wing margin such that, as tissue contraction proceeds, tension along the P-D axis elongates the wing and also draws the two wing surfaces together. Indeed, manipulating the pattern of Dp localization at this stage leads to dramatic changes in wing shape that reflect the underlying change in tissue anchorage. In the legs and antennae, Dp is found in a dense plaque at the distal tip of the appendage, and, as in the wing, tissue contraction results in tapering and elongation of the structure. Thus, this study has identified a genetic mechanism that determines shape by regulating the pattern of global tensile forces that the epithelium experiences during tissue contraction (Ray, 2015).

While the mechanism that this study has uncovered is clearly important for proper anchorage of the wing epithelium to the cuticle, it is only one part of the regulatory mechanism that leads to the localized attachment. Indeed, in a nw mutant, while the distribution of Dp is altered, it is still localized to the margin, thus other inputs must be involved in defining where Dp is localized during pupal development. In the wing, the localization of Dp to the margin is reminiscent of the expression of genes controlled by the Notch and Wingless pathways that define the dorsal-ventral compartment boundary. Indeed, the gene Dll is a downstream target of Wg, and knocking down dp in the cells that express Dll phenocopies the dp loss-of-function phenotype. Moreover, the notching associated with mutations in the Notch and Wg pathways, as well as their targets such as cut, are, in essence, defects in the anchorage of the margin during pupal development: the failure to specify the margin results in a gap in the expression of Dp which produces a phenotype not unlike that which is observed in dpp-Gal4>dpRNAi. Consistent with this, it has previously been shown that the notching associated with cut arises during pupal development during the period of hinge contraction. Thus, it may be that these phenotypes arise from a failure to localize Dp to specific regions of the margin rather than to cell death, as has been suggested previously (Ray, 2015).

In the leg and antenna, the localization of Dp to the extreme distal tip of the appendage is presumably under control of the P-D patterning system that operates in these tissues. Indeed, as in the wing, this study hasa shown that the retraction of the leg and antenna is produced by knocking down dp with Dll-Gal4, which is expressed in the most distal segments of both appendages. Moreover, the observed phenotypes are also associated with mutations in genes that affect specification of the distal most tarsal segments. Classical loss-of-function mutations of the Paired-type homeodomain protein Aristaless result in the same shortening of the arista as is observed with dp-RNAi. Similarly, loss-of-function alleles of the transcription factor Lim1 result in a shortening of the arista and the distal leg segments, which presumably reflects the failure to anchor the distal tip of the appendage to the pupal cuticle. Thus, it is speculated that for wings, legs, and antennae, the localized function of Dp appears to depend on cues from the developmental programs that pattern the appendages (Ray, 2015).

While Dp localization clearly depends on the positional cues set down in the imaginal discs, how these signaling molecules and transcription factors result in a localized pattern of Dp protein in the pupa remains unclear. Previous studies have shown that the dp mRNA is expressed throughout the developing wing in early pupal development (Wilkin, 2000), suggesting that the localized pattern of Dp expression that is evident at 18 hr APF results by a post-transcriptional mechanism. Throughout development, Dp functions as a link between the ectodermal epithelium and the cuticle, and, in order for the animal to molt, this connection must be periodically broken. During larval development, molting (or ecdysis) is initiated by apolysis, a process that separates epidermal cells from the old cuticle by secretion of a complex mixture of chitinases and proteases that degrade the carbohydrate and protein components of the exoskeleton, respectively. Given that Dp is essential for the link between epidermis and cuticle, it is a key target of the apolytic machinery. In the pupa, apolysis of the pupal cuticle occurs just prior to the onset of tissue contraction and is a prerequisite for generating global forces by differential anchorage. Given this, the localization of Dp and the localized anchorage of the appendage tissues to the cuticle presumably arise by protection of Dp at the margin and its degradation throughout the rest of the wing blade. These results suggest that in the wing, Nw acts to extend this protection more proximally to give rise to the wild-type shape of the wing (Ray, 2015).

The results show that coordination of the behavior of thousands of individual cells in the pupal wing is achieved by the precise regulation of global extrinsic forces that determine appendage shape. In this system, the wing hinge undergoes apical constriction to generate a pulling force that is transmitted through the tissue. As shown in this study, resistance to this force is provided by anchorage of the wing margin to the overlying pupal cuticle, resulting in anisotropic tension oriented predominantly along the P-D axis. As has been shown previously, cells respond to this tension via cell shape changes, oriented cell divisions, and cell rearrangements to drive tissue elongation and determine the final shape the wing. Thus, as a mechanical process, tissue shaping during pupal development depends on the magnitude of the force generated by the constricting cells, the strength of the anchorage resisting this force, and the fluidity of the tissue to relieve the tension (Ray, 2015).

Several lines of evidence suggest that these different components of the system operate toward an equilibrium state where opposing forces come to balance. For instance, if the force produced by the cell constriction exceeds the resistance, the anchorage ruptures, releasing the tissue. When this occurs, as in wings mutant for the hypomorphic allele dp^ov1, the margin collapses and the cells in the middle of the wing blade constrict further than they would otherwise. Similarly, the range of phenotypes observed in the interactions between nw^D and dp^ov1 suggest that if the level of Dp falls below a certain threshold, the anchorage ruptures after hinge contraction has started, resulting in intermediate phenotypes that are flattened and retain some taper, but have nevertheless retracted. These observations suggest that cells in the wing blade actively respond to the pattern of tensile force with significant consequences on wing shape (Ray, 2015).

The data also indicate that hinge cells respond to the tension in the blade and constrict accordingly. Morphometric analysis of the nw mutant shows that the narrowing and extension of the wing blade is accompanied by a isometric contraction of the hinge, such that in the adult wing, the hinge is significantly smaller than in wild-type. Similarly, in the various dp mutants this study had examined, any change in the wing shape is accompanied by a corresponding change in hinge size and shape. The implication from these observations is that in the absence of sufficient resistance, hinge cells continue to undergo constriction, resulting in a corresponding change in hinge shape. Indeed, in a computational model, whenever the anchorage is released by any significant amount, the hinge is also found to contract further than in the wild-type simulation (Ray, 2015).

Taken together, these observations suggest that the system operates toward an equilibrium point where the force generated by hinge contraction is balanced with the resistance coming from the distal anchorage and the deformation properties of the cell matrix. Initially, the force generated by hinge contraction is offset by cell division and cell rearrangement, but when the cell division ceases, resistance in the epithelium feeds back on the hinge and eventually blocks further constriction. At this point, the system comes to equilibrium and the tension along the cell junctions equalizes. Notably, this model is consistent with previous reports, which have shown that after the initial phase of hinge contraction is completed, equalization of junction tension initiates the shift toward the hexagonal packing geometry that is characteristic of late stage pupal wings. The attractiveness of this kind of model is that it ensures robustness of the shaping mechanism and avoids the complications of tears or buckles that might arise from stochastic perturbations occurring during development. The nature of these feedbacks and how the equilibrium is achieved remain a question for further study (Ray, 2015).

As orthologs of Dp and Nw have been found, and in all sequenced insect genomes and in the crustacean Daphnia, yhr results suggest that the Nw-Dp system may be a key target for evolution of appendage shape in Arthropods. Indeed, given the tremendous variety of wing shapes that are found in insects, it is tempting to speculate that some of this variation is achieved by modulation of the Nw-Dp system, resulting in changes in the force patterns in the developing wing. For instance, in a recent report, wing shape differences in males of Nasonia vitripennis and Nasonia giraulti were attributed to differences in transcriptional regulation of an unpaired-like gene that was proposed to regulate proliferation in the developing wing. Significantly, the expression of this gene in both species was confined to the margin at the distal tip of the wing and strikingly prefigures the radius of curvature of the adult wing. Given these results, it is plausible that the difference in wing shape in these two species may arise from differences in anchorage of the wing tip, with the difference in cell number arising as a secondary consequence as in the case of nw. Similarly, the evolution of the diverse, specialized wing shapes in butterflies and moths has been attributed to defined expression of margin specific genes that prefigure the adult wing shape. While little is known of the elaboration of this prepattern during pupal development, the scalloping of the adult wing margin observed in many species—reminiscent of notching in the Drosophila wing—may well arise from precise deployment of the Nw-Dp system. Exploration of how Dp is regulated in different species may shed light on how the variety of insect wing shapes has evolved (Ray, 2015).

In all metazoans, assembly of the aECM is dependent on proteins that contain a common protein motif, the Zona Pellucida (ZP) domain, which is thought to act as a polymerization module promoting the formation of homo and heterotypic filaments. Genetic studies have implicated ZP-domain proteins in a variety of morphogenetic processes that typically involve shaping or remodeling of the apical domain of the cells. In Drosophila, ZP-domain proteins shape the embryonic denticles and hairs and the wing trichomes that form on the apical surface of the cells, and in C. elegans, the Cuticlulins are ZP-domain proteins involved in the formation of the alae. Similarly, in flies and nematodes, aECM proteins have been implicated as anchors in the cellular morphogenesis of sensory neurons, with Drosophila NompA functioning to anchor neural dendrites to the cuticular structures in sensory organs while in the nematode, the aECM proteins DEX-1 and DYF-7 anchor the dendritic tips during cell body migration to shape the amphid sense organs. Among these examples, these findings represent a different paradigm for how aECM proteins can influence morphogenesis: rather than affecting the behavior of individual cells, and consequently the shape of the tissue, the Nw-Dp mechanism defines global force patterns across the tissue to which the individual cells respond to give rise to appendage shape (Ray, 2015).

In vertebrates, the most well studied ZP-domain proteins are the eponymous zona pellucida proteins that form the extracellular coat of mammalian ova and the α- and β-tectorins that are required for the formation of the tectorial membrane in the ear. However, despite their importance for fertility and hearing, respectively, these proteins do not affect morphogenesis of the tissue per se. On the other hand, in the human kidney, the ZP-domain protein hensin/DMPT1 regulates morphogenesis of α- and β-Intercalated cells in the collecting tubules, and results from at least one study have found that global deletion of hensin results in embryonic lethality, suggesting a more general role in epithelial differentiation. Thus, there is mounting evidence from both invertebrates and vertebrates that the aECM plays an important role in morphogenesis and further studies on aECM proteins will undoubtedly reveal other roles they play in development and disease (Ray, 2015).

Search PubMed for articles about Drosophila Notopleural

Diaz-de-la-Loza, M. D., Loker, R., Mann, R. S., Thompson, B. J. (2020). Control of tissue morphogenesis by the HOX gene Ultrabithorax. Development, 147(5) PubMed ID: 32122911

Drees, L., Konigsmann, T., Jaspers, M. H. J., Pflanz, R., Riedel, D., Schuh, R. (2019). Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis. PLoS Genet, 15(1):e1007882 PubMed ID: 30601807

Drees, L., Schneider, S., Riedel, D., Schuh, R., Behr, M. (2023). The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila. Elife, 12 PubMed ID: 37872795

Lai, Y. J., Chang, H. H., Lai, H., Xu, Y., Shiao, F., Huang, N., Li, L., Lee, M. S., Johnson, M. D., Wang, J. K., Lin, C. Y. (2015). N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin. PLoS One, 10(7):e0132163 PubMed ID: 26171609

Ray, R. P., Matamoro-Vidal, A., Ribeiro, P. S., Tapon, N., Houle, D., Salazar-Ciudad, I., Thompson, B. J. (2015). Patterned Anchorage to the Apical Extracellular Matrix Defines Tissue Shape in the Developing Appendages of Drosophila. Dev Cell, 34(3):310-322 PubMed ID: 26190146

Rios-Barrera, L. D., Sigurbjornsdottir, S., Baer, M., Leptin, M. (2017). Dual function for Tango1 in secretion of bulky cargo and in ER-Golgi morphology. Proc Natl Acad Sci U S A, 114(48):E10389-E10398 PubMed ID: 29138315

date revised: 2 September 2024

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