The Interactive Fly

Zygotically transcribed genes

Extracellular matrix and the basement membrane

  • Proteins expressed in the extracellular matrix and basement membrane

    Extracellular matrix Basement membrane in oogenesis Basement membrane in the fat body and lymph gland Basement membrane in the heart

    A moving source of matrix components is essential for de novo basement membrane formation

    The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly, this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components, including Col IV, Perl and LanA appears to be hierarchical, yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types, and it is unclear how large, possibly insoluble protein complexes are delivered into the matrix. This study exploited the ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, it was shown that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components (Matsubayashi, 2017).

    To analyze de novo basement membrane (BM) formation, developing Drosophila embryos were used. The developmental profile of BM components was analyzed from the Drosophila modENCODE project. This revealed that, while Laminin mRNAs are observed early in development, extracellular matrix (ECM) components associated with a mature BM, such as Collagen IV (Vkg in Drosophila) and Perlecan (Trol in Drosophila), are expressed later, suggesting that there is a temporal hierarchy of BM production during embryogenesis (Matsubayashi, 2017).

    Embryonic BM protein production was examined using endogenously tagged BM fly lines. Homozygous viable GFP-protein traps were used in Collagen IV (Col IV) and Perlecan (Perl) as well as a recently generated line containing GFP-tagged Lamininα (LanA). This LanA-GFP is capable of biochemically interacting with other Laminin subunits to form a mature Laminin trimer, and it rescued LanA mutant embryos. Furthermore, when expressed in a Lamininβ (LanB1) mutant background, LanA levels were severely reduced, suggesting that subunit trimerization is indeed essential for Laminin production and secretion. Using these GFP-tagged lines, the dynamics of BM production were analyzed by quantifying GFP intensity over time during development. This revealed that expression of BM components peaked immediately prior to embryonic hatching. Furthermore, components showed precise temporal regulation with LanA expressed first, followed by Col IV, and finally Perl. A second GFP-tagged construct was examined of the sole Drosophila Lamininβ isoform (LanB1), which was previously confirmed to be fully functional, and this also revealed Laminin expression to occur prior to Col IV or Perl (Matsubayashi, 2017).

    In Drosophila embryos, hemocytes are known to produce BM. However, it has been unclear what proportion of the embryonic BM is hemocyte dependent. When GFP-tagged BM proteins were expressed in a mutant background in which hemocytes failed to develop, it was revealed that BM components are differentially hemocyte dependent. This showed that 70% of Col IV and 50% of Perl are dependent on hemocytes. In contrast, hemocytes contribute only 30% of embryonic LanA, with most of the hemocyte-derived Laminin induced at later stages of development. As the mesoderm expresses LanA, it was hypothesize that its early expression is likely dependent on this tissue. For Col IV and Perl, the remaining protein was expressed in the fat body at late stages of development, which is known to be the major source of larval BM (Pastor-Pareja, 2011; Matsubayashi, 2017 and references therein).

    To investigate the functional importance of the temporal hierarchy of BM component expression, embryos were generated expressing the GFP-tagged LanA, Col IV, and Perl in all possible mutant backgrounds of opposite components. This revealed that, while LanA incorporation or levels were unaffected by the absence of subsequent components, Col IV and Perl formed disorganized extracellular deposits in the absence of Laminin. It was hypothesized that these aggregates are the specific result of Col IV aggregation, as the Perl deposits were absent in a Col IV/Laminin double mutant. Finally, Perl, which is expressed last in the temporal hierarchy, required prior production of Laminin and Col IV for proper expression and incorporation into the BM, which is similar to what was previously reported (Hollfelder, 2014). These results suggest that proper de novo BM formation requires temporal regulation of component production. A similar temporal hierarchy of BM production may be critical for BM formation in other species, as disorganized ECM deposits have also been observed in laminin mutant mice (Smyth, 1999) and C. elegans (Huang, 2003; Matsubayashi, 2017).

    Differences were observed in the appearance of Col IV and Laminin in the wild-type background, with Laminin showing a much more diffuse distribution. These differences were investigated by time-lapse microscopy during hemocyte migration along the ventral nerve cord (VNC), which is a known migratory route that is readily amenable to live imaging. Both LanA and LanB1 subunits were observed to form 'halos' of graded expression surrounding migrating hemocytes, with trails of Laminin forming as cells moved within the acellular fluid-filled cavity of the embryo (hemocoel). These halos of Laminin were identical to expression of secreted-GFP, suggesting that Laminin is simply filling the hemocoel. In contrast, while Col IV and Perl decorated the surface of the VNC, there was no observable fluorescence filling the hemocoel. Whether the differences in BM component localization were the result of their differing diffusive characteristics was examined by performing fluorescence recovery after photobleaching (FRAP) analysis. This showed that LanA had a significant mobile fraction unlike Col IV, which failed to show any recovery. To understand why Laminin formed halos surrounding hemocytes along the VNC, the ventral hemocoel was examined by transmission electron microscopy (TEM). This revealed that the ventral hemocoel is highly confined, with the VNC in physical contact with the overlying epithelium. Therefore the halos of Laminin and its trails following hemocyte movement represent hemocytes separating the VNC from the overlying epithelium, allowing Laminin diffusion. These data highlight that different BM components have distinct diffusive properties within the developing embryo (Matsubayashi, 2017).

    The apparent absence of soluble Col IV in the hemocoel suggested that Col IV might require a local mechanism of deposition by hemocytes. However, while it was possible to observe some BM material deposited beneath migrating hemocytes by TEM, it was difficult to examine the dynamics of Col IV deposition beneath hemocytes by standard confocal microscopy due to the low level of fluorescence and small size of the deposits. Therefore, lattice light-sheet microscopy, which allows for enhanced spatiotemporal resolution with reduced phototoxicity, was used. Indeed, hemocyte motility within the ventral hemocoel was highly amenable to lattice light-sheet imaging at early stages of hemocyte dispersal with minimal photobleaching (Matsubayashi, 2017).

    Imaging by lattice light-sheet microscopy revealed that, at the stage when hemocytes are aligned on the ventral midline, Col IV is primarily localized beneath hemocytes on the surface of the nerve cord and in the segmentally spaced dorsoventral channels of the VNC. Subsequently, when hemocytes left the midline and migrated laterally, they appeared to deposit Col IV in a local fashion leaving puncta of matrix that eventually developed into longer fibrils. Additionally, simultaneous imaging of Col IV and the hemocyte actin cytoskeleton showed that Col IV colocalized with actin fibers within lamellae, suggesting hemocyte secretion of Col IV may require release along actin fibers or that recently released Col IV is rapidly remodelled by hemocytes using their actin network. Indeed, tracking movements in the Col IV matrix at high temporal resolution by particle image velocimetry revealed strong regions of ECM deformation beneath hemocyte lamellae, suggesting hemocyte traction forces are being exerted on the developing BM (Matsubayashi, 2017).

    As time-lapse imaging suggested that hemocytes are 'plastering' embryonic surfaces with Col IV, it was hypothesized that hemocyte developmental dispersal may be a critical part of the BM deposition process. Hemocytes develop in the anterior of the embryo, and after stage 10 of embryogenesis they disperse within the hemocoel using a combination of external guidance cues and contact inhibition of locomotion, resulting in an evenly tiled cellular distribution. Therefore, how the timing of BM component production correlated with the dispersal of hemocytes was examined. While LanA was expressed during initial stages as hemocytes migrated from their source in the head of the embryo, Col IV production lagged behind by approximately 5 hr. As the induction of Col IV expression occurred largely after hemocyte dispersal, this suggested that hemocyte spreading within the embryo might be a prerequisite for Col IV delivery (Matsubayashi, 2017).

    It was previously proposed that hemocytes were required for BM deposition specifically around the renal tubules during embryogenesis (Bunt, 2010); however, this was only interrogated in mutant embryos that were defective in both hemocyte migration and their survival. To directly examine the role of hemocyte migration in BM component deposition, aberrant hemocyte dispersal was caused by misexpression of Pvf2, a platelet-derived growth factor (PDGF)-like chemotactic cue for hemocytes. Overexpressing Pvf2 during hemocyte dispersal caused hemocytes to aggregate in the embryonic head, which was likely due to a distraction of hemocytes from their normal Pvf source. LanA in wild-type embryos initially spread down the midline of the VNC, and this was unaffected by the inhibition of hemocyte migration. Subsequently, in control embryos, a sheet-like structure containing Laminin extended from the middle of the VNC to lateral positions. These nascent Laminin sheets were stable in time compared to the halos/trails of Laminin following migrating hemocytes, which fluctuated on the order of seconds. Therefore, it was hypothesize that the extension of the Laminin sheets reflects the incorporation and growth of the polymerized matrix from a soluble source of Laminin residing predominantly on the midline. The initiation of Laminin incorporation was unaffected by Pvf2 overexpression. However, in Pvf2-expressing embryos, the Laminin sheets failed to continue extending, leaving large gaps that increased in size by later stages of development. This apparent breakdown of the Laminin matrix was similar to embryos lacking hemocytes. Therefore, Laminin produced by hemocytes may be critical for proper Laminin incorporation or that hemocyte movement, which opens up spaces between tissues, could be aiding the growth of the Laminin matrix by enhancing its diffusion in the hemocoel. In contrast, despite an increase in Col IV upon Pvf2 overexpression, confocal microscopy and lattice light-sheet imaging revealed that there was an uneven coverage of Col IV within the embryo, with most Col IV surrounding hemocytes in the head. A similar local deposition of Col IV around hemocytes was also observed when hemocyte migration was disturbed by the expression of dominant-negative Rac (RacN17) or constitutively active Rac (RacV12). These results further suggest that Laminin deposition requires its diffusion within the embryonic hemocoel while Col IV is locally deposited by hemocytes (Matsubayashi, 2017).

    While these data suggested a highly local mechanism of Col IV delivery by migrating hemocytes, a more complex picture emerged over longer time periods of imaging. At later stages of development, Col IV appeared to spread at a distance from hemocytes and fill the hemocoel. Therefore Col IV was imaged within embryos over a longer period of approximately 12 hr, which represents the time frame just prior to embryonic hatching. Inducing hemocyte aggregation in the anterior of the embryo through overexpression of Pvf2 or RacN17/RacV12 revealed an accumulation of Col IV around hemocytes approximately 6 hr after Col IV induction. However, by 12 hr the fluorescence of Col IV was distributed throughout the embryo despite a continued aggregation of hemocytes. These data suggest that Col IV is eventually capable of spreading within the hemocoel but suffers from very slow effective diffusion (Matsubayashi, 2017).

    Whether hemocyte migration and even BM deposition are functionally important for embryogenesis was subsequently tested. Therefore VNC condensation, a known morphogenetic event that requires hemocytes and BM, was examined. As the BM surrounds the outer surface of the VNC, it is readily accessible to ultrastructural analysis. Fillet preparations of the embryonic VNC were generated, and the developing BM was examined by scanning electron microscopy (SEM). At stage 14 of development, the matrix surrounding the VNC was surprisingly fibrillar in appearance. However, by stage 15 these matrix fibrils were rapidly remodelled into a contiguous sheet containing holes that progressively closed during VNC condensation. Next the distribution of the BM surrounding the VNC was examined after inhibition of hemocyte migration, which severely affected the condensation process and led to a reduced embryonic viability. This revealed that, while the wild-type VNC showed a relatively even distribution of BM, Pvf2 overexpression led to a dense matrix in the head region with a sparse matrix surrounding the VNC in the tail. This highlights that uniform hemocyte dispersal is indeed essential for even incorporation of BM and that the catching up in fluorescence levels upon the inhibition of hemocyte migration is likely the result of diffusing Col IV within the hemocoel rather than proper incorporation (Matsubayashi, 2017).

    Whether the severity of hemocyte migration defects correlated with embryonic lethality was examined. Hemocytes are completely essential for embryogenesis, as killing off hemocytes led to 100% lethality as measured by the frequency of embryonic hatching. Varying degrees of hemocyte migration defects were examined. Expression of a dominant-negative Myosin II specifically in hemocytes led to minor clumping defects but no obvious effects on embryonic lethality. In contrast, Pvf2 overexpression or hemocyte-specific expression of RacN17 led to intermediate migration defects and resulted in approximately 50% embryonic lethality. Finally, hemocyte-specific expression of RacV12, which induced severe migration defects with hemocytes failing to disperse from their origin in the head, led to the most severe embryonic phenotype with 96% lethality. Importantly, these differences in lethality were not correlated with levels of Col IV expression, indicating that the lethality was not related to a change in Col IV levels. These data show that hemocyte migration is indeed essential for embryonic viability (Matsubayashi, 2017).

    Finally, whether a genetic interaction could be observed between hemocyte migration defects and BM mutant alleles was examined. Causing aberrant hemocyte migration in the presence of a heterozygous colIV mutant allele, which led to a 50% reduction in Col IV expression, abolished VNC condensation and induced a synergistic effect on embryonic lethality with 100% of embryos failing to hatch. This lethality was higher than homozygous colIV mutants, showing that the synergy between hemocyte migration and Col IV reduction is not simply the result of a loss of Col IV expression; it also suggests that uneven Col IV deposition may be worse for the embryo than a complete loss of Col IV. In contrast, combining hemocyte migration defects with heterozygous laminin mutants led to a slight increase in lethality, which was similar to homozygous laminin mutant embryos. These data further show that Col IV deposition is more dependent on hemocyte migration than other BM components, such as Laminin (Matsubayashi, 2017).

    This study has shown that during Drosophila embryogenesis, a subset of BM components requires local deposition by migrating hemocytes. This highlights that the ability of hemocytes to evenly spread throughout the embryo, part of a wider mechanism to uniformly deliver ECM. Therefore, as is increasingly realized for vertebrate macrophages, which are also involved in morphogenetic processes that involve matrix remodelling, hemocytes have important non-immune roles critical for development. Interestingly, mammalian macrophages have recently been revealed to produce various ECM components; along with the current data, this suggests that a critical role for macrophage-derived ECM may be more ubiquitous than previously recognized (Matsubayashi, 2017).

    It is unclear why embryonic BM components like Col IV require local delivery by hemocytes, while in larvae they are thought to diffuse from the fat body. This may be related to physiological differences between embryo and larva. In larvae, the heart pumps hemolymph around the animal, which may aid in the spreading of BM proteins. In contrast, the embryonic heart does not begin beating until stage 17, which is after the start of Col IV deposition; in lieu of flowing hemolymph, BM factors with low effective diffusion may therefore require a moving source. Interestingly, recent work has revealed that at least one larval tissue, the developing ovary, requires hemocyte-specific production of Col IV, and it is possible that tissues not in direct contact with hemolymph require other mechanisms of BM deposition. However, it is unclear whether hemocytes associated with the ovary plaster Col IV in a manner similar to embryonic hemocytes or shed soluble Col IV similarly to the larval fat body (Matsubayashi, 2017).

    It is also likely that there are differences between the mechanisms of de novo BM formation in the embryo versus homeostatic mechanisms involved in BM growth in the larva; when Col IV is first deposited in the embryo, its binding sites in the nascent Laminin matrix will be completely unsaturated leading to its rapid capture, thus preventing it from spreading far from its source. As Col IV saturates the BM at later stages of development, this would allow for its subsequent long-distance diffusion in older embryos and larvae. The larva may also have specific mechanisms that aid in Col IV solubility. Indeed, Sparc mutant larvae have abnormal extracellular BM deposits, and recent data from both Drosophila and C. elegans suggest that Sparc is a carrier for components like Col IV. It is interesting to note that there is no embryonic phenotype in Drosophila in the absence of Sparc, suggesting that embryonic Col IV does not need to be solubilized, which is hypothesized to be due to its specific hemocyte-dependent mechanism of delivery during de novo BM formation (Matsubayashi, 2017).

    Basement membrane manipulation in Drosophila wing discs affects Dpp retention but not growth mechanoregulation

    Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. This study manipulated BM composition to cause dramatic changes in tissue tension. Increased tissue compression was found when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor beta ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. These results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment (Ma, 2017).

    Basement membranes (BMs) are laminar polymers of extracellular matrix proteins which underlie epithelia and surround organs in all animals. The main components of BMs are collagen IV, nidogen, laminin, and perlecan, all conserved from insects to humans. Despite long-known conservation, ubiquity in animal tissues, and extensive biochemical knowledge, understanding of the developmental roles of BMs is comparatively poor. Nonetheless, significant progress has been made in recent years with the help of model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, thanks to limited genetic redundancy of BM components in these systems. In this way, it has been shown in the fruit fly Drosophila that collagen IV is required for full Dpp activity in dorsal cells of the embryo and for the response to Dpp of renal tubules. In addition, BMs are now known to play an essential role in mechanically shaping tissues: in the absence of a BM, tissues such as the egg follicleand the larval imaginal discs uffer profound deformations (Ma, 2017).

    Drosophila adult wings develop from the pouch region of the wing imaginal disc, a widely studied model for tissue growth regulation. The wing pouch of the third instar larva (L3 stage) is a highly columnar monolayered epithelium where each cell attaches to the BM. Recently, the hypothesis that mechanical factors contribute to the regulation of wing growth has gathered considerable momentum. The observations that cell compression is higher at the center of the pouch and that compression increases during larval development have led to several models postulating a negative effect of compression on growth. This negative effect of compression on growth is invoked to solve the apparent paradox that combined concentration of growth promoters Dpp and Wingless (Wg) is higher at the center of the pouch, yet the distribution of cell proliferation is roughly homogeneous throughout the disc. In this context, the Hippo signaling pathway, known to respond to cell contact, cell crowding, and cytoskeletal tension has been postulated as a mediator of mechanical inputs into wing growth. However, the difficulty of experimentally changing tissue constriction in an internally developing organ has precluded definitive testing of this hypothesis (Ma, 2017).

    To investigate the developmental role of the BM and explore the influence of mechanical factors on wing growth, this study subjected wing discs to different BM manipulations changing tissue constriction in order to assess their effect on disc development and adult wing size. The results show a lack of effect of mechanical constriction on Hippo signaling and wing growth. In contrast, BM was foudn to contribute to tissue growth by enhancing tissular retention of Dpp (Ma, 2017).

    The results of the experiments changing tissue constriction through BM manipulation are difficult to reconcile with a physiological role of cell compression in regulation of normal wing growth, a central tenet of wing growth mechanoregulation models. Increase in compression when perlecan was knocked down, and decreased compression when the BM was degraded, both failed to produce the predicted effects: smaller and larger wings, respectively. In contrast to the results in the larval wing, tissue size regulation by cell crowding and apoptosis has been shown to occur in the notum during metamorphosis. Since both the wing and the notum derive from the same imaginal disc, it follows that mechanical effects on size must be highly dependent on the specific developmental context (Ma, 2017).

    The failure to observe changes in Hippo activity after dramatic changes in tissue shape also challenges the role of Hippo signaling in regulating wing growth in response to compression. Nonetheless, several manipulations of cytoskeletal components clearly influence Hippo signaling in the wing, affecting growth. Because the actin-rich zonula adherens is the physical locus where Hippo signaling complexes assemble, Hippo signaling may act as a critical sensor of cell polarity or cell contact. According to the current results, however, it does not act in the wing as a tension-growth feedback regulator slowing growth in response to cell crowding (Ma, 2017).

    Discs made of larger, fewer cells have long been known to give rise to normally sized adult wings, indicating that some parameter different from cell numbers contributes to defining final wing size, for instance some physical dimension of the tissue such as planar area or tissue volume. BM manipulations dramatically changed apical area and height of individual cells and of the tissue as a whole, but they may not have changed cell size, as suggested by the fact that cell density in the adult wing did not change. These findings, therefore, would be consistent with a model in which tissue mass or volume contributes to determination of final wing size. Normally sized discs and adult wings made of larger, fewer cells, in addition, offer a further argument against mechanical regulation of wing growth, as these larger cells would display very different physical properties in terms of their apical areas and the tensions supported by their membranes and cytoskeletons (Ma, 2017).

    Even though no mechanical effects on Hippo signaling or wing growth were detected following profound tissue deformations, it cannot be completely rule out that BM manipulations caused secondary effects that negated putative effects of mechanical signals. Such is the case, it is arguee, of the discs flattened by BM elimination. These discs gave rise to smaller adult wings, an effect that further experiments indicate is a result of the specific requirement of the BM in Dpp signaling. Nonetheless, this study also failed to detect changes in cell proliferation or adult wing size when discs were flattened in vivo through direct application of force. Importantly, a contribution of the directionality of compression is also a possibility that cannot be rule out, as cells in the periphery of both act > troli and rn > Mmp2 discs change their apical discs change their apical area, but maintain the tendency of the wild-type to align their major axis tangentially to the center of the disc. Therefore, if the vector of the compression rather than its magnitude is readable by a cell or its neighbors, the results cannot rule out a role for this in regulating wing growth. This pattern of cell orientation has been attributed to a slightly higher proliferation rate in the center of the wing pouch, a fact overlooked in the past and possibly responsible in the first place for the higher cell compression in the center of the wing. BM modifications, therefore, would not affect this intrinsically different proliferation rate in the central and peripheral wing regions. The results, finally, do not rule out the possibility that more extreme mechanical inputs could impact wing growth, for instance in wound healing or damage-stimulated growth (Ma, 2017).

    Despite the lack of influence on Hippo signaling in the BM manipulations, the data show that the BM itself is required to preserve a growth-promoting environment by hindering diffusion of Dpp out of the disc. Collagen IV, the main component of BMs, physically interacts with Dpp through the C-terminal NC1 domains of both collagen IV chains. The effects of collagen IV loss on Dpp signaling in the wing, the dorsal blastoderm and germarium, and renal tubules are all consistent with a role of collagen IV in Dpp concentration. Elimination of the BM, however, did not seem to affect signaling by the other diffusible ligands Wg and Hh, which are, unlike Dpp, quite hydrophobic and may not require a mechanism preventing their escape from the tissue. The role of the BM in maintaining the concentration of extracellular ligands, therefore, may not be general, but ligand specific or specific to Dpp (Ma, 2017).

    A role has been attributed to Dpp signaling in modulating cell height in the wing epithelium. Even though the current experiments eliminating the BM caused both a Dpp deficit and decreased cell height, it is unlikely that the effects on cell height in this experiment are caused by the Dpp deficit. First, the effects of collagenase treatment on disc morphology are immediate, which is difficult to explain as a deficit in Dpp signaling, specially a transcriptionally mediated effect. Second, discs in which the BM was simultaneously degraded and Dpp signaling was activated were still flattened, supporting the idea that effects on tissue shape elicited by BM degradation are not due to a Dpp deficit (Ma, 2017).

    Since Dpp does not seem to accumulate basally in the wing disc, it is hypothesized that transient binding of Dpp allows the wing BM to act as a semipermeable barrier hindering Dpp diffusion, although not completely preventing it. This is a function that other BMs are long known to serve in the vertebrate kidney or the blood-brain barrier. Indeed, the results showing homogeneously high levels of Dpp signaling in the disc when Dpp was expressed in the fat body demonstrate an ability of Dpp to cross the BM. This result has also implications for understanding of Dpp signaling in the wing, as it shows that Dpp presentation by apical cytonemes is not absolutely required for signaling. A function of the BM in limiting basal escape of Dpp is, in addition, highly consistent with recent findings showing that a Dpp.GFP fusion could be immobilized at the BM, with effects on patterning and growth similar to the ones observed when the BM was eliminated. The findings support a critical role for basolaterally diffusing Dpp against a competing hypothesis stating that the functional Dpp gradient forms apically. It must be noted, however, that the role of the medial Dpp stripe in regulating growth has been called into question during the third larval instar, when a non-stripe source in the anterior compartment would serve this growth-promoting function instead. Because BM elimination reduces not just medial spalt and pMad, but also growth, it follows that the BM is required to maintain the concentration of Dpp from both sources: the medial stripe and the unknown anterior non-stripe source (Ma, 2017).

    Given the conservation of BM components and Dpp, BM degradation and epithelial-to-mesenchymal transitions may enhance BMP/TGF-β signaling across tissue layers in development. The results also suggest a way in which tumoral BM degradation could contribute to tissue signaling misregulation in cancer by allowing escape of these diffusible signals. Finally, the visualization of an apico-basal gradient of Dpp in this highly columnar epithelium calls for the inclusion of the apico-basal dimension in future quantitative studies of Dpp gradient formation (Ma, 2017).

    Variations in basement membrane mechanics are linked to epithelial morphogenesis

    The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, an atomic force microscopy-based method was developed to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. These data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. This study proves that BM softens around the squamous cells and that this softening depends on the TGFbeta pathway (the ligands Gbb and Dpp signalling to follicle cells). It was also demonstrated that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes (Chlasta, 2017).

    Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan

    Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation. The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals. Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues. Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless. Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. This study used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs). This study reports that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, this study provides evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche (Diaz-Torres, 2021).

    Extracellular chloride signals collagen IV network assembly during basement membrane formation

    Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. This study reports that extracellular Cl(-) ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl(-) in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins. Phylogenetic evidence indicates this conformational switch is a fundamental mechanism of collagen IV network assembly throughout Metazoa. Using recombinant triple helical protomers, this study proves that NC1 domains direct both protomer and network assembly and shows in Drosophila that NC1 architecture is critical for incorporation into basement membranes. These discoveries provide an atomic-level understanding of the dynamic interactions between extracellular Cl(-) and collagen IV assembly outside cells, a critical step in the assembly and organization of basement membranes that enable tissue architecture and function. Moreover, this provides a mechanistic framework for understanding the molecular pathobiology of NC1 domains (Cummings, 2016).

    The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland

    The heparin sulfate proteoglycan Terribly Reduced Optic Lobes (Trol) is the Drosophila melanogaster homolog of the vertebrate protein Perlecan. Trol is expressed as part of the extracellular matrix (ECM) found in the hematopoietic organ, called the lymph gland. In the normal lymph gland, the ECM forms thin basement membranes around individual or small groups of blood progenitors. The pattern of basement membranes, reported by Trol expression, is spatio-temporally correlated to hematopoiesis. The central, medullary zone which contain undifferentiated hematopoietic progenitors has many, closely spaced membranes. Fewer basement membranes are present in the outer, cortical zone, where differentiation of blood cells takes place. Loss of trol causes a dramatic change of the ECM into a three-dimensional, spongy mass that fills wide spaces scattered throughout the lymph gland. At the same time proliferation is reduced, leading to a significantly smaller lymph gland. Interestingly, differentiation of blood progenitors in trol mutants is precocious, resulting in the break-down of the usual zonation of the lymph gland. which normally consists of an immature center (medullary zone) where cells remain undifferentiated, and an outer cortical zone, where differentiation sets in. Evidence is presented that the effect of Trol on blood cell differentiation is mediated by Hedgehog (Hh) signaling, which is known to be required to maintain an immature medullary zone. Overexpression of hh in the background of a trol mutation is able to rescue the premature differentiation phenotype. These data provide novel insight into the role of the ECM component Perlecan during Drosophila hematopoiesis (Grigorian, 2013).

    Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate

    The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. This study identifies phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. It is proposed that PIP2, through the control of Crag's subcellular localization, restricts the secretion of BM proteins to the basal side (Devergne, 2014).

    Organ sculpting by patterned extracellular matrix stiffness

    How organ-shaping mechanical imbalances are generated is a central question of morphogenesis, with existing paradigms focusing on asymmetric force generation within cells. This study shows that organs can be sculpted instead by patterning anisotropic resistance within their extracellular matrix (ECM). Using direct biophysical measurements of elongating Drosophila egg chambers, this study documents robust mechanical anisotropy in the ECM-based basement membrane (BM) but not the underlying epithelium. Atomic force microscopy (AFM) on wild-type BM in vivo reveals an A-P symmetric stiffness gradient, which fails to develop in elongation-defective mutants. Genetic manipulation of ECM components Collagen IV, Laminin, and Perlecan showed that the BM is instructive for tissue elongation and the determinant is relative rather than absolute stiffness, creating differential resistance to isotropic tissue expansion. The stiffness gradient requires morphogen-like signaling to regulate BM incorporation, as well as planar-polarized organization to homogenize it circumferentially. These results demonstrate how fine mechanical patterning in the ECM can guide cells to shape an organ (Crest, 2017).

    Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles

    During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually dissociates into individual cells. Via a fat body-specific RNAi screen this study found that two matrix metalloproteinases (MMPs), Mmp1 and Mmp2, are both required for fat body cell dissociation. As revealed through a series of cellular, biochemical, molecular, and genetic experiments, Mmp1 preferentially cleaves DE-cadherin-mediated cell-cell junctions, while Mmp2 preferentially degrades basement membrane (BM) components and thus destroy cell-BM junctions, resulting in the complete dissociation of the entire fat body tissues into individual cells. Moreover, several genetic interaction experiments demonstrated that the roles of Mmp1 and Mmp2 in this developmental process are cooperative. In conclusion, Mmp1 and Mmp2 induce fat body cell dissociation during Drosophila metamorphosis in a cooperative yet distinct manner, a finding that sheds light on the general mechanisms by which MMPs regulate tissue remodeling in animals (Jia, 2014).

    Distinct functions of the laminin beta LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

    The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos, multiple mutants were obtained for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin beta and collagen IV alpha1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV alpha2 chain is detected during late embryogenesis. It is concluded that assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. The data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the beta6/beta3/beta8 interface of the laminin beta LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases (Hollfelder, 2014).

    Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila

    Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis. The Drosophila egg chamber transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber. This study identified the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation and shows that SPARC down-regulation is necessary for the increase in Col IV levels to occur. SPARC was found to interact with Col IV prior to secretion and it is proposed that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. A decrease was observed in Perlecan levels during elongation, and Perlecan was shown to be a negative regulator of this process. These data provide mechanistic insight into SPARC's conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis (Isabella, 2015).

    SPARC-dependent cardiomyopathy in Drosophila

    The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte-cardiomyocyte system. A Kruppel-Like Factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analysed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally-conditional knock-down of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein SPARC (Secreted Protein Acidic and Rich in Cysteine), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. The data implicate SPARC in the non-cell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans (Hartley, 2016).

    The impact of SPARC on age-related cardiac dysfunction and fibrosis in Drosophila

    Tissue fibrosis, an accumulation of extracellular matrix proteins such as collagen, accompanies cardiac ageing in humans and this is linked to an increased risk of cardiac failure. The mechanisms driving age-related tissue fibrosis and cardiac dysfunction are unclear, yet clinically important. Drosophila is amenable to the study of cardiac ageing as well as collagen deposition; however it is unclear whether collagen accumulates in the ageing Drosophila heart. This work examined collagen deposition and cardiac function in ageing Drosophila, in the context of reduced expression of collagen-interacting protein SPARC (Secreted Protein Acidic and Rich in Cysteine) an evolutionarily conserved protein linked with fibrosis. Heart function was measured using high frame rate videomicroscopy. Collagen deposition was monitored using a fluorescently-tagged collagen IV reporter (encoded by the Viking gene) and staining of the cardiac collagen, Pericardin. The Drosophila heart accumulated collagen IV and Pericardin as flies aged. Associated with this was a decline in cardiac function. SPARC heterozygous flies lived longer than controls and showed little to no age-related cardiac dysfunction. As flies of both genotypes aged, cardiac levels of collagen IV (Viking) and Pericardin increased similarly. Over-expression of SPARC caused cardiomyopathy and increased Pericardin deposition. The findings demonstrate that, like humans, the Drosophila heart develops a fibrosis-like phenotype as it ages. Although having no gross impact on collagen accumulation, reduced SPARC expression extended Drosophila lifespan and cardiac health span. It is proposed that cardiac fibrosis in humans may develop due to the activation of conserved mechanisms and that SPARC may mediate cardiac ageing by mechanisms more subtle than gross accumulation of collagen (Vaughan, 2017).

    Inter-adipocyte adhesion and signaling by Collagen IV intercellular concentrations in Drosophila

    Sheet-forming Collagen IV is the main component of basement membranes, which are planar polymers of extracellular matrix underlying epithelia and surrounding organs in all animals. Adipocytes in both insects and mammals are mesodermal in origin and often classified as mesenchymal. However, they form true tissues where cells remain compactly associated. Neither the mechanisms providing this tissue-level organization nor its functional significance are known. This study shows that discrete Collagen IV intercellular concentrations (CIVICs), distinct from basement membranes and thicker in section, mediate inter-adipocyte adhesion in Drosophila. Loss of these Collagen-IV-containing structures in the larval fat body caused intercellular gaps and disrupted continuity of the adipose tissue layer. Integrin and Syndecan matrix receptors attach adipocytes to CIVICs and direct their formation. Finally, Integrin-mediated adhesion to CIVICs was shown to promote normal adipocyte growth and prevents autophagy through Src-Pi3K-Akt signaling. These results evidence a surprising non-basement membrane role of Collagen IV in non-epithelial tissue morphogenesis while demonstrating adhesion and signaling functions for these structures (Dai, 2017).

    The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland

    The heparin sulfate proteoglycan Terribly Reduced Optic Lobes is the Drosophila melanogaster homolog of the vertebrate protein Perlecan. Trol is expressed as part of the extracellular matrix (ECM) found in the hematopoietic organ, called the lymph gland. In the normal lymph gland, the ECM forms thin basement membranes around individual or small groups of blood progenitors. The pattern of basement membranes, reported by Trol expression, is spatio-temporally correlated to hematopoiesis. The central, medullary zone which contain undifferentiated hematopoietic progenitors has many, closely spaced membranes. Fewer basement membranes are present in the outer, cortical zone, where differentiation of blood cells takes place. Loss of trol causes a dramatic change of the ECM into a three-dimensional, spongy mass that fills wide spaces scattered throughout the lymph gland. At the same time proliferation is reduced, leading to a significantly smaller lymph gland. Interestingly, differentiation of blood progenitors in trol mutants is precocious, resulting in the break-down of the usual zonation of the lymph gland. which normally consists of an immature center (medullary zone) where cells remain undifferentiated, and an outer cortical zone, where differentiation sets in. Evidence is presented that the effect of Trol on blood cell differentiation is mediated by Hedgehog (Hh) signaling, which is known to be required to maintain an immature medullary zone. Overexpression of hh in the background of a trol mutation is able to rescue the premature differentiation phenotype. These data provide novel insight into the role of the ECM component Perlecan during Drosophila hematopoiesis (Grigorian, 2013).

    Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila

    Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).

    Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

    Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, this study observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, attempts were made to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan (Sessions, 2017).

    Matrix metalloproteinases regulate ECM accumulation but not larval heart growth in Drosophila melanogaster

    If major cardiac extracellular matrix (ECM) components are secreted remotely, how is ECM "self assembly" regulated? This study explored whether ECM proteases were required to maintain the morphology of a growing heart while the cardiac ECM expanded. An increase in expression of Drosophila's single tissue inhibitor of metalloproteinase (TIMP), or reduced function of metalloproteinase MMP2, resulted in fibrosis and ectopic deposition of two ECM Collagens; type-IV and fibrillar Pericardin. Significant accumulations of Collagen-IV (Viking) developed on the pericardium and in the lumen of the heart. Congenital defects in Pericardin deposition misdirected further assembly in the larva. Reduced metalloproteinase activity during growth also increased Pericardin fibre accumulation in ECM suspending the heart. Although MMP2 expression was required to remodel and position cardiomyocyte cell junctions, reduced MMP function did not impair expansion of the heart. A previous study revealed that MMP2 negatively regulates the size of the luminal cell surface in the embryonic heart. Cardiomyocytes align at the midline, but do not adhere to enclose a heart lumen in MMP2 mutant embryos. Nevertheless, these embryos hatch and produce viable larvae with bifurcated hearts, indicating a secondary pathway to lumen formation between ipsilateral cardiomyocytes. MMP-mediated remodelling of the ECM is required for organogenesis, and to prevent assembly of excess or ectopic ECM protein during growth. MMPs are not essential for normal growth of the Drosophila heart (Hughes, 2020).

    Basement membrane and cell integrity of self-tissues in maintaining Drosophila immunological tolerance

    The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. This study established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. Genes for the two collagen IV subunits (see viking and Collagen type IV) and the four laminin subunits (see Laminin A) were nocked down individually via UAS-RNAi using ubiquitous and tissue-specific GAL4 drivers. The basement membrane was found to endow otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and it was further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, cell integrity, as determined by cell-cell interaction and apicobasal polarity, was found to function as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity (Kim, 2014).

    A cell migration tracking tool supports coupling of tissue rotation to elongation

    Cell migration is indispensable to morphogenesis and homeostasis. Live imaging allows mechanistic insights, but long-term observation can alter normal biology, and tools to track movements in vivo without perturbation are lacking. This study developed a tool called M-TRAIL (matrix-labeling technique for real-time and inferred location), which reveals migration histories in fixed tissues. Using clones that overexpress GFP-tagged extracellular matrix (ECM) components, motility trajectories are mapped based on durable traces deposited onto basement membrane. M-TRAIL was applied to Drosophila follicle rotation, comparing in vivo and ex vivo migratory dynamics. The rate, trajectory, and cessation of rotation in wild-type (WT) follicles measured in vivo and ex vivo were identical, as was rotation failure in fat2 mutants. However, follicles carrying intracellularly truncated Fat2, previously reported to lack rotation ex vivo, in fact rotate in vivo at a reduced speed, thus revalidating the hypothesis that rotation is required for tissue elongation. The M-TRAIL approach could be applied to track and quantitate in vivo cell motility in other tissues and organisms (Chen, 2017).

    Stratum, a homolog of the human GEF Mss4, partnered with Rab8, controls the basal restriction of basement membrane proteins in epithelial cells

    The basement membrane (BM), a sheet of extracellular matrix lining the basal side of epithelia, is essential for epithelial cell function and integrity, yet the mechanisms that control the basal restriction of BM proteins are poorly understood. In epithelial cells, a specialized pathway is dedicated to restrict the deposition of BM proteins basally. This study reports the identification of a factor in this pathway, a homolog of the mammalian guanine nucleotide exchange factor (GEF) Mss4, which is named Stratum (CG7787). The loss of Stratum leads to the missecretion of BM proteins at the apical side of the cells, forming aberrant layers in close contact with the plasma membrane. This study found that Rab8 GTPase acts downstream of Stratum in this process. Altogether, these results uncover the importance of this GEF/Rab complex in specifically coordinating the basal restriction of BM proteins, a critical process for the establishment and maintenance of epithelial cell polarity (Devergne, 2017).

    One of the common characteristics of epithelial tissues is the presence of a specialized sheet of extracellular matrix (ECM) at their basal side, called the basement membrane (BM). BMs are cell-adherent extracellular scaffolds composed of proteins such as type IV Collagen (Coll IV), laminins, and heparan sulfate proteoglycans such as Perlecan (Pcan). BMs interact with the basal side of epithelial cells via cellular receptors such as Integrin and Dystroglycan. In addition to providing tissue support, BMs are essential for embryonic and organ morphogenesis and adult functions. The BM has been shown to act as a signaling platform for the regulation of epithelial polarity. The BM can direct the orientation of the apico-basal axis of epithelial cells, resulting in the formation of a basal domain on the side contacting the BM and an apical domain on the opposite side. The loss of integrity and misregulation of the BM have been associated with tumor metastasis. Despite the significance of the BM in both normal and abnormal epithelial cells, the molecular mechanisms ensuring accurate basal secretion of BM proteins remain largely elusive (Devergne, 2017).

    Epithelial cells exhibit a pronounced apico-basal polarity. Polarized intracellular trafficking is a critical process required to establish and maintain epithelial cell polarity by delivering newly synthesized and recycled proteins to their correct destinations. In polarized epithelial cells, a pathway is specifically dedicated to the basal restriction of BM components. It is composed of the guanine nucleotide exchange factor (GEF) Crag (Calmodulin-binding protein related to a Rab3 guanosine diphosphate [GDP]/guanosine triphosphate [GTP] exchange protein) and its guanosine triphosphatase (GTPase) Rab10, as well as the phosphoinositide phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and the protease-like protein Scarface (Devergne, 2017 and references therein).

    To study the mechanisms leading to the basal restriction of BM proteins in polarized epithelial cells, the highly polarized follicular epithelium (FE) of the Drosophila melanogaster ovary was used as a model system. The FE consists of a monolayer epithelium composed of highly polarized cells, called follicle cells (FCs), which surrounds the germline cells. As is typical of epithelial cells, FCs contain different membrane domains: an apical domain facing the germline, a basolateral domain, and junctional domains. Components of the BM, such as Pcan and Coll IV, are actively secreted basally by FCs during egg chamber maturation, thus establishing the FE as an excellent model for the basal restriction of BM proteins in epithelial cells (Devergne, 2017).

    Using this model system, a GEF/RabGTPase complex has been identified, composed of the GEF Stratum (Strat) and the Rab8GTPase, which controls the basal restriction of BM proteins in polarized epithelial cells. The loss of one of these partners leads to the apical mislocalization of BM components. Although Rab8GTPase has a diffuse cytoplasmic localization in the FE, Strat is basally enriched; this suggests that Strat restricts Rab8GTPase activation basally, leading to basal secretion of BM proteins. In addition, this study shows that other factors involved in polarized BM deposition, including PI(4,5)P2 and Crag, control intracellular levels of Strat (Devergne, 2017).

    The GEF Crag and its RabGTPase partner Rab10 play critical roles in directing the basal secretion of BM proteins in polarized epithelial cells (Denef, 2008, Lerner, 2013). To identify factors that control the polarized intracellular trafficking and secretion of BM proteins, a Drosophila protein interaction map (DPiM) was used to find Rab10 interacting partners. One strong interactor was a putative GEF encoded by the gene CG7787. Because GEFs are critical for the control of intracellular trafficking, its role in BM secretion was investigated (Devergne, 2017).

    To test the involvement of CG7787 in BM polarity, the expression of CG7787 in FCs was knocked down by RNAi. The distribution of BM proteins was monitored using the GFP-protein trap lines Pcan-GFP and Coll IV-GFP that reflect the endogenous localization of these proteins. CG7787-depleted epithelial cells present an accumulation of Pcan and Coll IV on both their basal and their apical surfaces, indicating that CG7787 is required for polarized BM deposition. Because BM proteins accumulate in an apical sheet in CG7787-depleted cells, the gene was named stratum (strat) (Devergne, 2017).

    The gene strat encodes a GEF, based on predicted conserved protein domains, that belongs to the MSS4 family of proteins. In particular, mammalian Mss4 (mammalian suppressor of yeast Sec4), also called RABIF (Rab interacting factor), has been shown to interact with Rabs belonging to the same subfamily, including Rab1, Rab3, Rab8, and Rab10, all of which are involved in secretion. To assess whether Strat is the Drosophila homolog of mammalian Mss4/RabIF, human Mss4/RabIF (hMss4)was expressed in strat-knocked down FCs. hMss4 expression partially rescues the defects associated with the loss of Strat, indicating that Strat is the functional homolog of human Mss4/RabIF (Devergne, 2017).

    To confirm the phenotype observed in strat RNAi-expressing FCs, four strat mutant lines were generated by ethyl methanesulfonate (EMS) mutagenesis. Homozygous mutant FC clones were generated using the flippase/flippase recognition target (Flp/FRT) system. In strat mutant FCs, Pcan (Trol) accumulates apically. Expression of a full-length strat transgene rescued this mislocalization phenotype, indicating that strat mutant alleles were generated. Overall, these data confirm Stratum as an essential factor for the basal restriction of BM proteins in epithelial cells. Moreover, quantification of the BM mislocalization phenotype observed in strat mutant clones indicates that the apical accumulation of BM components progressively increases during egg chamber maturation (Devergne, 2017).

    However, Strat does not globally control the apico-basal polarity of FCs. The polarized distribution of other classes of proteins that undergo polarized intracellular trafficking localize normally, indicating that Strat is a member of a pathway specifically dedicated to the polarized sorting of BM proteins in epithelial cells (Devergne, 2017).

    A better understanding of this biological pathway requires a careful analysis of its different members. To do so, it as decided to better characterize the apical mislocalization of BM components observed in strat mutant FCs. Super-resolution three-dimensional structured illumination microscopy (3D-SIM) was used and the plasma membrane marker mCD8-RFP (red fluorescent protein) was expressed exclusively in the FE to observe the distribution of Coll IV-GFP in relation to mCD8-RFP. 3D-SIM has a resolution of 120 nm in xy axes, allowing the distribution of BM proteins to be uprecisely determined with respect to the FE plasma membrane. The spatial distribution and levels of these components were quantified by measuring fluorescence intensity with an optical section through FCs. The basal and apical membranes can be visualized by the two most extreme red mCD8-RFP peaks. As expected in wild-type (WT) FCs, only one peak of Coll IV (Coll IV-GFP, green) was observed on the basal side, and it co-localizes tightly with the basal plasma membrane of the cells; in addition, no apical Coll IV peak was observed (Devergne, 2017).

    In contrast, in strat-knocked down FCs, an additional Coll IV peak was observed at the apical membrane that is also tightly associated with the plasma membrane. Moreover, the Coll IV peak is apical to the apical plasma membrane peak, indicating that Coll IV is also found outside of the apical plasma membrane. Thus, the loss of Strat leads to the apical secretion of BM proteins. These data, confirmed by 3D reconstruction, suggest that the putative GEF Strat is not required for secretion per se but rather for the directionality of secretion. The same observation was made in Crag-knocked down FCs, suggesting that Crag and Strat are both involved in directionality of secretion (Devergne, 2017).

    3D-SIM imaging also revealed that the apical deposition of Coll IV is different from its basal deposition. The pixel distribution shows that the 'sheet' of Coll IV is thicker apically (between 300 and 600 nM) than basally (less than 100 nM, below the resolution of SIM). The apical FC membrane contains microvilli and is therefore topologically thicker than the basal membrane. Moreover, it is unknown whether the mechanisms needed to establish a properly assembled BM are present apically. Altogether, these data suggest that the loss of Strat leads to the apical secretion of Coll IV, which associates with the apical plasma membrane with an aberrant organization (Devergne, 2017).

    Next, attempts were made to identify the RabGTPase or RabGTPases that function with Stratum in polarized BM deposition. Because Rab10 is involved in polarized BM secretion in epithelial cells, Rab10 was tested as a potential Strat interactor (Devergne, 2017).

    To assess whether Rab10 functions downstream of Stratum, a constitutively active form of Rab10 (Rab10CA), was used. Constitutively active forms of RabGTPases remain bound to GTP and thus do not require a GEF for activation. If Strat functions as a GEF for Rab10 in polarized BM deposition, the expression of Rab10CA (yellow fluorescent protein [YFP]-Rab10CA) may rescue the apical mislocalization of BM proteins observed in strat-deficient cells. The expression of Rab10CA in strat-knocked FCs did not rescue the BM mislocalization phenotype. Although a negative result is difficult to interpret, these data might suggest one of the following: (1) Strat is not a GEF for Rab10 during polarized BM deposition, (2) Strat functions as a GEF for another Rab or other Rabs that also control this process, or (3) the expression of Rab10CA is not strong enough to suppress the strat phenotype. Because the expression of YFP-tagged Rab10CA could not be detected in the FE, the latter hypothesis seems unlikely (Devergne, 2017).

    To determine whether Strat interacts with other RabGTPases during BM polarity, Rab8 was examined. In Drosophila, Rab8 and Rab10 are paralogs, sharing an amino acid sequence identity of 67%. In addition, mammalian Mss4/RabIF can act as a GEF for Rab8a. First, the effects of Rab8 on BM proteins were examined. In FCs mutant for Rab8, knocked down for Rab8, or expressing a dominant-negative form of Rab8 (Rab8DN), mislocalization was observed of Pcan or Coll IV, indicating that Rab8 is also involved in polarized BM secretion in epithelial cells. To assess whether Rab8 acts downstream of Strat, a constitutively active (CA) form of Rab8 (YFP-Rab8CA) was expressed in strat knockdown FCs. This resulted in a partial rescue of the phenotype associated with the loss of strat, suggesting that Rab8 acts downstream of Strat in the process of polarized BM deposition. In addition, the expression of wild-type full-length Rab8 did not rescue the phenotype associated with the loss of Strat, suggesting that Strat activates the GTPase activity of Rab8 (Devergne, 2017).

    Finally, to determine whether Strat and Rab8 interact physically, co-immunoprecipitation (coIP) was performed of tagged Rab8 (YFPMYC-Rab8) and Stratum (Strat-HA [C-term HA tagged Stratum]), and they were found to interact in ovary extracts. Altogether, these results suggest that Strat acts as a GEF for Rab8 during the basal restriction of BM deposition. This conclusion is supported by the data that Rab8a interacts with Mss4 in mammalian cells and has weak GEF activity for Rab8a in vitro. It was also shown that Strat phenotype is rescued by hMss4, suggesting that Strat and hMss4 share similar activities. Thus, another GEF/Rab complex, Strat/Rab8, was identified in addition to Crag/Rab10, that is involved in BM deposition in epithelial cells (Devergne, 2017).

    Three non-exclusive mechanisms have been proposed to explain the basal secretion of BM proteins: (1) BM-containing vesicles are directly targeted to the basal side of polarized cells, (2) BM-containing vesicles are blocked apically, and (3) BM proteins are secreted on both sides of epithelial cells but are degraded or endocytosed apically. The intracellular localization of components involved in this process, such as Stratum and Rab8, may provide insight into how these factors restrict BM proteins basally. First, this study assessed the subcellular localization of Rab8 using endogenously tagged YFP-Myc-tagged-Rab8 (YFPMyc-Rab8). In the FE, YFPMyc-Rab8 is detected diffusely throughout the cytoplasm and is non-polarized during early and mid-stages of oogenesis. YFPMyc-Rab8 accumulates in intracellular puncta, which may represent endosomes and/or vesicles. More specifically, Rab8 partially co-localizes with early and recycling endosome and Golgi markers. This subcellular localization is consistent with the known role of Rab8 in regulating vesicular transport from the Golgi to the plasma membrane. YFPMyc-Rab8 becomes slightly enriched at the basal side of the FCs starting in stages 9 to 10. In contrast to Rab8, Strat (Strat-HA) has a diffuse intracellular localization earlier in oogenesis but quickly assumes a pronounced polarized distribution, accumulating basally in FCs. This observation suggests that Strat restricts the activity of Rab8 basally to allow proper basal deposition of BM proteins. Alternatively, because mammalian Mss4 protein has been shown to have only weak GTPase activity compared to other GEFs, Mss4 may act as a chaperone, allowing interacting Rab proteins to be properly activated where and when they are needed in the cell. Therefore, Strat may play one or both of these roles to restrict Rab8 activity basally and thus direct BM protein-containing vesicles toward the basal side of the cell (Devergne, 2017).

    The polarized localization of Strat differs from Crag, which accumulates at apical and lateral membranes, suggesting that Crag blocks the apical secretion of BM proteins. Both Crag and Stratum GEFs have critical roles to restrict BM proteins basally; however, they are structurally, and perhaps functionally, different. Crag is a 187 kDa multidomain protein composed of three differentially expressed in normal and neoplastic cells (DENN) domains with GEF activity, a Calmodulin binding domain, and a conserved C-terminal domain. In contrast, Stratum is 14 kDa and composed of a single Mss4 domain with weak GEF activity. In view of these structural differences, it is unlikely that Crag and Stratum have the same interactors, regulators, and effectors. In addition, the strikingly different localization of these factors suggests independent roles in this process, because Crag is localized to lateral and apical membranes and Stratum is localized to the basal side of cells. Yet altogether, these proteins allow the specific basal restriction of BM components in epithelial cells (Devergne, 2017).

    Recently studies have shown that the proper intracellular distribution of Crag is dependent on the phosphoinositide PI(4,5)P2. A decrease in PI(4,5)P2 levels leads to a loss of Crag apico-basal distribution and the mislocalization of BM proteins. To assess the role of other members of the pathway, such as PI(4,5)P2 and Crag, on Strat localization, the distribution of Strat-HA was determined in Phosphatidylinositol synthase (Pis) and Crag mutant FCs. As was previously observed for Crag, a decrease in PI(4,5)P2 levels in Pis mutant FCs leads to reduced levels of Strat. This phenotype is observed in 49% of mutant clones. The same decrease of Strat can be observed in Crag mutant FCs (in 47% of mutant clones). These data suggest that both PI(4,5)P2 levels and Crag control the levels and distribution of Strat. Because previous work has shown that PI(4,5)P2 controls Crag localization, the decrease of Strat observed in Pis mutant FCs might be due to the loss of Crag. However, the distribution and levels of Crag are not significantly affected in strat mutant FCs. Overall, the loss of Strat observed in the mutant backgrounds highlights the existence of a regulatory mechanism between the two GEF/Rab complexes dedicated to the polarized secretion of BM proteins and should be investigated further (Devergne, 2017).

    In conclusion, this study has identified Strat, the homolog of mammalian GEF Mss4/RabIF, and Rab8GTPase as essential regulators in the basal sorting of BM proteins in polarized epithelial cells. This GEF/Rab complex partners to correctly deliver BM protein-containing vesicles basally, an essential process for epithelial cell function. Previous work identified an apical complex involved in this process containing Crag/Rab10 and depending on PI(4,5)P2. This study has found a more basally localized complex, consisting of Strat and Rab8, also required for the exclusive basal localization of BM proteins. These complexes do not function redundantly but both complexes are required independently. A third complex involving Rab10 and Ehbp1 has been described to deliver BM proteins to the basolateral side of the follicle cells in a late differentiation process involved in egg chamber elongation. These findings reveal that the proper positioning of BM proteins is handled by the cell in more complex regulatory pathways than was previously realized (Devergne, 2017).

    Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation

    How extracellular matrix participates to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. This study found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell autonomous control of F-actin fibers orientation relies on the previous BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a critical organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation (Cerqueira Campos, 2020).

    Deciphering the mechanisms underlying tissue morphogenesis is crucial for fundamental understanding of development and also for regenerative medicine. Building organs generally requires the precise modeling of a basement membrane extracellular matrix (ECM), which in turn can influence tissue shape. However, the mechanisms driving the assembly of a specific basement membrane (BM) and how this BM then feeds forward on morphogenesis are still poorly understood. Drosophila oogenesis offers one of the best tractable examples in which such a morphogenetic process can be studied. Each ovarian follicle, which is composed of a germline cyst surrounded by the somatic follicular epithelium, undergoes a dramatic growth, associated with tissue elongation, starting from a little sphere and ending with an egg in which the anteroposterior (AP) axis is 3-fold longer than the mediolateral (ML) axis. This elongation is roughly linear from the early to the late stages, but can be separated in at least two mechanistically distinct phases. The first phase (from stage 3 to stage 8; hereby 'early stages') requires a double gradient of JAK-STAT pathway activity that emanates from each pole and that controls myosin II-dependent apical pulsations. In the second phase, from stage 7-8, elongation depends on the atypical cadherin Fat2 that is part of a planar cell polarity (PCP) pathway orienting the basal domain of epithelial follicle cells. Earlier during oogenesis, Fat2 gives a chirality to the basal domain cytoskeleton in the germarium, the structure from which new follicles bud. This chirality is required to set up a process of oriented collective cell migration perpendicularly to the elongation axis that induces follicle revolutions from stage 1 to stage 8. From each migrating cell, Fat2 also induces, in the rear adjacent cell, the formation of planar-polarized protrusions that are required for rotation. These rotations allow the polarized deposition of BM fibrils, which involves a Rab10-dependent secretion route targeted to the lateral domain of the cells. These BM fibrils are detectable from stage 4 onwards and persist until late developmental stages. Follicle rotation also participates in the planar cell polarization of integrin-dependent basal stress fibers that are oriented perpendicularly to the AP axis. Moreover, at stage 7-8, a gradient of matrix stiffness controlled by the JAK-STAT pathway and Fat2 contributes to elongation. Then, from stage 9, the epithelial cell basal domain undergoes anisotropic oscillations, as a result of periodic contraction of the oriented stress fibers, which also promotes follicle elongation . To explain the impact of fat2 mutations on tissue elongation, it is generally accepted that oriented stress fibers and BM fibrils act as a molecular corset that constrains follicle growth in the ML axis and promotes its elongation along the AP axis. However, the exact contribution of F-actin versus BM to this corset is still unclear, as is whether the orientations of stress fibers and of BM fibrils are causally linked (Cerqueira Campos, 2020).

    This analyzed the function of Dystrophin (Dys) and Dystroglycan (Dg) during follicle elongation. Dys and Dg are the two main components of the Dystrophin-associated protein complex (DAPC), an evolutionarily conserved transmembrane complex that links the ECM (via Dg) to the F-actin cytoskeleton (via Dys). This complex is expressed in a large variety of tissues and is implicated in many congenital degenerative disorders. Loss-of-function studies in model organisms have revealed an important morphogenetic role for Dg during development, usually linked to defects in ECM secretion, assembly or remodeling . A developmental role for Dys is less clear, possibly because of the existence of several paralogs in vertebrates. As Drosophila has only one Dg and one Dys gene, it is a promising model for their functional study during development and morphogenesis (Cerqueira Campos, 2020).

    Dg and Dys were found to be required for follicle elongation and proper BM fibril formation early in fly oogenesis. During these early stages, DAPC loss and hypomorphic fat2 conditions similarly delay stress fiber orientation. However, DAPC promotes this alignment more locally than Fat2. Moreover, DAPC genetically interacts with fat2 in different tissues, suggesting that they belong to a common morphogenetic network. Later in oogenesis, Dg and Dys are required for stress fiber orientation in a cell-autonomous manner. This is the period when the main elongation defect is seen in these mutants, arguing for a more determinant role for stress fibers compared with BM fibrils in the elongation process. Nonetheless, this latter function depends on the earlier DAPC function in BM fibril deposition. It is proposed that BM fibrils serve as a PCP memory for the late stages that are used as a template by the DAPC for F-actin stress fiber alignment (Cerqueira Campos, 2020).

    Genetic data has already demonstrated that follicle elongation relies on at least two different and successive mechanisms. The first is controlled by JAK-STAT and involves the follicle cell apical domain, whereas the second is controlled by Fat2 and involves the basal domain and the BM. Between these phases, around stage 7-8, JAK-STAT and Fat2 seem to be integrated in a third mechanism based on a BM stiffness gradient. Interestingly, a very recent report suggests that this gradient may not directly influence tissue shape but rather do so by modifying the properties of the follicle cells underneath. This study shows that the DAPC influences elongation mainly at very late stages, suggesting the existence of a fourth mechanistic elongation phase. Of note, elongation at these late stages is also defective in fat2 mutants. This is consistent with the fact that rotation is required for polarized BM fibril deposition, and that this deposition depends on and is required for DAPC function. The existence of multiple and interconnected mechanisms to induce elongation, a process that initially appeared to be very simple, highlights the true complexity of morphogenesis, and the necessity to explore it in simple models (Cerqueira Campos, 2020).

    Fat2 is clearly part of the upstream signal governing the basal planar polarization. However, how this polarization leads to tissue elongation is still debated. It has been proposed that elongation relies on a molecular corset that could be formed, non-exclusively, by BM fibrils or F-actin stress fibers. The initial observation that rotation is required for both elongation and BM fibrils favored a direct mechanical role for these structures. Recent data showing that BM fibrils are stiffer than the surrounding ECM supports this view. Moreover, increasing the BM fibril number and size can lead to hyper-elongation. Finally, addition of collagenase induces follicle rounding, at least at some stages, and genetic manipulation of the ECM protein levels also influences elongation. However, these experiments did not discriminate between the function of the fibril fraction and a general BM effect. Moreover, they do not demonstrate whether their impact on elongation is direct and mechanic or, indirect by a specific response of the epithelial cells. Fat2 and rotation are also required for the proper orientation of the stress fibers. The F-actin molecular corset is dynamic with follicle cells undergoing basal pulsations, and perturbation of both these oscillations and of the stress fiber structure affect elongation. In the DAPC mutants, a faint but significant elongation defect was observed during mid-oogenesis and a stronger one after stage 12. These defects are clearly correlated with the stress fiber orientation defects observed in the same mutants, both temporally and in terms of intensity. Moreover, although overexpression of Rab10 in a Dys loss-of-function mutant restores BM fibrils, it does not rescue elongation, indicating that stress fiber orientation is instrumental (Cerqueira Campos, 2020).

    Thus, if the role of the BM fibrils as a direct mechanical corset appears limited, what is their function? One possibility could have been that they promote rotation, acting by positive feedback and explaining the speed increase over time. However, the rotation reaches the same speed in WT and DAPC mutants, excluding this possibility. Similarly, increasing the fibril fraction also has no effect on rotation speed (Cerqueira Campos, 2020).

    The results strongly argue that BM fibrils act as a cue for the orientation of stress fibers, which then generate the mechanical strain for elongation. This appears clear in late stages when the function of the DAPC for stress fiber orientation is dependent on the previous BM fibril deposition. Although it is unknown why the cells lose their orientation from stages 10 to 12, the BM fibrils provide the long-term memory of the initial PCP of the tissue, allowing stress fiber reorientation. Such a mechanism appears to be a very efficient way to memorize positional cues, and could represent a general BM function in many developmental processes (Cerqueira Campos, 2020).

    DAPC was found to impact the two key actors at the basal domain of the follicle cells: the BM and the stress fibers linked to the BM. All the defects of Dg null mutants were also observed in Dys mutants, demonstrating a developmental and morphogenetic role for this gene. In vertebrates, at least in some tissues, Dg presence appears to be essential for BM assembly. However, BM formation on the follicular epithelium does not require Dg, suggesting the existence of alternative platforms for its general assembly. The genetic data suggest that Rab10 is epistatic to Dg for BM fibril deposition. The usual interpretation of such a result would be that Dg is involved in the targeting of ECM secretion upstream of Rab10 rather than in ECM assembly in the extracellular space. In Caenorhabditis elegans, Dg acts as a diffusion barrier to define a precise subcellular domain for ECM remodeling. One could imagine that the DAPC has a similar function in follicle cells, by defining the position where the Rab10 secretory route is targeted. However, in DAPC loss of function, some ECM is still secreted between cells, suggesting that the lateral Rab10 route is not affected. Moreover, ECM proteins do not abnormally accumulate between cells in such mutants, suggesting that they are able to leave this localization but without forming BM fibrils. Therefore, the functional interplay between Rab10 and the DAPC is still unclear (Cerqueira Campos, 2020).

    As mentioned before, Dg has often been proposed to act as a scaffold to promote BM assembly in mice. Deletion of the Dg intracellular domain is only sub-lethal in mice, whereas complete loss of this protein is lethal very early during development, indicating that the abolishment of Dg's interaction with Dys affects its function only partially. In these mice, laminin assembly can still be observed, for instance in the brain and retina. Similar results were also obtained in cultured mammary epithelial cells. Thus, despite the existence of Dys paralogs that could mask some effects on ECM and the fact that the same ECM alteration was observed in Dg or Dys mutant fly follicles, not all the Dg functions related to ECM assembly or secretion involve Dys. It is possible that Dys is required when Dg needs a very specific subcellular targeting for its function, whereas a more general role in ECM assembly would be independent of Dys. The results suggest that some specific effects of Dys on ECM could have been underestimated and this could help to explain the impact of its loss of function on tissue integrity maintenance. For instance, as it has been reported that Dg influences ECM organization in fly embryonic muscles, it would be interesting to determine whether this also involves Dys (Cerqueira Campos, 2020).

    The DAPC is involved in planar polarization of the basal stress fibers and its ability to read ECM structure to orchestrate integrin-dependent adhesion could play a role in many developmental and physiological contexts. The link between the ECM and F-actin provided by this complex is likely required for this function, although this remains to be formally demonstrated. BM fibrils could provide local and oriented higher density of binding sites for Dg, and the alignment could then be transmitted to the actin cytoskeleton. Alternatively, DAPC function could rely on sensing the mechanical ECM properties. The hypothesis that the DAPC could act as a mechanosensor is a long-standing proposal, partly due to the presence of spectrin repeats in Dys. The basal domain of the follicle cells may offer an amenable model to combine genetics and cell biology approaches to decipher such function (Cerqueira Campos, 2020).

    Altogether, this work provides important insights on the role of the BM during morphogenesis, by acting as a static PCP cue retaining spatial information while cells are highly dynamic. It also reveals important functions of the DAPC, including Dys, that may be broadly involved during animal development and physiology (Cerqueira Campos, 2020).

    Rab10-mediated secretion synergizes with tissue movement to build a polarized basement membrane architecture for organ morphogenesis

    Basement membranes (BMs) are planar protein networks that support epithelial function. Regulated changes to BM architecture can also contribute to tissue morphogenesis, but how epithelia dynamically remodel their BMs is unknown. In Drosophila, elongation of the initially spherical egg chamber correlates with the generation of a polarized network of fibrils in its surrounding BM. This study used live imaging and genetic manipulations to determine how these fibrils form. BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber's epithelial cells and undergo oriented insertion into the BM by directed epithelial migration. It was found that a Rab10-based secretion pathway promotes pericellular BM protein accumulation and fibril formation. Finally, by manipulating this pathway, it was shown that BM fibrillar structure influences egg chamber morphogenesis. This work highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape (Isabella, 2016).

    Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles

    T antigen (Galβ1-3GalNAcalpha1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcalpha1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. This study has unraveled biological roles of glucuronylated T antigen. The data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). dGlcAT-P null mutants were created, and it was found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. It was also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, a genetic interaction was shown between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles (Itoh, 2018).

    Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly

    Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen in addition to the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg in morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from cultured embryonic tissue experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival strongly questioned this proposed linking role. This study has isolated mutations in the only Ndg gene present in Drosophila. While, similar to C. elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. Alike in mice, tissue-specific requirements of Ndg were found for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, a thorough functional analysis of the different Ndg domains was performed in vivo. These results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of these findings, it is propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen (Dai, 2018).

    Basement membranes (BM) are specialized thin extracellular matrices underlying all epithelia and endothelia, and surrounding many mesenchyme cells. This thin layer structure, which appears early in development, plays key roles in the morphogenesis, function, compartmentalization and maintenance of tissues (Dai, 2018).

    All BMs contain at least one member of the Laminin, Type IV Collagen (Col IV), proteoglycan Agrin and Perlecan, and Nidogen (Entactin) families. Nidogen is a 150-kDa glycoprotein highly conserved in mammals, Drosophila, Caenorhabditis elegans (C. elegans) and ascidians. Nidogens have been proposed to play a key role in BM assembly by providing a link between the Laminin and Col IV networks and by integrating other ECM proteins, such as Perlecan, into this specialized extracellular matrix. While invertebrates possess only one Nidogen, two Nidogen isoforms, Nid1 and Nid2, have been identified in vertebrates. The individual knock out of either Nid1 or Nid2 in mice does not affect BM formation or organ development. In fact, these Nid1 or Nid2 null animals appear healthy, fertile and have a normal life span. However, simultaneous elimination of both isoforms results in perinatal lethality, with defects in the lung, heart and limb development that are not compatible with postnatal survival. In addition, BM defects are only observed in certain organs, which strongly suggests tissue-specific roles for Nidogens in BM assembly and function. Like in mice, loss of the only Nidogen-encoding gene in C. elegans, NID-1, is viable with minor defects in egg laying, neuromuscular junctions and position of longitudinal nerves, but no defects in BM assembly. Altogether, these studies reveal that Nidogen may play important roles in specific contexts, consistent with its evolutionary conservation. However, the different requirements for Nidogens in BM assembly and organogenesis in mice and C. elegans suggest that new functions may have arisen in vertebrates. The isolation of mutants in Nidogen in other organisms will help to shed light on the role of the Nidogen proteins in vivo and its conservation throughout evolution (Dai, 2018).

    All Nidogens comprise three globular domains, namely G1, G2 and G3, one flexible linker connecting G1 and G2, and one rod-shaped segment, composed primarily of epidermal growth factor repeats, separating the G2 and G3 domains. A number of studies using recombinant fragments of Nidogens have provided a wealth of information on the structure and binding properties of the different Nidogen domains in vitro. Thus, key roles have been proposed for the globular domains G3 and G2 in mediating interactions of Nidogen with the Laminin network and with the Collagen IV network, respectively. Despite this, the relevance of these interactions in vivo remains to be established. Furthermore, some of the predictions from cell culture and in vitro experiments do not hold when tested in model organisms. For example, deletion of the G2 domain in C. elegans is viable and does not affect organogenesis. Furthermore, it has been shown that Ndg1 and Ndg2 do not form molecular cross-bridges between the Laminin and Collagen IV networks in the epidermal BM of human skin. These results in animal models are inconsistent with a role for Nidogen as a generally essential linker between the Collagen IV and Laminin networks, leaving open the question of whether in vivo Nidogen functions at all as a linker (Dai, 2018).

    Drosophila BMs are analogous to the vertebrate ones. They cover the basal surface of all epithelia and surround most organs and tissues, including muscles and peripheral nerves. Even though their composition might vary according to tissues and developmental stages, all Drosophila BMs contain Col IV, Laminin, Perlecan and Nidogen. However, in contrast to the three Col IV, sixteen Laminins and two Nidogens found in humans, Drosophila only produces one Col IV, two distinct Laminins and one Nidogen (Ndg). The reduced number of ECM components, which limits the redundancy among them, and their high degree of conservation with their mammalian counterparts, makes Drosophila a perfect model system to dissect their function in vivo. Drosophila Col IV has been identified as a homolog of mammalian Type IV Collagen, which is a long helical heterotrimer that consists of two α1 chains and one α2 chain encoded by the genes Collagen at 25 C (Cg25C) and viking (vkg), respectively. The C terminal globular non-collagenous (NC1) domain and the N terminal 7S domain interact to form the Col IV network. Loss of function mutations in either of the two Col IV genes in flies affect muscle development, nerve cord condensation, germ band retraction and dorsal closure, causing embryonic lethality. In addition, mutations in Col IV have been associated with immune system activation, intestinal dysfunction and shortened lifespan in the Drosophila adult. Finally, while Col IV deposition in wing imaginal discs and embryonic ventral nerve cord (VNC) BMs is not required for localization of Laminins and Nidogens, it is essential for Perlecan incorporation. The Drosophila Laminin αβγ trimer family consists of two members comprised of two different α subunits encoded by Laminin A and wing blister, one β and one γ subunits encoded by Laminin B1 and Laminin B2, respectively. Same as Col IV, Laminin trimers can also self-assemble into a scaffold through interactions of the N-terminal LN domains located in their short arms. Elimination of Laminins in Drosophila affects the normal morphogenesis of most organs and tissues, including the gut, muscles, tracheae and nervous system. In addition, abnormal accumulation of Col IV and Perlecan was observed in Laminin mutant tissues. Perlecan, encoded by the trol (terribly reduced optic lobes) gene, is subdivided into five distinct domains. Interactions with Laminins and Col IV occur through domains I and V. Mutations in trol affect postembryonic proliferation of the central nervous system, plasmatocytes and blood progenitors. Loss of trol also affects the ultrastructure and deposition of Laminins and Col IV in the ECM around the lymph gland. Altogether, these results suggest that BM components Laminin, Col IV and Perlecan are all essential for proper development. In addition, they also reveal a hierarchy for their incorporation into BMs that seems to be tissue-specific and required for proper BM assembly and function. In this context, however, the role of Ndg in Drosophila morphogenesis and BM assembly has remained elusive. This may be in part due to the lack of mutations in this gene (Dai, 2018).

    This work describes the role of Ndg in Drosophila. Using a newly generated anti-Ndg antibody, Ndg was shown to accumulate in the BMs of embryonic, larval and adult tissues. By isolating several mutations in the single Drosophila Ndg gene, it was found that while, similar to C. elegans and mice, Ndg is not required for overall organogenesis or viability, it is required for fertility. Also similar to the tissue-specific defects in mice and C. elegans, the BMs surrounding the larval fat body and flight muscles of the notum were found to be disrupted in the absence of Ndg. Furthermore, uncoupling of laminin and Collagen IV was observed in the fat body of Ndg mutants, indeed supporting a role of Ndg as a linker between the two networks. In addition, a thorough functional analysis of the different Ndg domains was performed in vivo, supporting an essential requirement of the G3 domain for Ndg function and, on the other hand, uncovering a new key role for the Rod domain in regulating Ndg incorporation into BMs. Finally, this study found that BM assembly is not universal but differs depending on the tissue and propose that this could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen (Dai, 2018).

    BMs are thin extracellular matrices that play crucial roles in the development, function and maintenance of many organs and tissues. Critical for the assembly and function of BMs is the interaction between their major components, Col IV, Laminins, proteoglycans and Ndg. Both the ability of Ndg to bind laminin and Col IV networks and the crucial requirements for Laminins and Col IV in embryonic development anticipated a key role for Ndg during morphogenesis. However, experiments showing that elimination of Ndg in mice and C. elegans are compatible with survival casted doubt upon the crucial role for Ndg in organogenesis as a linker of the crucial Laminin and Col IV networks within the BM. This study has isolated mutations in the single Drosophila Ndg gene and found that, as it is the case in mammals and C. elegans, Ndg is not generally required for BM assembly and viability. However, Ndg mutant flies display mild motor or behavioral defects. In addition, similar to mammals, this study shows that the Nidogen-deficient flies show BM defects only in certain organs, suggesting tissue-specific roles for Ndg in BM assembly and maintenance. Finally, functional study of the different Ndg domains challenges the significance of some interactions derived from in vitro experiments while confirming others and additionally revealing a new key requirement for the Rod domain in Ndg function and incorporation into BMs (Dai, 2018).

    Results from cell culture and in vitro experiments led to the proposal of a crucial role for Ndg in BM assembly and stabilization. Recombinant Ndg promotes the formation of ternary complexes among BM components. In addition, incubation with recombinant Ndg or antibodies interfering with the ability of Ndg to bind Laminins results in defects in BM formation and epithelial morphogenesis in cultured embryonic lung, submandibular glands and kidney. However, elimination of Ndg in model organisms has shown that Ndg is not essential for BM formation per se but required for its maintenance in some tissues. Thus, while the early development of heart, lung and kidney prior to E14 is not affected in Nidogen-deficient mice, defects in deposition of ECM components and BM morphology were observed at E18.5. Similarly, whereas BM components localized normally in Nidogen-deficient mice during the early stages of limb bud development, this BM breaks down at later stages. In contrast, removal of Ndg does not impair assembly or maintenance of any BM in C. elegans. This study shows that in Drosophila, as it is the case in mammals, different BMs have different requirements for Ndg. Thus, while elimination of Ndg in Drosophila does not impair embryonic BM assembly or maintenance, it results in discontinuity of the BM in fat body and flight muscles. The basis for this tissue-specificity of Ndg requirements is currently unknown. Recent experiments have shown that there is a tissue-specific hierarchy of expression and incorporation of BM proteins in the Drosophila embryo, with Laminins being expressed first followed by Col IV and finally Perlecan. Laminins and Col IV can reconstitute polymers in vitro that resemble the networks seen in vivo. In this context, Laminins and Col IV could self-assemble into networks in the embryo as they are produced, being this sufficient to assemble a BM capable of sustaining embryonic development in the absence of the two subsequent components, Ndg and Perlecan. This study also shows that, while fat body and blood cells are the source of the majority of the proteins in larval BMs, there are notable exceptions, a fact that highlights a diversity in the origins of BM components in different tissues. Thus, fat body produces entirely all its BM, the larval heart receives it all from the hemolymph, imaginal discs produce a portion of their Laminins and similarly for tracheae with respect to Perlecan. These differences in the source of BM components for different tissues (incorporated vs. self-produced) may impose different assembly mechanisms, a possibility to study in more detail in the near future. In addition, although BM components are universally present in numerous tissues and organs, they are diverse depending on tissue and developmental stage. This heterogeneity arises from variations in protein subtypes, such as the two alternative Laminin α chains or the numerous Perlecan isoforms. Heterogeneity may also stem from differences in relative amounts of each component and posttranslational modifications thereof. In this respect, it is possible that BM assembly of the Drosophila fat body and adult flight muscles of the notum is such that is more dependent on Ndg function for its formation and stability than BMs found in other tissues. Finally, dynamics of BMs can orchestrate organ shape changes. Reciprocally, the associated tissues can control properties of BMs by, for instance, expressing a specific repertoire of ECM receptors or remodeling factors. In this context, it is also possible that fat body or adult flight muscles sculpt BMs with properties demanding a high requirement of Ndg function (Dai, 2018).

    This study finds that Ndg mutant flies are less fertile and behave differently with respect to wild type in ChillComa Recovery Time assays. The physiological mechanisms underlying the response in insects to critical thermal limits remain largely unresolved. The onset and recovery of chill coma have been attributed to defects in neuromuscular function due to depolarization of muscle fiber membrane potential. Interestingly, flight muscle fiber membrane is strongly depolarized upon exposure to low temperatures in Drosophila. In this context, the defects observed in the BM of adult flight muscles in the absence of Ndg could be behind the defective response of Ndg mutant flies to chill coma recovery assays. Altogether, these results show that, though not critical for survival, Ndg is required for overall fitness of the fly (Dai, 2018).

    All Nidogen proteins consist of three globular domains (G1 to G3) and two connecting segments; one Rod domain separating G2 and G3, and a flexible linker between G1 and G2. Crystallographic and binding epitope analyses using recombinant domains of the mouse Nidogen-1 protein have demonstrated high affinity binding of domain G2 to Col IV and Perlecan, of domain G3 to the Laminin γ1 chain and Col IV, and no activity for the Rod domain. In addition, recent physicochemical studies analyzing the solution behavior of full length purified Nidogen-1 confirmed the formation of a high affinity complex between the G3 domain of Nidogen-1 and the Laminin γ1 chain, and excluded cooperativity effects engaging neighboring domains of both proteins . However, little is known about the functional meaning of the binding abilities of Ndg on its localization and function in BM assembly in vivo. In fact, mutant C. elegans animals carrying a deletion removing the entire G2 domain of NID-1 are viable and show no defects on Ndg or Col IV localization in BMs. These results demonstrate that, despite the strong sequence conservation between C. elegans and mammalian G2 domains, C. elegans NID-1 localization appears to occur independently of this domain. This study shows that, as it is the case in C. elegans, the Drosophila G2 domain is not essential for neither Ndg localization nor function. A possible explanation for this result is that although some of the modules present in BM components are conserved, there might be variations in sequence and structure that might be sufficient to confer binding specificity to the different proteins. For instance, the IG3 domain of mouse Perlecan, which binds to a β-barrel in the G2 domain of Nidogen, is strikingly conserved in all mammals, but not in Drosophila or C. elegans. This result suggests that either the Perlecans present in these organisms are too distant in evolution from the mouse proteins for these domains to be conserved or that Perlecans may only bind Nidogen in mammals. Previous studies aimed to characterize the biological significance of the Nidogen-Laminin interactions have targeted the Nidogen-binding module of the Laminin γ1 chain, showing that this domain is required for kidney and lung organogenesis. However, the role of the Nidogen G3 domain has not yet been addressed directly. This study show sthat the G3 domain is essential for Ndg localization, supporting a role for Nidogen-Laminin interactions on Ndg function. In addition, in contrast to what has been shown in mammals, the current results unravel a key role for the Rod domain in Nidogen localization. Again, an explanation for this result could hinge on variations in Nidogen between species. In fact, one of the major differences between Drosophila and mammalian Nidogen lies on the Rod domain. Thus, while vertebrates have four EGF repeats and one or two thyroglobulin repeats, Drosophila and C. elegans have 12 and 11 EGF repeats, respectively. Alternatively, conclusions derived from in vitro studies may not be always applicable to the circumstances occurring in the living organism. Furthermore, the appearance of new in vitro studies combining different techniques has revealed the existence of multiple Nidogen-1/Laminin γ1 interfaces, which include, besides the known interaction sites, the Rod domain (Dai, 2018).

    Different BM assembly models have been proposed over the last thirty years. Based upon biochemical studies and rotary shadow electronic microscopic visualization, the BM assembly model firstly proposed that Collagen IV self-assembles into an initial scaffold, followed by Laminin polymerization structure attachment mediated by Perlecan. However, more recent studies have postulated a contradicting model for in vivo systems. The most widely endorsed model states that the polymer structure is initiated by a Laminin scaffold built through self-interaction, bridged by Nidogen and Perlecan and finally completed by another independent network formed by Col IV self-interaction. This study examined in detail the hierarchy of BM assembly in the Drosophila larval fat body. Thus, while the requirements for Drosophila Laminins in the incorporation of other ECM components into BMs are preserved between tissues, this is not the case for Collagen IV. For instance, absence of Col IV does not completely prevent deposition of Laminin in the fat body, but remarkably reduces it; in contrast, no such drastic effect has been observed in wing discs or embryonic BMs, suggesting that Collagen IV does not affect Laminin incorporation in these other tissues to the same degree or that it does not affect it at all. In addition, this study found that BM assembly in Drosophila also differs from that in mammals and C. elegans. In this case, the divergences may arise during evolution, when different organisms might have incorporated novel ways to assemble ECM proteins to serve new specialized functions (Dai, 2018).

    Nidogen has been proposed to play a key role in BM assembly based on results from in vitro experiments and on its ability to serve as a bridge between the two most abundant molecules in BMs: Laminin and Type IV Collagen. However, phenotypic analysis of its knock out in mice and C. elegans have called into question a general role for Nidogen in BM formation and maintenance. This study shows that although Ndg is dispensable for BM assembly and preservation in many tissues, it is absolutely required in others. These differences on Ndg requirements stress the need to analyze its function in vivo and in a tissue-specific context. In fact, it is believed that this should also be the case when analyzing the requirements of the other ECM components for proper BM assembly, as this study shows they also differ between species and tissues. One has to be cautious when inferring functions of different BM proteins or their domains based on experiments performed in vitro or in a tissue-specific setting. This might be especially relevant when trying to apply conclusions derived from these studies to understanding of the pathogenic mechanisms of BM-associated diseases or to the development of innovative therapeutic approaches (Dai, 2018).

    Integrins Modulate Extracellular Matrix Organization to Control Cell Signaling during Hematopoiesis

    During hematopoiesis, progenitor cells receive and interpret a diverse array of regulatory signals from their environment. These signals control the maintenance of the progenitors and regulate the production of mature blood cells. Integrins (see Myospheroid) are well known in vertebrates for their roles in hematopoiesis, particularly in assisting in the migration to, as well as the physical attachment of, progenitors to the niche. However, whether and how integrins are also involved in the signaling mechanisms that control hematopoiesis remains to be resolved. This study shows that integrins play a key role during fly hematopoiesis in regulating cell signals that control the behavior of hematopoietic progenitors. Integrins can regulate hematopoiesis directly, via focal adhesion kinase (FAK) signaling, and indirectly, by directing extracellular matrix (ECM) assembly and/or maintenance. ECM organization and density controls blood progenitor behavior by modulating multiple signaling pathways, including bone morphogenetic protein (BMP) and Hedgehog (Hh). Furthermore, this study shows that integrins and the ECM are reduced following infection, which may assist in activating the immune response. These results provide mechanistic insight into how integrins can shape the signaling environment around hematopoietic progenitors (Khadilkar, 2020).

    Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix

    Epithelial sheets define organ architecture during development. This study employed an iterative multiscale computational modeling and quantitative experimental approach to decouple direct and indirect effects of actomyosin-generated forces, nuclear positioning, extracellular matrix, and cell-cell adhesion in shaping Drosophila wing imaginal discs. Basally generated actomyosin forces generate epithelial bending of the wing disc pouch. Surprisingly, acute pharmacological inhibition of ROCK-driven actomyosin contractility does not impact the maintenance of tissue height or curved shape. Computational simulations show that ECM tautness provides only a minor contribution to modulating tissue shape. Instead, passive ECM pre-strain serves to maintain the shape independent from actomyosin contractility. These results provide general insight into how the subcellular forces are generated and maintained within individual cells to induce tissue curvature. Thus, the results suggest an important design principle of separable contributions from ECM prestrain and actomyosin tension during epithelial organogenesis and homeostasis (Nematbakhsh, 2020).

    Mechano-chemical enforcement of tendon apical ECM into nano-filaments during Drosophila flight muscle development

    Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the muscular force is transmitted to the exoskeleton through the tendon cells and tendon apical extracellular matrix (ECM). In Drosophila, tendon cells were found to secrete Dumpy (Dpy), a zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. Dpy undergoes filamentous conversion in response to the tension increment during indirect flight muscle development. Another ZPD protein Quasimodo (Qsm) was found to protect the notum epidermis from collapsing under the muscle tension by enhancing the tensile strength of Dpy filaments. Qsm is co-transported with Dpy in the intracellular vesicles and diffuses into the exuvial space after secretion. Tissue-specific qsm expression rescued the qsm mutant phenotypes in distant tissues, suggesting Qsm can function in a long-range, non-cell-autonomous manner. In the cell culture assay, Qsm interacts with Dpy-ZPD and promotes secretion and polymerization of Dpy-ZPD. The roles of Qsm underlies the positive feedback mechanism of force-dependent organization of Dpy filaments, providing new insights into apical ECM remodeling through the unconventional interaction of ZPD proteins (Chu, 2021).

    Tweedle proteins form extracellular two-dimensional structures defining body and cell shape in Drosophila melanogaster

    Tissue function and shape rely on the organization of the extracellular matrix (ECM) produced by the respective cells. Understanding of the underlying molecular mechanisms is limited. This study shows that extracellular Tweedle (Twdl) proteins in the fruit fly Drosophila melanogaster form two adjacent two-dimensional sheets underneath the cuticle surface and above a distinct layer of dityrosinylated and probably elastic proteins enwrapping the whole body. Dominant mutations in twdl genes cause ectopic spherical aggregation of Twdl proteins that recruit dityrosinylated proteins at their periphery within lower cuticle regions. These aggregates perturb parallel ridges at the surface of epidermal cells that have been demonstrated to be crucial for body shaping. In one scenario, hence, this disorientation of epidermal ridges may explain the squatty phenotype of twdl mutant larvae. In an alternative scenario, this phenotype may be due to the depletion of the dityrosinylated and elastic layer, and the consequent weakening of cuticle resistance against the internal hydrostatic pressure. According to Barlow's formula describing the distribution of internal pressure forces in pipes in dependence of pipe wall material properties, it follows that this reduction in turn causes lateral expansion at the expense of the antero-posterior elongation of the body (Zuber, 2020).

    Resilin matrix distribution, variability and function in Drosophila

    Elasticity prevents fatigue of tissues that are extensively and repeatedly deformed. Resilin is a resilient and elastic extracellular protein matrix in joints and hinges of insects. For its mechanical properties, Resilin is extensively analysed and applied in biomaterial and biomedical sciences. However, there is only indirect evidence for Resilin distribution and function in an insect. Commonly, the presence of dityrosines that covalently link Resilin protein monomers (Pro-Resilin), which are responsible for its mechanical properties and fluoresce upon UV excitation, has been considered to reflect Resilin incidence. Using a GFP-tagged Resilin version, Resilin was directly identified in pliable regions of the Drosophila body, some of which were not described before. Interestingly, the amounts of dityrosines are not proportional to the amounts of Resilin in different areas of the fly body, arguing that the mechanical properties of Resilin matrices vary according to their need. For a functional analysis of Resilin matrices, applying the RNA interference and Crispr/Cas9 techniques, flies were generated with reduced or eliminated Resilin function, respectively. These flies are flightless but capable of locomotion and viable suggesting that other proteins may partially compensate for Resilin function. Indeed, localizations of the potentially elastic protein Cpr56F and Resilin occasionally coincide. Thus, Resilin-matrices are composite in the way that varying amounts of different elastic proteins and dityrosinylation define material properties. Understanding the biology of Resilin will have an impact on Resilin-based biomaterial and biomedical sciences (Lerch, 2020).

    Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster

    SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. It was previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. This study sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency was found to lead to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell autonomous accumulation of BL components around mutant cells that include Collagen IV (Col IV), Laminin and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. These data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV (Shahab, 2014).

    The emergence of multicellular organisms was co-incident with the appearance of genes coding for extracellular matrix (ECM) molecules that gave rise to two major classes of ECMs: interstitial matrices and basal laminae (BL)/basement membranes. In contrast to vertebrate tissues where interstitial matrices predominate, BL are the principal ECMs in animals of lower phyla. Universal components of BLs include network-forming Collagen IV (Col IV), Laminin, Perlecan, and Nidogen, which are assembled into 2D sheet-like networks. In addition to serving as tissue boundaries and an adhesive substratum for cell anchoring and migration, BLs make diverse regulatory contributions to the development of tissues and organs (Hohenester, 2013; Shahab, 2014).

    Col IV imparts tensile strength to BL and provides an anchoring substratum for cell adhesion, migration, and secreted signaling molecules. Much of what is known about Col IV is derived from vertebrate studies. Vertebrates express six Col IV α-chains [α1(ΙV)-α6(ΙV)] that are assembled in the endoplasmic reticulum into different combinations of heterotrimeric protomers. Upon secretion, the C-terminal globular domain of these trimeric protomers form head-to-head dimers Flexible non-helical interruptions separating collagenous domains of the protomers promote lateral associations during supramolecular assembly of 2D Col IV networks. Further contributing to the stability of these networks, the N-terminal globular domain of the heterotrimers form anti-parallel tetramers. As with fibril-forming collagens, purified Col IV protomers can self-assemble into polymeric networks. In contrast to vertebrates, the Drosophila genome codes for only two Col IV α-chains: Dcg 1/Cg25C and Viking (Vkg). The primary sources of BL components produced within Drosophila embryos and larvae are hemocytes and the fat body (Olofsson, 2005); however, how Col IV and the other BL components are assembled into a stereotypic 2D sheet of precise thickness is unknown (Shahab, 2014).

    Previously studies have shown that SPARC (Secreted Protein, Acidic and Rich in Cysteine), a highly conserved matricellular glycoprotein, is a major component of embryonic Drosophila BL (Martinek, 2002; Martinek, 2008). SPARC, also known as osteonectin/ BM40, binds to fibril-forming collagens and Col IV via epitopes located within the C-terminal domain. The absence of interstitial matrices in Drosophila makes it an ideal developmental and genetic model to decipher the role of SPARC in BL assembly and maturation (Shahab, 2014).

    Using imprecise P-element excision to generate a mutation/deletion of SPARC in Drosophila, a previous study reported decreased Col IV and BL stability and neural defects resulting in embryonic lethality in the absence of SPARC. However, attempts to rescue embryonic lethality by expressing exogenous SPARC were unsuccessful (Martinek, 2008), raising the possibility that aspects of this phenotype were due to a second site mutation on the 3rd chromosome. The present study, determined that both the neural phenotype and embryonic lethality reported previously, result from a disruption of the neurogenic gene, neutralized. The disruption of SPARC alone leads to larval lethality characterized by compromised fat body homeostasis. The fat body is crucial for development. It acts as the primary source of energy, and fat body together with hemocytes are the principle sources of BL components during larval development. Formed during embryonic development, the larval fat body is a bilateral, multi-lobed organ consisting of a monolayer of about 2,200 polygonal cells called adipocytes. The larval fat body is entirely surrounded by hemolymph, but does not directly interface with it owing to the presence of a BL that covers the entire surface of the fat body. The adipocytes within the fat body have no classical apical-basal polarity. Instead, cell-cell adhesion and shape is mediated by BL surrounding the adipocytes (Pastor-Pareja, 2011). This study reports that a reduction of SPARC leads to defective fat body BL assembly, inducing the resident polygonal adipocytes to round up and accumulate BL components within their microenvironment in a cell-autonomous manner. These findings define a pivotal role for SPARC in the proper assembly of BL surrounding the adipocytes of the Drosophila fat body (Shahab, 2014).

    The results of this study demonstrate that loss or knockdown of SPARC expression in Drosophila result in arrest during larval development and disruption of fat body architecture and function. Based upon the SPARC mutation Df(3R)nm136, it was previously reported that loss of SPARC resulted in embryonic lethality associated with severe defects in nervous system development. This study now provide evidence that a second-site mutation present in the neuralized locus, a key regulator of Notch/Delta signalling, is the cause of the Df(3R)nm136 neural phenotype and embryonic lethality. Hence, SPARC is not required for nervous system development (Shahab, 2014).

    The new Df(3R)nm136 H2AvD::GFP line, from which the neuralized mutation has been removed, demonstrates that loss of SPARC in Drosophila results in larval lethality and morphological changes of the fat body. The larval fat body is a multifunctional organ essential to fly development. Principle functions of the organ are nutrient storage and regulation of energy availability, functions that may become compromised in SPARC-deficient larvae. SPARC-deficient larvae appear transparent, which is consistent with reduced lipid or energy stores. While it is possible that knockdown of SPARC in hemocytes was responsible for the lethality and fat body morphological defects, knockdown of SPARC selectively within hemocytes using a hemolectin promoter did not result in larval lethality or a fat body phenotype, indicating that the phenotype reported in this study is due to loss of fat body SPARC expression. Moreover, larval lethality and the fat body phenotype of SPARC mutant larvae were rescued by a SPARC transgene that was expressed under the control of either endogenous SPARC or Col IV promoters (Shahab, 2014).

    SPARC reduction led to a marked accumulation of BL components in the extracellular microenvironment of affected adipocytes. Temporal expression data from modENCODE indicate that maximum levels of SPARC and Col IV expression occur during the 1st and 2nd instar stages, with expression decreasing during the 3rd larval instar prior to pupariation. Consistent with the idea that SPARC effects are largely mediated prior to the late 3rd instar stage, knockdown of SPARC in 3rd instar had no impact on survival or fat body remodeling (Shahab, 2014).

    Pastor-Pareja (2011) showed that knockdown of SPARC results in extracellular assembly of Col IV into thick fibers in the fat body, leading them to speculate that SPARC is required for Col IV secretion and solubility. However, the impact of SPARC knockdown on Col IV secretion, BL integrity, or adipocyte morphology was not addressed in that study. The current study suggests that SPARC deficiency does not prevent Col IV secretion. Consistent with the results of Pastor-Pareja (2011), this study shows extracellular accumulation of Col IV, suggestive of decreased solubility. Moreover, this study shows that Laminin, Perlecan, and Nidogen also accumulate at the surface of SPARC-deficient adipocytes, indicating that all BL components are affected by the loss or knockdown of SPARC (Shahab, 2014).

    Biochemical studies have shown that SPARC binds to the triple-helical domains of purified invertebrate and vertebrate Col IV, an interaction that is mediated by two collagen-binding epitopes located in the C-terminal region of SPARC. Col IV is a primary regulator of cell shape and adhesion; thus, alterations in the availability or structure of Col IV fibrils impact cell morphology. Several studies have shown that SPARC has counter-adhesive activity in vitro that causes cells to detach from their substrate and round up. The current data appear paradoxical as loss of SPARC results in cell rounding but does not lead to adipocyte dissociation. However, the impact on cell shape in this instance is likely due to the dysregulation of Col IV polymerization and BL homeostasis, rather than directly to the effect of SPARC on cell-cell or cell-matrix interactions (Shahab, 2014).

    A previous studies suggested that SPARC co-localizes with Col IV within secretory vesicles of adipocytes, but it remains to be determined whether SPARC and Col IV directly bind to one another intracellularly. Upon exocytosis, close proximity of SPARC with Col IV enables immediate physical association such that SPARC can regulate Col IV polymerization and sequester Col IV from its cellular receptors. Bradshaw (2009) demonstrated such a relationship between SPARC and Collagen I in mammalian cells. SPARC deficiency does not lead to an increase in intracellular Col IV, demonstrating that the impact of a lack of SPARC on Col IV assembly into BL likely occurs extracellularly. Upon secretion, SPARC may act to maintain solubility of Col IV, preventing it from immediately undergoing polymerization. In the absence of SPARC, Col IV release to the fat body extracellular space occurs; however, without SPARC to delay its polymerization, Col IV may rapidly assemble into a dense meshwork. Other ECM proteins, such as Laminin, Perlecan, and Nidogen, are synthesized and secreted; they encounter polymerized Col IV and are incorporated into the assembled structure as they would in a normal BL. This causes accumulation of multiple BL proteins on the surface of adipocytes. As ECM material accumulates, it promotes the rounding of the cells. The formation of a dense ECM meshwork likely impedes normal adipocyte function and could interfere with a variety of physiological processes such as feeding behavior and energy metabolism (Shahab, 2014).

    In light of the diffusible nature of SPARC, the finding of a cell-autonomous phenotype with fat body SPARC knockdown clones was unexpected. The failure of SPARC secreted from adjacent wild-type adipocytes to compensate for the lack of production by SPARC-deficient cell clones indicates that SPARC was not able to diffuse across the BL in sufficient quantities. To date, no study that has addressed the ability of SPARC to diffuse across the BL, but the current data raise the possibility of a charge-dependent barrier that retains SPARC within the microenvironment of a cell. Alternatively, the more immediate interaction of SPARC with Col IV afforded by their intracellular co-localization may be required to effectively prevent premature polymerization of Col IV. Hence, an intracellular interaction between SPARC and Col IV may be required to regulate the kinetics of Col IV polymerization immediately upon its secretion (Shahab, 2014).

    SPARC may also regulate BL deposition and remodelling through cell surface receptors. Expression of the cell-matrix adhesion molecules Dg and the βPS integrin subunit was observed on the plasma membrane of wild-type adipocytes. RNAi knockdown of SPARC did not alter the expression or localization of either of these transmembrane receptors in fat body cells indicating that it is unlikely that ECM accumulation around SPARC mutant adipocytes is associated with dysregulation of ECM receptors. However, the possibility that the interaction of BL components with these ECM receptors may have been affected cannot be excluded (Shahab, 2014).

    Randomly distributed pits were observed on the surface of adipocytes, which increased in number with the knockdown of SPARC. However, the majority of the pits associated with a SPARC knockdown exhibited thickened circumferential borders underlaid by intracellular lipid-like vesicles. It is conceivable that the pits represent sites of lipid exocytosis. However, preliminary data indicates that the knockdown of SPARC does not affect protein or vesicular endocytosis and exocytosis. Moreover, differences in lipid content between wild-type and SPARC-deficient adipocytes were not observed. Hence the molecular basis of the dramatic difference in the surface topography between wild-type and SPARC-deficient adipocytes remains unknown (Shahab, 2014).

    Analysis of the evolutionary history of SPARC revealed a conservation of the collagen-binding epitopes from cnidarians to mammals, which enable SPARC to bind to fibril-forming and network-forming Col IV. While SPARC-null mice develop normally, ultrastructural analysis revealed that interstitial Col IV fibrils are less abundant, smaller and more uniform in size, resulting in fibrils with decreased tensile strength. Biochemical studies indicate that SPARC increases the length of the first stage/lag phase of collagen fibrillogenesis by decreasing the rate of nucleation (Bradshaw, 2009). SPARC is also concentrated in the basal laminae of the nematode C. elegans. RNAi knockdown of SPARC leads to larval lethality for a large percentage of the progeny with a deficiency in gut granules and reduction in body size (Fitzgerald, 1998). It remains to be determined if aberrations in BL lamina assembly is the underlying cause of the phenotype (Shahab, 2014).

    Hence, these findings support an emerging concept of SPARC as a critical extracellular collagen chaperone. A detrimental loss of BL homeostasis is evident in the absence of SPARC. The evolutionary conservation of SPARC parallels the advent of BL in multi-cellular organisms, indicating that this chaperone activity of SPARC is important for the maintenance of ECM homeostasis in all metazoans (Shahab, 2014).


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    Martinek, N., Shahab, J., Sodek, J. and Ringuette, M. J. (2007). Is SPARC an evolutionarily conserved collagen chaperone? J. Dent Res. 86: 296-305. PubMed ID: 17384023

    Matsubayashi, Y., Louani, A., Dragu, A., Sanchez-Sanchez, B. J., Serna-Morales, E., Yolland, L., Gyoergy, A., Vizcay, G., Fleck, R. A., Heddleston, J. M., Chew, T. L., Siekhaus, D. E. and Stramer, B. M. (2017). A moving source of matrix components is essential for de novo basement membrane formation. Curr Biol 27(22): 3526-3534. PubMed ID: 29129537

    Nematbakhsh, A., Levis, M., Kumar, N., Chen, W., Zartman, J. J. and Alber, M. (2020). Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix. PLoS Comput Biol 16(8): e1008105. PubMed ID: 32817654

    Olofsson, B. and Page, D. T. (2005). Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity. Dev Biol 279: 233-243. PubMed ID: 15708571

    Pastor-Pareja, J. C. and Xu, T. (2011). Shaping cells and organs in Drosophila by opposing roles of fat body-secreted Collagen IV and perlecan. Dev Cell 21: 245-256. PubMed ID: 21839919

    Pastor-Pareja, J. C. and Xu, T. (2011). Shaping cells and organs in Drosophila by opposing roles of fat body-secreted Collagen IV and perlecan. Dev Cell 21(2): 245-256. PubMed ID: 21839919

    Sessions, A. O., Kaushik, G., Parker, S., Raedschelders, K., Bodmer, R., Van Eyk, J. E. and Engler, A. J. (2017). Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan. Matrix Biol 62: 15-27. PubMed ID: 27793636

    Shahab, J., Baratta, C., Scuric, B., Godt, D., Venken, K. J. and Ringuette, M. J. (2014). Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster. Dev Dyn [Epub ahead of print]. PubMed ID: 25529377

    Smyth, N., Vatansever, H. S., Murray, P., Meyer, M., Frie, C., Paulsson, M. and Edgar, D. (1999). Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation. J Cell Biol 144(1): 151-160. PubMed ID: 9885251

    Vaughan, L., Marley, R., Miellet, S. and Hartley, P. S. (2017). The impact of SPARC on age-related cardiac dysfunction and fibrosis in Drosophila. Exp Gerontol [Epub ahead of print]. PubMed ID: 29032244

    Zuber, R., Wang, Y., Gehring, N., Bartoszewski, S. and Moussian, B. (2020). Tweedle proteins form extracellular two-dimensional structures defining body and cell shape in Drosophila melanogaster. Open Biol 10(12): 200214. PubMed ID: 33292106

    Zygotically transcribed genes

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