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• W2093746797 abstract "Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, we used a GFP-based RNAi method (iGFPi) designed to knock down any GFP-trapped protein in Drosophila. We found with this method that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. We also show that incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. Our results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form. Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, we used a GFP-based RNAi method (iGFPi) designed to knock down any GFP-trapped protein in Drosophila. We found with this method that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. We also show that incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. Our results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form. In vivo GFP RNA interference (iGFPi) can be used to knock down GFP-trapped proteins Fat body-secreted Collagen IV is incorporated to larval basement membranes (BMs) Collagen IV shapes cells and organs by exerting a mechanical constricting tension Perlecan, recruited into BMs by Collagen IV, counters Collagen IV constriction Basement membranes (BMs) are layered polymers of extracellular matrix proteins that underlie epithelia in all animals and surround organs, including muscles, fat, endothelium, and nervous tissue (Timpl, 1989Timpl R. Structure and biological activity of basement membrane proteins.Eur. J. Biochem. 1989; 180: 487-502Crossref PubMed Scopus (788) Google Scholar, Yurchenco and Schittny, 1990Yurchenco P.D. Schittny J.C. Molecular architecture of basement membranes.FASEB J. 1990; 4: 1577-1590Crossref PubMed Scopus (770) Google Scholar). Among BM components, Collagen IV is the most abundant, comprising 50% of the proteins of the BM (Kalluri, 2003Kalluri R. Basement membranes: structure, assembly and role in tumour angiogenesis.Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1233) Google Scholar). Collagen IV molecules consist of α chains bound in long helical trimers that assemble into a network through lateral and end-domain interactions (Yurchenco and Ruben, 1987Yurchenco P.D. Ruben G.C. Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network.J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (235) Google Scholar). Stacked layers of this polymer provide the main structural feature of BMs. The biogenesis of functional collagen is very complex. Deficient or aberrant production is crucially involved in many diseases, including syndromes caused by mutations in collagens and collagen-modifying enzymes (Myllyharju and Kivirikko, 2004Myllyharju J. Kivirikko K.I. Collagens, modifying enzymes and their mutations in humans, flies and worms.Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar). Among the first steps in collagen biogenesis, α chains undergo extensive posttranslational modification in the endoplasmic reticulum (ER). A number of chaperones and enzymes assist folding and trimerization, including lysyl- and prolyl-hydroxylases, which require vitamin C as a cofactor (Lamandé and Bateman, 1999Lamandé S.R. Bateman J.F. Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones.Semin. Cell Dev. Biol. 1999; 10: 455-464Crossref PubMed Scopus (159) Google Scholar). CopII coated vesicles, which mediate ER-to-Golgi transport of secreted proteins, are typically 60–80 nm in diameter, whereas collagen trimers are 300 nm long. Consequently, studies on collagen secretion have fuelled a controversy as to whether alternative mechanisms exist for the secretion of large proteins (Fromme and Schekman, 2005Fromme J.C. Schekman R. COPII-coated vesicles: flexible enough for large cargo?.Curr. Opin. Cell Biol. 2005; 17: 345-352Crossref PubMed Scopus (79) Google Scholar). It is not clear either how collagen trimers avoid self-aggregation and aggregation with other BM components inside the producing cell or at the plasma membrane. Therefore, while collagens are the most abundant proteins in the human body (30% of its protein mass), many of the steps of their biogenesis are poorly understood. Collagen IV is found in all animals, from sponges to humans, indicating a central role of BMs in the development of complex body plans (Hynes and Zhao, 2000Hynes R.O. Zhao Q. The evolution of cell adhesion.J. Cell Biol. 2000; 150: F89-F96Crossref PubMed Google Scholar). It is the ancestral type of collagen, from which the 27 remaining vertebrate types have evolved. Type IV Collagens are divided into two subfamilies, α1-like and α2-like, split already in Cnidaria (Aouacheria et al., 2006Aouacheria A. Geourjon C. Aghajari N. Navratil V. Deléage G. Lethias C. Exposito J.Y. Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.Mol. Biol. Evol. 2006; 23: 2288-2302Crossref PubMed Scopus (66) Google Scholar). Drosophila has two genes encoding α chains of Collagen IV, named viking (vkg) and Collagen at 25C (Cg25C) (Le Parco et al., 1986Le Parco Y. Knibiehler B. Cecchini J.P. Mirre C. Stage and tissue-specific expression of a collagen gene during Drosophila melanogaster development.Exp. Cell Res. 1986; 163: 405-412Crossref PubMed Scopus (26) Google Scholar, Natzle et al., 1982Natzle J.E. Monson J.M. McCarthy B.J. Cytogenetic location and expression of collagen-like genes in Drosophila.Nature. 1982; 296: 368-371Crossref PubMed Scopus (35) Google Scholar, Rodriguez et al., 1996Rodriguez A. Zhou Z. Tang M.L. Meller S. Chen J. Bellen H. Kimbrell D.A. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.Genetics. 1996; 143: 929-940PubMed Google Scholar, Yasothornsrikul et al., 1997Yasothornsrikul S. Davis W.J. Cramer G. Kimbrell D.A. Dearolf C.R. viking: identification and characterization of a second type IV collagen in Drosophila.Gene. 1997; 198: 17-25Crossref PubMed Scopus (68) Google Scholar), belonging to the α2-like and α1-like subfamilies respectively. vkg and Cg25C are adjacently located head-to-head in the genome, an arrangement conserved in the three α1-like/α2-like pairs of Collagen IV genes in mammals. Drosophila Collagen IV genes are essential, as loss of function of either of them causes embryonic lethality (Borchiellini et al., 1996Borchiellini C. Coulon J. Le Parco Y. The function of type IV collagen during Drosophila muscle development.Mech. Dev. 1996; 58: 179-191Crossref PubMed Scopus (39) Google Scholar, Rodriguez et al., 1996Rodriguez A. Zhou Z. Tang M.L. Meller S. Chen J. Bellen H. Kimbrell D.A. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.Genetics. 1996; 143: 929-940PubMed Google Scholar). In addition, Cg25C and Vkg modulate TGF-β gradient formation in the blastoderm embryo before a BM exists (Wang et al., 2008Wang X. Harris R.E. Bayston L.J. Ashe H.L. Type IV collagens regulate BMP signalling in Drosophila.Nature. 2008; 455: 72-77Crossref PubMed Scopus (227) Google Scholar). Early lethality, although informative of the importance of Collagen IV for normal development, has precluded further analysis of its function in later stages, when organs and systems increase in size and complexity. Apart from Collagen IV, the three other major components of BMs are Laminin, Nidogen, and the heparan-sulfate proteoglycan Perlecan, all conserved in Drosophila as well (Hynes and Zhao, 2000Hynes R.O. Zhao Q. The evolution of cell adhesion.J. Cell Biol. 2000; 150: F89-F96Crossref PubMed Google Scholar). Multiple interactions between BM components have been mapped in vitro. However, little evidence exists in vivo to show which interactions are crucial for BM assembly, maintenance and regeneration. Furthermore, a study of mice homozygous for a targeted deletion of the Collagen IV α1/α2 pair reported embryonic lethality but no defect in deposition of other BM components (Pöschl et al., 2004Pöschl E. Schlötzer-Schrehardt U. Brachvogel B. Saito K. Ninomiya Y. Mayer U. Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development.Development. 2004; 131: 1619-1628Crossref PubMed Scopus (518) Google Scholar). This led to the proposal that Collagen IV is a terminal BM component, dispensable for BM assembly. In this study, we have used a GFP protein trap inserted into the Drosophila vkg gene, producing a functional GFP-tagged version of Vkg, to study Collagen IV biogenesis and function. We developed an approach (in vivo GFP interference, iGFPi) to specifically knock down Vkg-GFP. Using iGFPi, we found that Collagen IV is synthesized in the larva by the fat body and continuously incorporated into BMs, where it exerts a constricting force on tissues. We additionally found that Collagen IV is not a terminal BM component, but, on the contrary, essential for deposition of Perlecan into BMs. Surprisingly, we found that Perlecan incorporation counters, rather than reinforces, constriction by Collagen IV. Protein trapping strategies have been developed in several model systems for large scale tagging of proteins and visualization of their subcellular localizations (Jarvik et al., 2002Jarvik J.W. Fisher G.W. Shi C. Hennen L. Hauser C. Adler S. Berget P.B. In vivo functional proteomics: mammalian genome annotation using CD-tagging.Biotechniques. 2002; 33 (856, 858–860 passim): 852-854PubMed Google Scholar). In Drosophila, several insertional trapping screenings have been conducted by mobilizing transposons carrying the GFP-coding sequence flanked by a splice acceptor and donor (Buszczak et al., 2007Buszczak M. Paterno S. Lighthouse D. Bachman J. Planck J. Owen S. Skora A.D. Nystul T.G. Ohlstein B. Allen A. et al.The carnegie protein trap library: a versatile tool for Drosophila developmental studies.Genetics. 2007; 175: 1505-1531Crossref PubMed Scopus (368) Google Scholar, Clyne et al., 2003Clyne P.J. Brotman J.S. Sweeney S.T. Davis G. Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements.Genetics. 2003; 165: 1433-1441PubMed Google Scholar, Morin et al., 2001Morin X. Daneman R. Zavortink M. Chia W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.Proc. Natl. Acad. Sci. USA. 2001; 98: 15050-15055Crossref PubMed Scopus (558) Google Scholar, Quiñones-Coello et al., 2007Quiñones-Coello A.T. Petrella L.N. Ayers K. Melillo A. Mazzalupo S. Hudson A.M. Wang S. Castiblanco C. Buszczak M. Hoskins R.A. Cooley L. Exploring strategies for protein trapping in Drosophila.Genetics. 2007; 175: 1089-1104Crossref PubMed Scopus (123) Google Scholar). As a result, more than 550 transgenic lines expressing GFP-tagged versions of proteins under control of their normal promoters are currently available. We found these trapping constructs are highly efficient (between 1.31 and 0.24% of transcripts skipped the artificial GFP exon in tested lines; see Figures S1A–S1C available online). We therefore reasoned that targeting through RNAi the GFP-encoding portion of the mRNA could be a versatile method to silence the expression of any GFP-trapped protein. To test the feasibility of this approach (in vivo GFP interference or iGFPi, Figure 1A ), we expressed under control of the Gal4/UAS system an RNA inverted repeat targeting the GFP coding sequence (Brand and Perrimon, 1993Brand A.H. Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.Development. 1993; 118: 401-415Crossref PubMed Google Scholar, Roignant et al., 2003Roignant J.Y. Carré C. Mugat B. Szymczak D. Lepesant J.A. Antoniewski C. Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila.RNA. 2003; 9: 299-308Crossref PubMed Scopus (196) Google Scholar). iGFPi efficiently silenced expression of GFP-trapped proteins (examples in Figures 1B–1M and Figures S1D and S1E; 99% of the GFP-trapped product is knocked down, Figure S2E). This reduction was tissue specific, dependent on the expression pattern of the Gal4 line used to drive iGFPi. Furthermore, iGFPi was able to elicit loss-of-function phenotypes when the only allele of the gene present was GFP-trapped (i.e., flies homozygous or hemizygous for the GFP-trap insertion) (Figures 1B–1O and Figures S1F–S1I). These results show that iGFPi silences expression of GFP-trapped alleles specifically and that the method can be used for functional studies of GFP-trapped proteins. In order to study the biogenesis and function of Collagen IV, we made use of a Collagen IV GFP-trap line, vikingG454 (vkgG454) (Morin et al., 2001Morin X. Daneman R. Zavortink M. Chia W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.Proc. Natl. Acad. Sci. USA. 2001; 98: 15050-15055Crossref PubMed Scopus (558) Google Scholar). Vkg-GFP localizes to BMs and has accordingly been used by us and others to monitor BM dynamics (Medioni and Noselli, 2005Medioni C. Noselli S. Dynamics of the basement membrane in invasive epithelial clusters in Drosophila.Development. 2005; 132: 3069-3077Crossref PubMed Scopus (41) Google Scholar, Srivastava et al., 2007Srivastava A. Pastor-Pareja J.C. Igaki T. Pagliarini R. Xu T. Basement membrane remodeling is essential for Drosophila disc eversion and tumor invasion.Proc. Natl. Acad. Sci. USA. 2007; 104: 2721-2726Crossref PubMed Scopus (152) Google Scholar, Pastor-Pareja et al., 2008Pastor-Pareja J.C. Wu M. Xu T. An innate immune response of blood cells to tumors and tissue damage in Drosophila.Dis Model Mech. 2008; 1: 144-154Crossref PubMed Scopus (177) Google Scholar, Haigo and Bilder, 2011Haigo S.L. Bilder D. Global tissue revolutions in a morphogenetic movement controlling elongation.Science. 2011; 331: 1071-1074Crossref PubMed Scopus (193) Google Scholar). iGFPi under control of the ubiquitous driver actin-Gal4 efficiently decreased Vkg-GFP expression in third instar (L3) heterozygous larvae (vkgG454/+; Figures 2A–2D and Figure S2E). iGFPi in vkgG454 homozygotes (vkgG454/vkgG454) caused in addition embryonic lethality (Figures 2E and 2F). This, together with the fact that vkgG454/vkgG454 flies are phenotypically wild-type, indicates that the Vkg-GFP fusion protein produced by the vkgG454 allele is functional. The previous result is consistent with embryonic lethality reported for vkg mutants (Rodriguez et al., 1996Rodriguez A. Zhou Z. Tang M.L. Meller S. Chen J. Bellen H. Kimbrell D.A. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.Genetics. 1996; 143: 929-940PubMed Google Scholar). It is not known, however, whether the function of Vkg, very abundant in BMs throughout development, is required after embryogenesis. To test a postembryonic requirement of Vkg, we made use of a thermosensitive form of the Gal4 repressor Gal80 (Gal80ts) to modulate Gal4-driven iGFPi. In presence of Gal80ts, vkgG454/vkgG454 animals developed into wild-type adults at 18°C (act-Gal4+Gal80ts>iGFPi; permissive temperature). In contrast, shifting from 18°C to 29°C after completion of embryonic development resulted in pupal lethality (Figure 2G). These results show that Vkg expression is required for normal development after embryonic stages and confirm that the vkgG454 GFP trap can be used to study Collagen IV biogenesis in vivo. Once established a role for Viking in postembryonic development, we asked where the expression of Viking takes place during larval stages. In the embryo, even though Collagen IV is found in all tissues, mRNA in situ and enhancer traps suggest high expression in mesodermal derivatives (Le Parco et al., 1986Le Parco Y. Knibiehler B. Cecchini J.P. Mirre C. Stage and tissue-specific expression of a collagen gene during Drosophila melanogaster development.Exp. Cell Res. 1986; 163: 405-412Crossref PubMed Scopus (26) Google Scholar, Rodriguez et al., 1996Rodriguez A. Zhou Z. Tang M.L. Meller S. Chen J. Bellen H. Kimbrell D.A. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.Genetics. 1996; 143: 929-940PubMed Google Scholar). In order to ascertain the source of Vkg protein, we made use of iGFPi in vkgG454/+ larvae and knocked down expression of GFP-trapped Viking using Gal4 drivers with restricted expression patterns. iGFPi under control of the wing disc drivers nub-Gal4 (data not shown) and Hh-Gal4 (Figures S2A–S2D) had no effect in the amount of Vkg-GFP present in the BM of the disc. In contrast, iGFPi driven by Cg-Gal4, expressed in hemocytes and fat body, drastically decreased Vkg-GFP expression in the whole animal, including imaginal discs (Figures 3A–3D and 3H ; quantified in Figure S2E). Similar results were obtained when iGFPi was induced with ppl-Gal4, a fat body-specific driver (Figures 3E and 3I). iGFPi under control of hemocyte-specific drivers, however, did not reduce Vkg-GFP accumulation in BMs (Figure S2E) and neither did hemocyte ablation (data not shown). These results show that the fat body is the main source of the Vkg protein present in larval BMs. We also induced iGFPi under control of two mesodermal drivers with temporally opposite expression patterns: c754-Gal4, expressed in second and third instar larvae (L2 and L3) (Figure 3F) and twi-Gal4, expressed in the embryo and first instar (L1; Figure 3G). iGFPi under control of c754-Gal4 did not affect Vkg-GFP expression in L1; however, Vkg-GFP expression greatly decreased in L3 (Figure 3J). iGFPi under control of twi-Gal4, in contrast to c754-Gal4, reduced Vkg-GFP expression in L1, but Vkg-GFP visibly reappeared in L3 (Figure 3K). All these results together indicate that during larval development Vkg is produced by fat body cells and continuously incorporated into the BMs of the animal. The fat body is an organ formed by large polyploid cells (adipocytes), with known roles in lipid storage, metabolic regulation and immunity. To confirm that the fat body is the source of Viking and further investigate Collagen IV biogenesis, we decided to knock down in fat body cells the expression of several genes and examine the effects on Vkg-GFP localization. Secretion of collagen in human cells involves components of the CopII coatomer, required for ER-to-Golgi transport in the secretory pathway (Stephens and Pepperkok, 2002Stephens D.J. Pepperkok R. Imaging of procollagen transport reveals COPI-dependent cargo sorting during ER-to-Golgi transport in mammalian cells.J. Cell Sci. 2002; 115: 1149-1160PubMed Google Scholar). It has been shown as well in human cells that Tango1, a CopII cargo adaptor, is required for secretion of Collagen VII (Saito et al., 2009Saito K. Chen M. Bard F. Chen S. Zhou H. Woodley D. Polischuk R. Schekman R. Malhotra V. TANGO1 facilitates cargo loading at endoplasmic reticulum exit sites.Cell. 2009; 136: 891-902Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). We knocked down expression of Tango1 and observed that this caused retention of Vkg-GFP in fat body cells (Figures 4A and 4D ). Confocal imaging revealed that Vkg-GFP accumulated in growing intracellular aggregates (Figures 4B and 4E). The aggregates eventually coalesced and occupied most cytoplasm between the lipid droplets, affecting cell viability (Figures 4E–4G). These larvae lacked Vkg-GFP in the BM of other organs, such as the wing disc (Figures 4C and 4H; quantified in Figure S3C). We obtained the same results with ppl-Gal4 (data not shown). Additionally, knockdown of the CopII coat components Sar1 or Sec23 caused similar intracellular accumulation of Vkg-GFP (Figures 4I and 4J). These results show that fat body cells produce large amounts of Vkg protein and indicate that secretion of Collagen IV in Drosophila requires CopII coated vesicles and Tango1. We next investigated the effect of SPARC, the Drosophila homolog of BM40/SPARC/osteonectin, required for Collagen IV secretion by embryonic blood cells (Martinek et al., 2008Martinek N. Shahab J. Saathoff M. Ringuette M. Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos.J. Cell Sci. 2008; 121: 1671-1680Crossref PubMed Scopus (82) Google Scholar). Knockdown of SPARC also caused retention of Vkg-GFP in the fat body (Figures 4K and 4L) and its absence in wing discs (Figure 4M). However, unlike loss of Tango1, sar1, or sec23, accumulation of Vkg-GFP occurred not intracellularly, but as thick fibers outside the plasma membranes of fat body cells (Figures 4B' and 4L'). These results show a requirement for SPARC in fat body cells for correct secretion of Collagen IV, suggesting that in its absence Collagen IV is not soluble. A critical step in Collagen IV biogenesis is catalyzed by Prolyl-4-Hydroxylase (PH4). In the absence of this enzyme or its cofactor ascorbate (vitamin C), collagen chains cannot form functional trimers (Lamandé and Bateman, 1999Lamandé S.R. Bateman J.F. Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones.Semin. Cell Dev. Biol. 1999; 10: 455-464Crossref PubMed Scopus (159) Google Scholar). Among ten predicted Drosophila PH4 genes, we knocked down PH4αEFB, abundantly expressed in the fat body (Abrams and Andrew, 2002Abrams E.W. Andrew D.J. Prolyl 4-hydroxylase alpha-related proteins in Drosophila melanogaster: tissue-specific embryonic expression of the 99F8-9 cluster.Mech. Dev. 2002; 112: 165-171Crossref PubMed Scopus (26) Google Scholar). In these larvae, we found most Vkg-GFP signal in the hemolymph that fills the body cavity (Figures 4N–4P). Western blotting of hemolymph in nonreducing conditions, known to preserve trimeric collagen, showed that wild-type Vkg migrated with a higher apparent weight than Vkg from PH4α-EFB-deficient larvae (Figures 4Q and 4R). These results show that in PH4α-EFB-deficient larvae Collagen IV is secreted into the hemolymph in monomeric form and cannot be incorporated into BMs. Finally, we created in wing discs clones of cells mutant for myospheroid (mys), encoding the βPS subunit of the transmembrane integrin receptor. We found that loss of mys function caused scars in the BM underlying the mutant clones (Figure 4S and Figures S3A and S3B). This shows that integrins are involved in the capture by tissues of Collagen IV from the contacting hemolymph. Altogether, our results, showing defective deposition of Vkg-GFP into BMs and accumulation at different locations, indicate that Collagen IV is synthesized by fat body cells in the larva, secreted as a trimer to the hemolymph and from there incorporated into BMs (Figure S3D). To address next the function of Collagen IV, we knocked down in the fat body the expression of vkg. We used Gal80ts again to limit knockdown to larval stages and thus avoid early lethality. At permissive temperature, CgGal4+Gal80ts>vkgi flies developed into normal adults, but pupal lethality resulted when larvae were transferred to restrictive temperature after completion of embryogenesis. Dissection of these larvae 96 hr after the temperature shift revealed highly aberrant shape in several organs. Imaginal discs, consisting of highly columnar epithelial cells, appeared flattened (Figures 5A and 5B ), The ventral nerve cord (VNC) portion of the central nervous system was strikingly elongated (Figures 5C and 5D), reminiscent of embryonic defects in VNC condensation (Olofsson and Page, 2005Olofsson B. Page D.T. Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity.Dev. Biol. 2005; 279: 233-243Crossref PubMed Scopus (92) Google Scholar). Additionally, the ducts and imaginal rings of the salivary glands were dilated, resulting in an expanded lumen (Figures 5E and 5F). Similar results were obtained when we knocked down in the same way expression of Cg25C, encoding a second Collagen IV chain (Figure 5G), and both vkg and Cg25C simultaneously (Figures 5N–5Q and Figures S4A–S4D). Given the similar vkg and Cg25C loss-of-function phenotypes and the fact that Collagen IV molecules in mammals are heterotrimers (Hudson et al., 1993Hudson B.G. Reeders S.T. Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J. Biol. Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar), we examined the effect of Cg25C knockdown on Vkg-GFP localization. Knockdown of Cg25C caused accumulation of Vkg-GFP in the hemolymph and prevented its deposition into BMs (Figures 5H–5M), similar to PH4-aEFB knockdown (Figures 4N–4R). This indicates that, in the absence of Cg25C, Vkg is secreted to the hemolymph in nonfunctional monomeric form, which supports the existence of all or most Drosophila Collagen IV as α1-like/α2-like heterotrimers despite suggestions to the contrary (Lunstrum et al., 1988Lunstrum G.P. Bächinger H.P. Fessler L.I. Duncan K.G. Nelson R.E. Fessler J.H. Drosophila basement membrane procollagen IV. I. Protein characterization and distribution.J. Biol. Chem. 1988; 263: 18318-18327Abstract Full Text PDF PubMed Google Scholar). The phenotypes obtained through inhibition of Collagen IV production suggested to us the possibility that a loss of tension provided by the BM could underlie the observed deformations. To explore this, we treated wing discs ex vivo with collagenase to degrade the BM (Figures S4E–S4H). We observed that 1 min of collagenase treatment was sufficient to elicit changes in cell and tissue shape that phenocopied loss of Collagen IV (Figures 5R and 5S). Furthermore, collagenase treatment of discs from Collagen IV-deficient larvae did not cause additional flattening (Figure 5T). These fast shape changes following collagenase treatment (apico-basal shortening and planar expansion; Figures 5U–5W) are consistent with the BM exerting a basal constricting force that contributes to the highly columnar shape of disc cells. We additionally confirmed tissue flattening after degradation of the BM by Matrix Metalloprotease 2 (Mmp2) overexpression (Domínguez-Giménez et al., 2007Domínguez-Giménez P. Brown N.H. Martín-Bermudo M.D. Integrin-ECM interactions regulate the changes in cell shape driving the morphogenesis of the Drosophila wing epithelium.J. Cell Sci. 2007; 120: 1061-1071Crossref PubMed Scopus (51) Google Scholar), with cell shape changes reverting upon BM healing (Figures S4I–S4Q). Altogether, our experiments indicate a role of fat body-secreted Collagen IV in maintaining cell and organ shape by producing a basally constricting force. Apart from Collagen IV, the other three main BM constituents are Laminin, Nidogen, and the heparan sulfate proteoglycan (HSPG) Perlecan. Having established a role of Collagen IV in maintenance of cell and organ shape, we decided to investigate the effect of lack of Collagen IV in the deposition of other BM components. To this end, we examined with antibodies the expression of Laminin, Nidogen, and Perlecan in larvae where expression of both vkg and Cg25C had been knocked down (Cg-Gal4+Gal80ts>vkgi+Cg25Ci). Presence of Nidogen and the Laminin B1 subunit was unaffected by the absence of Collagen IV (Figures 6A, 6B, 6D, and 6E ). Basal localization of Myospheroid, the βPS subunit of the integrin receptor, was similarly unaffected (Figures S5J and S5K). In contrast, Perlecan, encoded by the gene trol, was absent from BMs in these animals (Figures 6C and 6F). These data show that Collagen IV deposition into BMs, while not required for localization of Nidogen or Laminin, is essential for Perlecan incorporation. Similar results were obtained in embryos homozygous for a deficiency deleting both vkg and Cg25C (Figures S5A–S5G), suggesting a requirement of Collagen IV for Perlecan incorporation also during initial BM assembly. Having determined a requirement for Collagen IV in the incorporation of Perlecan to BMs, we finally decided to investigate the effects in larval tissues and BMs of both loss and excess of Perlecan. To reduce Perlecan expression, we knocked down trol expression through RNAi controled by actin-Gal4. This effectively abrogated the presence of Perlecan in BMs (anti-Trol staining; Figures 7A and 7B ). To overexpress Perlecan, we used a unidirectional Gene Search Vector insertion (GSV2) upstream of trol (Figure 7C). Perlecan consists of a large core protein decorated by heparan-sulfate chains. Consistent with anti-Trol st" @default.
• W2093746797 created "2016-06-24" @default.
• W2093746797 creator A5017035217 @default.
• W2093746797 creator A5070139845 @default.
• W2093746797 date "2011-08-01" @default.
• W2093746797 modified "2023-10-16" @default.
• W2093746797 title "Shaping Cells and Organs in Drosophila by Opposing Roles of Fat Body-Secreted Collagen IV and Perlecan" @default.
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