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

Abstract

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. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our 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 our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.

Atypical basement membranes and basement membrane diversity – what is normal anyway?

“…Two different laminin heterotrimers can form with alternative α chains encoded by genes Laminin A (LanA) and wing blister (wb), whereas unique β and γ chains are encoded by the Laminin B1 (LanB1) and Laminin B2 (LanB2) genes (Fessler et al, 1987;Kusche-Gullberg et al, 1992;Martin et al, 1999;Goodman, 1988, 1989). One perlecan protein encoded by terribly reduced optic lobes (trol) (Park et al, 2003;Voigt et al, 2002) and the product of Nidogen (Ndg) (Dai et al, 2018;Wolfstetter et al, 2019;Wolfstetter et al, 2009) complete the Drosophila LNCP set. For all four, transgenically expressed tagged versions, mutants and tissue-specific RNAi tools are available and have been used in multiple studies.…”

“…4A), but also two general tendencies. First, mesodermal tissues are more capable of secreting matrix, as is the case with blood cells and fat body, the major sources of BM components for the embryo and larva, respectively (Dai et al, 2018;Fessler and Fessler, 1989;Matsubayashi et al, 2017;Pastor-Pareja and Xu, 2011). The follicular epitheliuma rare example of an epithelium of mesodermal origin (Brookman et al, 1992;King et al, 1968)is indeed able to secrete all four main BM components (Isabella and Horne-Badovinac, 2015b).…”

“…2F), which secrete their own Laminin, but receive Collagen IV, Nidogen and Perlecan from the fat body through the hemolymph (Dai et al, 2018;Henchcliffe et al, 1993;Urbano et al, 2009). In some cases, a certain BM component may have a mixed origin, as it is in the case of the BM surrounding the larval central nervous system, which receives its Collagen IV from both intrinsic ( perineural glia) and extrinsic (fat body) sources (Dai et al, 2018;Petley-Ragan et al, 2016).…”

“…Examples of this are fat body and follicle cells, where short and long-range modes of deposition coexist, each mode producing a different type of matrix. Fat body adipocytes produce the four main components for their BM and CIVICs (Dai et al, 2018;Kim and Choe, 2014;Pastor-Pareja and Xu, 2011;Zang et al, 2015). In CIVICs, Collagen IV is deposited at short range by contacting cells, whereas Collagen IV in the surrounding BM comes from the common soluble pool in the hemolymph (Dai et al, 2017).…”

Characterization ofDrosophila Nidogen/entactinreveals roles in basement membrane stability, barrier function and nervous system patterning

“…Two adjacent loci encode for type IV collagens (Col4a1 and viking): one for the ColXV/XVIII homolog Multiplexin and one Nidogen/ entactin gene. The terribly reduced optic lobes (trol) locus encodes Perlecan (Borchiellini et al, 1996;Dai et al, 2018;Datta and Kankel, 1992;Friedrich et al, 2000;Henchcliffe et al, 1993;Lindsley and Zimm, 1992;Martin et al, 1999;Meyer and Moussian, 2009;Urbano et al, 2009;Voigt et al, 2002;Wolfstetter and Holz, 2012;Yarnitzky and Volk, 1995;Yasothornsrikul et al, 1997). In 1983, Timpl et al (1983 reported the identification of an 80 kDa protein from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells that was named Nidogen due to its ability to selfaggregate into 'nest-like structures'.…”