Nidogen/entactin: Biological Overview | References
Gene name - Nidogen/entactin
Cytological map position - 47A1-47A1
Function - secreted basement membrane component
Keywords - basement membrane component, required for proper assembly and maintenance of certain basement membranes of the adipose tissue and flight muscles, binds to and couples Collagen IV and Laminin networks
Symbol - Ndg
FlyBase ID: FBgn0026403
Genetic map position - chr2R:10,313,891-10,322,104
FlyBase gene group - G2 nidogen domain and fibulin, Extracellular domain of unknown function in nidogen (entactin), Low-density lipoprotein-receptor YWTD domain, Calcium-binding EGF domain, EGF-like domain, NHL repeat unit of beta-propeller proteins
Cellular location - secreted
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 (Ackley, 2003; Kang, 2000; Kim, 2000). 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 (Mann, 1988; Lossl, 2014). 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 (Patel, 2014). 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).
Search PubMed for articles about Drosophila Nidogen
Ackley, B. D., Kang, S. H., Crew, J. R., Suh, C., Jin, Y. and Kramer, J. M. (2003). The basement membrane components nidogen and type XVIII collagen regulate organization of neuromuscular junctions in Caenorhabditis elegans. J Neurosci 23(9): 3577-3587. PubMed ID: 12736328
Dai, J., Estrada, B., Jacobs, S., Sanchez-Sanchez, B. J., Tang, J., Ma, M., Magadan-Corpas, P., Pastor-Pareja, J. C. and Martin-Bermudo, M. D. (2018). Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly. PLoS Genet 14(9): e1007483. PubMed ID: 30260959
Kang, S. H. and Kramer, J. M. (2000). Nidogen is nonessential and not required for normal type IV collagen localization in Caenorhabditis elegans. Mol Biol Cell 11(11): 3911-3923. PubMed ID: 11071916
Kim, S. and Wadsworth, W. G. (2000). Positioning of longitudinal nerves in C. elegans by nidogen. Science 288(5463): 150-154. PubMed ID: 10753123
Mann, K., Deutzmann, R. and Timpl, R. (1988). Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays. Eur J Biochem 178(1): 71-80. PubMed ID: 2462498
Lossl, P., Kolbel, K., Tanzler, D., Nannemann, D., Ihling, C. H., Keller, M. V., Schneider, M., Zaucke, F., Meiler, J. and Sinz, A. (2014). Analysis of nidogen-1/laminin gamma1 interaction by cross-linking, mass spectrometry, and computational modeling reveals multiple binding modes. PLoS One 9(11): e112886. PubMed ID: 25387007
Patel, T. R., Bernards, C., Meier, M., McEleney, K., Winzor, D. J., Koch, M. and Stetefeld, J. (2014). Structural elucidation of full-length nidogen and the laminin-nidogen complex in solution. Matrix Biol 33: 60-67. PubMed ID: 23948589
date revised: 20 October 2018
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