Laminin B1: Biological Overview | References
Gene name - Laminin B1
Cytological map position - 28C4-28D1
Function - ligand
Symbol - LanB1
FlyBase ID: FBgn0261800
Genetic map position - 2L: 7,811,440..7,820,774 [+]
Classification - Laminin-type epidermal growth factor-like domain
Cellular location - secreted
Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin α chains (see Drosophila LanA), one β and one γ, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, the gene encoding the sole lamininβ chain in Drosophila, LanB1 was mutated, so that no trimers can be made. LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, these results unravel novel functions in cell adhesion, migration and rearrangement. It is proposed that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution (Urbano, 2009).
Basement membranes (BMs) are specialised layers of extracellular matrix (ECM) covering the basal side of all epithelia and endothelia, and surrounding muscles, peripheral nerves and other tissues. BMs provide mechanical stability and physical barriers between different cell types and are important for tissue morphogenesis in metazoans. They have been implicated in many processes such as cell differentiation, shape, adhesion, survival and migration (Urbano, 2009).
Even though the composition of BMs varies according to tissues and developmental stages, they are mainly composed of two meshworks formed by laminins and collagen IV. They also contain other ECM components, such as Nidogen and proteoglycans. Laminins are a family of large heterotrimeric glycoproteins composed of three non-identical chains. The laminin trimer forms a cross-shaped structure consisting in three short arms, each formed by a different chain, and a long arm composed of the three assembled coiled chains. Data from mammalian cell culture has shown that while the α subunit can be secreted alone as a monomer, secretion of the β and γ chains requires simultaneous expression of all three chains and their assembly into α-β-γ heterotrimers (Yurchenco, 1997). Once secreted, laminins form a meshwork by self-assembly through interactions between the three short arms. In developing tissues, laminin assembly also requires the long arm to be tethered to receptors on the cell surface, such as integrins and dystroglycan (Urbano, 2009).
Laminins are common to tissues of most multicellular metazoans and they are highly conserved across evolution. However, whereas invertebrates possess one to two laminin heterotrimers, mammals possess at least 15, which are formed through combinations of 5α, 4β and 3γ subunits (Aumailley, 2005). They differ with respect to their tissue distribution, presumably reflecting diverse biological functions. Thus, whereas null mutations in laminin α1 results in early embryonic lethality (day E7) (Miner, 2004), lack of laminin α2 and α3 chains causes, respectively, severe muscular dystrophy and skin blistering both in mice and in humans. Mice failing to express the laminin β1 or γ1 subunits lack embryonic BMs and do not survive beyond embryonic day 5.5 (E5.5), suggesting some type of compensation between different α subunits (Urbano, 2009).
The Drosophila genome encodes only four laminin chains: two α chains (α1,2 and α3,5), one β chain and one γ chain. These form two trimers, lamininA (α3,5; β1; γ1) and lamininW (α1,2; β1; γ1). The first laminin α chain described in Drosophila, α3,5 (encoded by Laminin A, LanA), is most similar to vertebrate α3 and β5 and is part of the lamininA trimer. Experiments in cell culture have shown that lamininA is likely to bind PS1 integrin (αPS1βPS) and not PS2 integrin (αPS2βPS). This is supported by the similarity of phenotypes of LanA mutant embryos and those lacking the PS1 integrin. The second laminin α chain described in Drosophila, α1,2 (encoded by wing blister, wb), is most similar to vertebrate α1 and α2 chains and is part of the lamininW trimer. α1,2 contains an RGD (Arg-Gly-Asp) motif, which is a recognition site for PS2 integrin. Indeed, experiments with Drosophila S2 cells in culture have shown that RGD-containing peptides derived from laminin α1,2 can serve as PS2 integrin ligands (Graner, 1998). This is further supported by studies showing that lamininW is not recruited to muscle attachment sites in embryos carrying a mutation in the αPS2 subunit affecting the RGD binding site (Devenport, 2007; Urbano, 2009 and references therein).
Only mutations in the α subunits have been described in Drosophila so far. Null mutations in the LanA gene result in embryonic lethality with defects in somatic muscles, dorsal vessel (heart) and endoderm. The α3,5 chain is also required for proper localisation of anteroposterior markers in the oocyte and for normal pathfinding of pioneer axons in the brain. In addition, hypomorphic mutants and trans-heteroallelic combinations of LanA give rise to adult escapers that have disorganised rhabdomeres and display vein defects and wing blistering. Mutations in wb have wing blisters and defects in the dorsal vessel, trachea, muscles and rhabdomeres. Mutations in the laminin β subunit (encoded by Laminin B1, LanB1) or β subunit (encoded by laminin B2, lanB2) have not been characterised (Urbano, 2009).
In this work, null mutations were isolated in LanB1, allowing the generation of embryos lacking all laminin function. These embryos develop until the end of embryogenesis, suggesting that in Drosophila, as it is the case in nematodes and contrary to mice, laminins are not required for early embryonic morphogenetic events. Analysis of LanB1 loss reveals that laminins are required for accumulation of major ECM components, such as Collagen IV and Perlecan (Trol), into BMs. Furthermore, in the absence of the β chain, BMs are absent and the remaining extracellular material appears disorganised and diffuse. In addition, the results reveal new functions for laminins in cell adhesion, migration and rearrangement, and identify laminins as essential regulators of the morphogenesis of most organs in Drosophila (Urbano, 2009).
The isolation of loss-of-function mutations in the single laminin β subunit encoded by the Drosophila genome has allowed a study laminin requirements during development. In absence of laminin β, other laminin subunits as well as major BMs components fail to assemble into BMs. This analysis reveals new functions for laminins in cellular adhesion, migration and rearrangement during organogenesis (Urbano, 2009).
Data from cell-culture experiments suggest that only laminin trimers are secreted extracellularly. The current model is that a transitional dimeric configuration composed of a β and γ chain is first assembled intracellularly before incorporation of an α chain allows secretion (Goto, 2001; Kumagai, 1997; Morita, 1985; Peters, 1985). This model predicts that in absence of the laminin β or γ subunits no functional laminin trimers could be exported. The experiments showing that in laminin β mutant embryos the laminin α3,5 and laminin α1,2 chains are not present at BMs or muscle attachment sites fully support this model (Urbano, 2009).
The network of collagen IV was thought to provide an initial scaffold that incorporates other BM components, including laminins, nidogens and perlecan. However, analysis of collagen IV mutants in mice and C. elegans has shown that collagen IV was in fact dispensable for deposition and initial assembly of BMs. Similarly, in Drosophila embryos lacking collagen IV or SPARC, a collagen IV interacting protein, the association of laminin and perlecan with cell surfaces was not affected until late embryogenesis (Martinek, 2008). However, when examined the other way around, genetic and developmental studies in the mouse and in C. elegans have demonstrated that laminins are essential for BM assembly in these two species. This study has shown, at both microscopic and ultrastructural levels, that this is also the case in Drosophila. Thus, the crucial role of laminins as a scaffold for recruitment of BM components is conserved throughout animal evolution (Urbano, 2009).
In contrast to BMs, this study found that laminins are not required for assembly of ECM components at the specialised matrix present at muscle attachment sites. This could be explained by the fact that assembly of this matrix clearly differs from assembly of BMs, as it is mainly mediated by cell-cell interactions. An alternative explanation might be that transmembrane receptors, such as integrins, could promote association of the tendon matrix to the cell surface independently of laminin networks. The findings that integrins become localised to muscle attachment sites in the absence of laminins and integrins are essential for lamininW recruitment (Devenport, 2007) support this idea (Urbano, 2009).
In mouse, laminin β1 is required for embryo implantation and gastrulation. Lamb1-/- embryos lack BMs and do not survive beyond embryonic day 5.5 (Miner, 2004). Similarly, in sea urchin embryos, injections with antibodies to laminin α chain inhibit gastrulation and spicule formation (Benson, 1999). However, RNAi inhibition of either β, γ or both α laminin genes in C. elegans did not impair embryonic development before the elongation stage (Kao, 2006), at which stage mutant embryos stopped developing and displayed severe defects in BM integrity and tissue development. These different requirements for laminins during early stages of embryonic development could be explained by considering that in sea urchins and amniotes the basal matrix forms shortly before gastrulation, whereas in nematodes, as well as in insects and amphibians, a basal lamina develops only at the end of gastrulation (Stern, 2004). An alternative explanation could be that laminins are required for epithelialisation and while in mammals this process precedes gastrulation, in nematodes, gastrulation precedes epithelialisation. In Drosophila mutations in either of the two laminin trimers result in late embryonic lethality. However, the role of laminins during gastrulation in Drosophila has remained an open question, as each trimer could compensate for the absence of the other. This study shows that removal of all laminins in Drosophila does not affect gastrulation, demonstrating that in Drosophila, and contrary to the mouse (Miner, 2004), the integrity of BMs is not crucial for this developmental event. Taken altogether, it is concluded that, although the late functions during organogenesis are well conserved, an early function for laminins is absent in Drosophila (Urbano, 2009).
During midgut morphogenesis, both the migration of endodermal cells and the subsequent transition to a polarised epithelium depend on the association of the endoderm with the visceral mesoderm. Laminins are deposited between these two cell layers, yet the overall morphogenesis of the midgut, including migration, midgut constriction, tube elongation and adhesion of the endoderm to the visceral mesoderm, was shown to occur normally in mutants for the Drosophila laminin α3,5 chain (Yarnitzky, 1995). This led to the proposal that these processes might be mediated by other substrates. This study has shown that this other substrate is the second laminin, since all these processes are affected in LanB1 mutant embryos. Whether this represents a unique function for the lamininW or requires both laminins awaits the analysis of these processes in embryos lacking just laminin α1,2 (Urbano, 2009).
Experiments in different model systems, such as the chick, axolotl and mouse, have demonstrated a role for laminins in the migration of different cell populations (Tzu, 2008). One of the best-studied processes is the migration of the neural crest, which appears to migrate in response to heterogeneity in the ECM that forms their migration substrate. Collectively, these studies propose that neural crest migration may be governed by the relative ratio of permissive ECM components, such as fibronectin and laminin, versus non-permissive ECM components, such as chondroitin sulphate proteoglycans. Other ECM molecules, such as vitronectin, perlecan and several collagen types, seem to play a neutral role in this process (Henderson, 1997; Perris, 2000). In Drosophila, despite the implication of integrins in most migratory processes during embryogenesis, a clear role for laminins in regulating cell migration has remained elusive. To date, only mutations in the laminin α1,2 were shown to result in gaps in the tracheal dorsal trunk (Martin, 1999). This study shows that laminins are required for all integrin-dependent migrations described so far, including that of the endoderm, macrophages, salivary glands and trachea visceral branches. These results identify the laminins as being either the key integrin ligands regulating cell migration during Drosophila embryogenesis, or essential for recruitment of key ligands into the migration substrate. In this new scenario, several questions now arise. What cells provide the laminins? Do other Drosophila ECM molecules, such as Collagen IV or Perlecan, permit or inhibit cell migration? Are other laminin receptors beside integrins involved in cell migration in Drosophila? This study shows that in the absence of laminins, other BM components, such as Collagen IV and Perlecan, are not deposited around the VNC, and macrophages no longer migrate along this path. Inhibition of haemocyte migration impairs ECM deposition around the VNC (Olofsson, 2005). Thus, as macrophages need matrix components to migrate and BM deposition around the VNC requires macrophage migration, it is tempting to speculate that macrophages might be able to deposit their own matrix molecules for migration, making them independent of the matrix of the environment. For example, human keratinocytes deposit laminin 332 to promote their linear migration (Urbano, 2009 and references therein).
Laminins can interact with different types of receptors, including integrins, α-dystroglycan, sulphated carbohydrates (sulphatides, heparin, heparan sulphates and HNK-1) and the Lutheran antigen (Yurchenco, 2004). Several of the defects seen in LanB1 mutants are remarkably similar to those reported for loss of integrin function, including defects in: adhesion between the wing surfaces; macrophage and tracheal cell migration; proventriculus morphogenesis, elongation and formation of constrictions during gut development; and adhesion between visceral mesoderm and endoderm (Bradley, 2003
The current results are consistent with the idea that in Drosophila, as in mouse, laminins play a central role in organising the specialised ECM present at BMs and that this may represent a first important step for BM formation (Li, 2002). The establishment of a laminin-based BM scaffold is then crucial for many different cellular processes governing morphogenesis of most organs and tissues. Future studies are needed to address how laminins can perform such different developmental functions, including strong adhesion between different layers but also weak adhesion to allow cell migration. The information derived from these studies should help to understand the pathology of diseases related to abnormal laminin functions (Urbano, 2009).
Search PubMed for articles about Drosophila Laminin
Aumailley M., et al. (2005). A simplified laminin nomenclature. Matrix Biol. 24: 326-332. PubMed ID: 15979864
Benson S., et al. (1999). Developmental characterization of the gene for laminin alpha-chain in sea urchin embryos. Mech. Dev. 81: 37-49. PubMed ID: 10330483
Bradley, P. L., Myat, M. M., Comeaux, C. A. and Andrew, D. J. (2003). Posterior migration of the salivary gland requires an intact visceral mesoderm and integrin function. Dev. Biol. 257: 249-262. PubMed ID: 12729556
Chanana, B., et al. (2007). AlphaPS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin. Mech. Dev. 124: 463-475. PubMed ID: 17482800
Devenport D., et al. (2007). Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly. Dev. Biol. 308: 294-308. PubMed ID: 17618618
Goto, A., Aoki, M., Ichihara, S. and Kitagawa Y. (2001). alpha-, beta- or gamma-chain-specific RNA interference of laminin assembly in Drosophila Kc167 cells. Biochem. J. 360: 167-172. PubMed ID: 11696004
Graner M. W., et al. (1998). Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2. J. Biol. Chem. 273: 18235-18241. PubMed ID: 9660786
Henderson D. J. and Copp A. J. (1997). Role of the extracellular matrix in neural crest cell migration. J. Anat. 191: 507-515. PubMed ID: 9449070
Kao G., et al. (2006). The role of the laminin beta subunit in laminin heterotrimer assembly and basement membrane function and development in C. elegans. Dev. Biol. 290: 211-219. PubMed ID: 16376872
Kumagai C., Kadowaki T. and Kitagawa Y. (1997). Disulfide-bonding between Drosophila laminin beta and gamma chains is essential for alpha chain to form alpha betagamma trimer. FEBS Lett. 412: 211-216. PubMed ID: 9257722
Li, S., et al. (2002). Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation. J. Cell Biol. 157: 1279-1290. PubMed ID: 12082085
Martin D., et al. (1999). wing blister, a new Drosophila Laminin a chain required for cell adhesion and migration during embryonic and imaginal development. J. Cell Biol. 145: 191-201. PubMed ID: 10189378
Martinek, N., et al. (2008). Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos. J. Cell Sci. 121: 1671-1680. PubMed ID: 18445681
Martin-Bermudo, M. D., Alvarez-Garcia I. and Brown N. H. (1999). Migration of the Drosophila primordial midgut cells requires coordination of diverse PS integrin functions. Development 126: 5161-5169. PubMed ID: 10529432
Miner J. H., et al. (2004). Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation. Development 131: 2247-2256. PubMed ID: 15102706
Morita, A., Sugimoto, E. and Kitagawa, Y. (1985). Post-translational assembly and glycosylation of laminin subunits in parietal endoderm-like F9 cells. Biochem. J. 229: 259-264. PubMed ID: 4038260
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
Pankratz, M. J. and Hoch M. (1995). Control by epithelial morphogenesis by cell signaling and integrin molecules in the Drosophila foregut. Development 121: 1885-1898. PubMed ID: 7601002
Perris, R. and Perissinotto, D.(2000). Role of the extracellular matrix during neural crest cell migration. Mech. Dev. 95: 3-21. PubMed ID: 10906446
Peters, B. P., et al. (1985). The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells. J. Biol. Chem. 260: 14732-14742. PubMed ID: 3840485
Stern, C. D. (2004). Gastrulation: From Cells to Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Stark, K. A., et al. (1997). A novel a integrin subunit associates with bPS and functions in tissue morphogenesis and movement during Drosophila development. Development 124: 4583-4594. PubMed ID: 9409675
Subramanian A., Wayburn, B., Bunch, T. and Volk, T. (2007). Thrombospondin-mediated adhesion is essential for the formation of the myotendinous junction in Drosophila. Development 134: 1269-1278. PubMed ID: 17314133
Tzu, J. and Marinkovich. M. P. (2008). Bridging structure with function: structural, regulatory, and developmental role of laminins. Int. J. Biochem. Cell Biol. 40: 199-214. PubMed ID: 17855154
Urbano, J. M., et al. (2009). Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis. Development 136(24): 4165-76. PubMed ID: 19906841
Yarnitzky, T. and Volk T. (1995). Laminin is required for heart, somatic muscles, and gut development in the Drosophila embryo. Dev. Biol. 169: 609-618. PubMed ID: 7781902
Yurchenco, P. D., Amenta, P. S. and Patton, B. L. (2004). Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol. 22: 521-538. PubMed ID: 14996432
date revised: 30 April 2010
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