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Evolutionarily conserved developmental pathways
SPARC is an evolutionarily conserved collagen-binding extracellular matrix (ECM) glycoprotein whose morphogenetic contribution(s) to embryonic development remain elusive despite decades of research. This study used Drosophila genetics to gain insight into the role of SPARC during embryogenesis. In Drosophila embryos, high levels of SPARC and other basal lamina components (such as network-forming collagen IV, laminin (see Laminin A and Laminin B1) and Trol, the Drosophila perlecan) are synthesized and secreted by haemocytes, and assembled into basal laminae. A SPARC mutant was generated by P-element mutagenesis that is embryonic lethal because of multiple developmental defects. Whereas no differences in collagen IV immunostaining were observed in haemocytes between wild-type and SPARC-mutant embryos, collagen IV was not visible in basal laminae of SPARC-mutant embryos. In addition, the laminin network of SPARC-mutant embryos appeared fragmented and discontinuous by late embryogenesis. Transgenic expression of SPARC protein by haemocytes in SPARC-mutant embryos restored collagen IV and laminin continuity in basal laminae. However, transgenic expression of SPARC by neural cells failed to rescue collagen IV in basal laminae, indicating that the presence of collagen IV deposition requires SPARC expression by haemocytes. A previous finding that haemocyte-derived SPARC protein levels are reduced in collagen-IV-mutant embryos and the observation that collagen-IV-mutant embryos showed a striking phenotypic similarity to SPARC-mutant embryos suggests a mutual dependence between these major basal laminae components during embryogenesis. Patterning defects and impaired condensation of the ventral nerve cord also resulted from the loss SPARC expression prior to haemocyte migration. Hence, SPARC is required for basal lamina maturation and condensation of the ventral nerve cord during Drosophila embryogenesis (Martinek, 2008).
Metazoan radiation gave rise to a complex variety of organisms with distinctive body plans, adaptations and survival strategies. This necessitated the co-evolution of specialized extracellular matrix (ECM) macromolecules capable of forming elaborate matrices that provide tissues with their unique biomechanical, biochemical and functional properties. Among the most ancient ECM molecules are those that comprise the basal lamina, a specialized, cell-surface-associated ECM sheet underlying epithelial and endothelial cells and surrounding muscle, neural and adipose tissues. In addition to serving as adhesive substrata for cell adhesion and migration, basal laminae regulate signal transduction pathways through interactions with cell-surface receptors, such as members of the integrin superfamily. Whereas the molecular complexity of basal laminae varies among tissues, the most broadly distributed components include laminin, collagen IV, perlecan, nidogen and SPARC. Mammalian genomes encode six genetically distinct collagen IV α chains. The major embryonic and most broadly distributed isoform of collagen IV is a heterotrimer composed of two α1(IV) and one α2(IV) chain, designated as α1(IV)2α2(IV). The folding and maturation of collagen IV is dependent on molecular chaperones such as the endoplasmic reticulum (ER)-resident 47-kDa heat shock protein (HSP47). Even though embryonic expression of collagen IV begins in mouse embryos at day 5 post-coitus, mutations in collagen IV do not lead to developmental arrest until embryonic day (E) 10.5-11.5 (Poschl, 2004). Since embryonic lethality is coincident with the onset of muscle contractions, it has been hypothesized that collagen IV is required at this stage of development to provide tensile strength to basal laminae, enabling them to withstand contractile forces associated with embryonic movements (Yurchenco, 2004). However, the underlying cause of lethality is likely to be more complex because dynamic interactions exist between collagen IV and other basal laminae components that affect multiple signaling pathways during embryogenesis (Martinek, 2008).
SPARC is a 32-35 kD Ca2+-binding matricellular glycoprotein whose modular organization is phylogenetically conserved (Martinek, 2002). Biochemical studies indicate that SPARC binds to several collagenous and non-collagenous ECM molecules, including a Ca2+-dependent interaction with network-forming collagen IV. The binding of SPARC to collagen IV might serve to concentrate SPARC in a subset of embryonic basal laminae and basal lamina EHS tumors. However, studies indicate that SPARC is either associated with the plasma membrane or concentrated at the interface between epithelial and basal lamina. Whereas the precise role of SPARC in vertebrate basal lamina assembly and maturation is poorly understood, in vivo studies indicate that the stability of the lens capsule is compromised in SPARC-null mice. The lens capsule (hereafter referred to as a basement membrane) is a continuous thick avascular collagen-IV-rich specialized basal-lamina-like matrix that surrounds the lens. In SPARC-null mice, cataract formation is preceded by disruptions in the ultrastructural organization of capsular collagen IV and laminin networks. Coincident with the altered matrix organization is the presence of filopodia-like cellular extensions in the lens capsule derived from cells that form the lens capsule (Martinek, 2008 and references therein).
SPARC is an integral component of most embryonic laminae in invertebrates. In the nematode Caenorhabditis elegans, SPARC protein is distributed in basal laminae body wall and sex muscles and overlaps with the distribution of collagen IV (Fitzgerald, 1998). The reduction of SPARC protein production by RNA interference results in embryonic and larval lethality. Previously studies have shown that SPARC is a component of embryonic basal laminae in Drosophila (Martinek, 2002). In collagen-IV-α1-mutant embryos, the level of SPARC immunostaining within haemocytes was dramatically decreased and present at very low levels in the basal laminae. This study now reports that inhibition of SPARC expression in Drosophila leads to several developmental anomalies, impaired ventral nerve cord (VNC) condensation and the absence of collagen IV from haemocyte-derived embryonic basal laminae (Martinek, 2008).
SPARC is required for normal embryonic development in Drosophila. In the absence of SPARC, haemocyte-derived collagen IV is not observed in basal laminae during mid- to late embryonic development. The absence of collagen IV leads to discontinuous laminin distribution during late embryonic development, indicative of decreased basal lamina stability. That SPARC selectively affects the presence of collagen IV in basal laminae is further supported by data demonstrating that collagen-IV-mutants have phenotypic similarities to SPARC-mutant embryos (Martinek, 2008).
Studies using vertebrates and invertebrates have shown that laminin is the first basal lamina component to be expressed and secreted during embryonic development. The expression and deposition of laminin along cell surfaces are promoted by its binding to cell-surface receptors such as α1-integrin and β-dystroglycan. In SPARC-mutant embryos, the association of laminin with cell surfaces is unaffected until late embryogenesis, a stage in development when collagen IV and SPARC have been integrated into basal laminae of wild-type embryos. In support of the proposal that the discontinuous laminin network observed in SPARC mutants is because collagen IV is absent from the basal lamina, discontinuous laminin networks are also observed in late-stage collagen-IV-mutant embryos. Laminin networks are likewise disrupted in mouse and C. elegans mutants that lack the expression of collagen IV (see Poschl, 2004). The data indicate that the compromised structural integrity of the laminin network is probably owing to the absence of collagen IV in basal lamina rather than a molecular interaction between SPARC and laminin. However, the presence of a thicker laminin network in lens capsules of SPARC-null mice might reflect a more complex relationship between laminin and SPARC (Martinek, 2008).
Molecular interactions have not been demonstrated between SPARC and perlecan or nidogen, two other universal components of basal laminae. The current data indicate that absence of SPARC does not affect the distribution of perlecan and nidogen in basal laminae during embryogenesis. A potential explanation is that nidogen and perlecan do not form extended crosslinked polymers such as laminin and collagen IV. Hence, they are expected to be less susceptible to distortion by mechanical forces associated with late embryonic development. Another possibility is that, whereas perlecan and nidogen bind to, and bridge with, laminin and collagen IV, their interactions with transmembrane receptors promotes pericellular associations that are independent of laminin and collagen IV networks (Martinek, 2008).
Whereas the current data indicate that SPARC and collagen IV are integral components of the majority of embryonic basal laminae in Drosophila, no SPARC was detected in basal laminae overlying the dorsal vessel and somatic muscles of wild-type embryos, which suggests that molecules other than SPARC promote the deposition of collagen IV molecules in these basal laminae. Interestingly, pericardial cells only express the α2 chain of collagen IV, raising the possibility that the basal lamina overlying the dorsal vessel is composed of collagen IV α2 homotrimers. Adding to the complexity of this basal lamina, Pericardin, a collagen-IV-like ECM molecule is also required for proper dorsal vessel formation (Chartier, 2002). Hence, diverse regulatory factors and mechanisms are likely to control collagen IV deposition and/or stability during development, consistent with cumulative data indicating that the precise molecular composition and function of basal laminae varies between tissues and at different stages of development (Martinek, 2008).
A direct Ca2+-dependent interaction has been demonstrated between collagen IV and the EC domain of SPARC. Phylogenetic analysis reveals a striking evolutionary conservation of amino acids in the EC domain essential for collagen binding in organisms ranging from nematodes to mammals. Site-directed mutagenesis of these conserved amino acids results in a loss of binding between SPARC and collagen triple helices (Maurer, 1995; Mayer, 1991; Martinek, 2002; Martinek, 2007; Pottgiesser, 1994). Since this study has demonstrated that the presence of collagen IV in basal laminae requires SPARC, whether mutations in collagen IV generate a similar phenotype as SPARC mutants was examined to further substantiate their proposed interrelationship (Martinek, 2008).
This study partially characterized alleles of the gene encoding the α1 subunit of collagen IV (DCg1412 and DCgl234) and a deficiency line that lacks both collagen IV genes (Df(2L)sc19-8). Mutant embryos homozygous for collagen IV show reduced protein expression of collagen IV and, similar to SPARC-mutant embryos, are embryonic lethal. As in SPARC-mutant embryos, ventral cuticle holes are observed in these collagen-IV-mutant embryos; however, the holes are smaller in the latter. In both SPARC- and collagen-IV-mutants, tracheal integrity is also compromised. A major function of collagen IV is to provide tensile strength to basal laminae, a biomechanical contribution that increases in importance during late embryogenesis due to an increase in the frequency and strength of muscle contractions. The discontinuous laminin network surrounding the ventral nerve cord and other organs by late embryogenesis in collagen IV and SPARC mutants is probably due to the absence of collagen IV from basal laminae (Martinek, 2008).
A similarity between SPARC-mutant and collagen-IV-mutant embryos during late embryogenesis is the absence of VNC condensation. VNC condensation has been shown by a variety of genetic approaches to be dependent on the deposition of collagen IV in basal laminae and on electrical conductivity (Olofsson, 2005). Hence, failure to undergo VNC condensation in SPARC-mutant embryos is probably because of the absence of collagen IV from basal lamina surrounding the VNC. Whereas the molecular and cellular events regulating VNC condensation are poorly understood, intracellular signaling events are affected by integrins binding to collagen IV during late embryogenesis (Fessler, 1989). These data suggest both a biomechanical and regulatory role for collagen IV that is crucial in VNC condensation. Transgenic expression of SPARC in haemocytes and glia (under the control of gcm-GAL4) as well transgenic expression only in haemocytes (under the control of SrpHemo-GAL4) in a SPARC mutant background, restored the presence of collagen IV in the basal lamina surrounding the VNC, but did not promote its condensation. The combined data indicate that SPARC plays a role in neural patterning that is independent of its contribution to the deposition of collagen IV in basal laminae (Martinek, 2008).
The coexpression of SPARC and collagen IV in haemocytes, combined with the direct demonstrated biochemical interactions (Maurer, 1995: Mayer, 1991: Pottgiesser, 1994), raises the possibility that SPARC and collagen IV form a complex in the ER that promotes the proper folding and secretion of collagen IV. In support of this hypothesis, the presence of collagen IV in basal laminae is restored when haemocyte expression of SPARC is rescued transgenically. Ectopic expression of SPARC by neuroblasts or glia in SPARC-mutant embryos does not induce collagen IV expression by neural and glial cells, nor does it induce the presence of haemocyte-derived collagen IV in basal laminae. Whereas collagen IV and SPARC colocalize in basal laminae of tissues that do not express either protein, their coexpression by haemocytes appears to be required for their proper integration into basal laminae (Martinek, 2008).
The data indicate that inhibition of SPARC expression leads to the absence of collagen IV in the basal laminae during Drosophila embryogenesis, without affecting the secretion and deposition of the other major basal lamina components. The combined data raise the possibility that SPARC functions intracellularly to promote correct folding and secretion of collagen IV and/or its stability in basal laminae during Drosophila embryogenesis. Consistent with a collagen-chaperone-like activity is the recent report that SPARC affects the processing of fibrillar collagen I at the plasma membrane, which could in part account for the distinct collagen phenotype between wild-type and SPARC-null mice (Rentz, 2007). Moreover, it is also possible that collagen IV is not properly assembled extracellularly into a stable network and is therefore rapidly degraded by matrix remodeling proteases. Whereas this possibility cannot be discounted on the basis of the current data, proteases capable of selectively degrading collagen IV during Drosophila embryogenesis have yet to be identified. Moreover, as stated above, the secretion of SPARC by non-haemocyte cells does not rescue the association of collagen IV with basal laminae, which indicates that the formation of a stable collagen IV network is not generated by an extracellular interaction with SPARC. Whereas a potential role for SPARC in regulating the maturation of collagen IV in extracellular membrane compartments cannot be eliminated, the vesicular colocalization of SPARC and collagen IV in haemocytes is indicative of an intracellular functional relationship (Martinek, 2008).
The folding, assembly and processing of collagens from cells via the secretory pathway is dependent on molecular chaperones. Misfolded or incompletely assembled proteins are retained in the ER and are eventually targeted for degradation. In vertebrates, heat shock protein 47 (Hsp47) is a 47 kD collagen-specific protein that binds to and promotes the maturation of collagen molecules (Ishida, 2006: Marutani, 2004: Nagata, 2003). In the absence of Hsp47, both fibril-forming collagen I, and network-forming collagen IV secretion and assembly into matrices are severely compromised, leading to embryonic lethality at ES10.5-ES11.5 in mice (Marutani, 2004). Immunoelectron microscopy shows that collagen IV accumulates within the dilated ER of mutant cells. The accumulation of misfolded or unfolded protein within the ER activates an ER-stress response, in which the expression of molecular chaperones is induced. In Hsp47-null mouse embryos, massive apoptotic cell death occurs just before the death of the embryo at ES10.5. Collagen molecules that bypass the ER-quality control in mouse Hsp47-null fibroblasts and embryonic stem (ES) cells show increased sensitivity to protease degradation, indicative of incorrectly folded procollagen molecules (Marutani, 2004: Matsuoka, 2004). Since an Hsp47 ortholog is not encoded by invertebrate genomes, it is possible that one or more alternative chaperones ensure correct collagen assembly, maturation and secretion (Martinek, 2008).
Studies have indicated that the basal lamina components are highly conserved in metazoans. These data and findings from other laboratories indicate that a functional relationship between SPARC and collagens is also evolutionarily conserved. Analyses of SPARC-null mice demonstrate that SPARC affects the supramolecular assembly of both network and fibrillar collagens (Bradshaw, 2003: Norose, 2000: Sangaletti, 2003). Two months after birth, SPARC-null mice develop early onset cataracts, which suggest of a role for SPARC in lens transparency. Ultrastructural analysis of the lens capsule revealed that cellular extensions from the lens epithelium penetrate and invade the overlying basal lamina, and that the lens capsule contains an altered distribution of collagen IV and laminin (Yan, 2002). Therefore, the early onset cataracts observed in SPARC-null mice probably result from compromised assembly and stability of the lens basal lamina. The data indicate that, in Xenopus, decreased SPARC expression during embryogenesis also leads to the formation of cataracts (Martinek, 2008).
In this study it was observed that early loss of SPARC expression in SPARC-mutant embryos and SPARC knockdown using da-GAL4 prior to haemocyte migration produces a variety of patterning defects within the developing nervous system that cannot be rescued by SPARC expression in haemocytes. Moreover, loss of tracheal, fat-body and ventral-epidermal integrity were observed by the end of embryogenesis together with disorganized neurons and glia. These observations suggest that SPARC has a non-cell-autonomous role in the development of the CNS that impacts on guidance of muscles, neurons, glia and the tracheal system (Martinek, 2008).
The novel neural phenotype observed in SPARC-mutant embryos points to a role for SPARC in CNS patterning that is independent of collagen IV. This is not surprising in light of vertebrate studies that lend strength to the idea that SPARC is a multifunctional glycoprotein with both extracellular and intracellular functions (Martinek, 2008).
Bradshaw, A. D., Puolakkainen, P., Dasgupta, J., Davidson, J. M., Wight, T. N. and Sage, E. H. (2003). SPARC-null mice display abnormalities in the dermis characterized by decreased collagen fibril diameter and reduced tensile strength. J. Invest. Dermatol. 120: 949-955. PubMed Citation: 12787119
Chartier, A., Zaffran, S., Astier, M., Semeriva, M. and Gratecos, D. (2002). Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure. Development 129: 3241-3253. PubMed Citation: 12070098
Fessler, J. H. and Fessler, L. I. (1989). Drosophila extracellular matrix. Annu. Rev. Cell Biol. 5: 309-339. PubMed Citation: 2557060
Fitzgerald, M. C. and Schwarzbauer, J. E. (1998). Importance of the basement membrane protein SPARC for viability and fertility in Caenorhabditis elegans. Curr. Biol. 8: 1285-1288. PubMed Citation: 9822581
Ishida, Y., Kubota, H., Yamamoto, A., Kitamura, A., Bächinger, H. P. and Nagata, K. (2006). Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis. Mol. Biol. Cell 17: 2346-2355. PubMed Citation: 9457905
Martinek, N., Zou, R., Berg, M., Sodek, J. and Ringuette, M. (2002). Evolutionary conservation and association of SPARC with the basal lamina in Drosophila. Dev. Genes Evol. 212: 124-133. PubMed Citation: 11976950
Martinek, N., Shahab, J., Sodek, J. and Ringuette, M. (2007). Is SPARC an evolutionarily conserved collagen chaperone? J. Dent. Res. 86(4): 296-305. PubMed Citation: 17384023
Martinek, N., Shahab, J., Saathoff, M. and Ringuette, M. (2008). Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos. J. Cell Sci. 121: 1671-80. PubMed Citation: 18445681
Marutani, T., Yamamoto, A., Nagai, N., Kubota, H. and Nagata, K. (2004). Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47-knockout mouse embryos via induction of CHOP. J. Cell Sci. 117: 5913-22. PubMed Citation: 15522896
Matsuoka, Y., et al. (2004). Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells. Mol. Biol. Cell. 15(10): 4467-75. PubMed Citation: 15282337
Maurer, P., Hohenadl, C., Hohenester, E., Gohring, W., Timpl, R. and Engel, J. (1995). The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV. J. Mol. Biol. 253: 347-357. PubMed Citation: 7563094
Mayer, U., et al. (1991). Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV. Eur. J. Biochem. 198(1): 141-50. PubMed Citation: 2040276
Nagata, K. (2003). HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development. Semin. Cell Dev. Biol. 14: 275-282. PubMed Citation: 14986857
Norose, K., Lo, W. K., Clark, J. I., Sage, E. H. and Howe, C. C. (2000). Lenses of SPARC-null mice exhibit an abnormal cell surface-basement membrane interface. Exp. Eye Res. 71: 295-307. PubMed Citation: 10973738
Pöschl, E., et al. (2004). Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development. Development 131: 1619-1628. PubMed Citation: 14998921
Pottgiesser, J., Maurer, P., Mayer, U., Nischt, R., Mann, K., Timpl, R., Krieg, T. and Engel, J. (1994). Changes in calcium and collagen IV binding caused by mutations in the EF hand and other domains of extracellular matrix protein BM-40 (SPARC, osteonectin). J. Mol. Biol. 238: 563-574. PubMed Citation: 8176746
Sangaletti, S., Stoppacciaro, A., Guiducci, C., Torrisi, M. R. and Colombo, M. P. (2003). Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma. J. Exp. Med. 198: 1475-1485. PubMed Citation: 14610043
Yan, Q., Clark, J. I., Wight, T. N. and Sage, E. H. (2002). Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice. J. Cell Sci. 115: 2747-2756. PubMed Citation: 12077365
date revised: 10 February 2012
Developmental Pathways conserved in Evolution
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