terribly reduced optic lobes
Unlike the mouse counterpart, domain V from Drosophila perlecan is not modified by glycosaminoglycans and endogenous proteolysis, due to the absence of the link region. It shows moderate affinities for heparin and sulfatides but does not bind to chick alpha-dystroglycan or to various mammalian basement membrane proteins. A single RGD sequence in LG3 of Drosophila domain V is incapable of mediating cell adhesion. Production of a proteoglycan form of perlecan (approximately 450 kDa) in one Drosophila cell line could be demonstrated by immunoblotting with antibodies against Drosophila domain V (Friedrich, 2000).
Mouse perlecan domain V was previously shown to be a cell-adhesive substrate and to bind to the alpha-dystroglycan receptor, several extracellular matrix proteins, and sulfatides, and weakly to heparin. A weak affinity for heparin was also found for Drosophila domain V, which could be displaced from a heparin affinity column with 0.15 m NaCl. Domain V of mouse and fly proteins also shows a comparable binding to immobilized sulfatides. However, no binding was observed between Drosophila domain V and chick alpha-dystroglycan, in contrast with the strong binding of mouse domain V. Drosophila domain V also showed no interaction in solid-phase assays with nidogen-1, laminin-1-nidogen complex, fibulin-1, fibulin-2, fibronectin and BM-40 of either mouse or human origin (Friedrich, 2000).
Mouse perlecan and its domain V, but not Drosophila domain V, are adhesive substrates for rat glioma Rugli cells. The same pattern was also observed with rat Schwannoma RN22 cells, which are known to bind to the RGD sequences of vitronectin. In solid-phase assays with immobilized human v3 integrin, fibronectin and vitronectin bind strongly, whereas no interaction was observed with Drosophila domain V (Friedrich, 2000).
The mPerlecans function as coreceptors for FGF signaling. Since identification of members of a signaling pathway by detection of dominant enhancement of a sensitized mutant background is well established, mutations in the FGF pathway were tested in combination with trol to determine whether trol also functions in the FGF pathway in Drosophila. Genetic interaction studies were done with strong alleles of branchless (bnlPI, bnl06916), breathless (btlLG19), and heartless (htlAB42) and a weak trol allele, trolb22. trolb22 mutants have barely sufficient trol activity to produce normal neuroblast division, and any further decrease in trol function results in a neuroblast proliferation phenotype (Park, 1998, 2001). btl and htl encode FGF receptor homologs, with btl encoding the receptor for Bnl. The ligand for htl is currently unknown. bnl06916 or btlLG19 but not htlAB42 dominantly enhance the trol phenotypes in trolb22 mutant animals as shown by the production of a neuroblast proliferation phenotype. As expected, all larvae mutant for trolb22 alone have normal neuroblast proliferation. In contrast, 68% of trolb22;bnl06916/+ samples and 89% of trolb22;btlLG19/+ samples have dramatically fewer S-phase neuroblasts. All of trolb22; htlAB42/+ samples, however, show normal neuroblast proliferation as do all control bnl06916/+, btlLG19/+ and htlAB42/+ samples (Park, 2003).
Dominant enhancement of the neuroblast proliferation phenotype of a second independent trol allele, trol4, was also observed upon heterozygosity by bnl06916 or I>btlLG19 but not htlAB42 (Park, 2003).
The ability of human FGF-2 to rescue the neuroblast phenotype of a trol mutant, trol8, was investigated by using explant cultures. trol8 has a neuroblast proliferation defect in vivo in approximately 23% of samples tested and normal neuroblast proliferation in the remaining 77% (Park, 2001). In culture, trol8 brains had normal proliferation in 8% of the samples, while CS controls had normal proliferation in 24% of the samples. When cultured in the presence of 10 ng/ml FGF-2, however, 28% of trol8 brains now show normal proliferation compared with 26% of CS brains also cultured with FGF-2 (Park, 2003).
The decreased number of S-phase cells when Bnl signaling was compromised could have been due to fewer neuroblasts being determined during embryogenesis. To eliminate this possibility, activation of the downstream effector MAPK was blocked in first instar wildtype animals and neuroblast proliferation was assayed. The MAPK inhibitor PD98059 blocks MAPK activation in Aplysia, mammals, and insects. To show PD98059 functions in Drosophila, third instar eye discs were cultured in 10 microM PD98059 and assayed for lack of MAPK activity by immunohistochemistry with the doubly phosphorylated-MAPK antibody. After a 2-h incubation, none of the discs cultured with PD98059 stained with the doubly phosphorylated-MAPK antibody, while control discs showed the expected pattern. When larval brains were dissected from wildtype CS larvae at 10 h posthatching and incubated without addition of PD98059, 27% of the samples had normal numbers of S-phase neuroblasts 16-20 h posthatching. In contrast, if CS brains were cultured in 10 microM PD98059 from 10-20 h posthatching, only 3% of the samples had normal numbers of S-phase neuroblasts at 16-20 h posthatching. These data show that blocking MAPK activation at first instar also blocks activation of cell division in arrested neuroblasts (Park, 2003).
To test whether FGF-2 binds to the Trol protein, coimmunoprecipitation studies with Trol and FGF-2 were performed. Third instar extracts were size-fractionated to produce a ~100-kDa aliquot enriched in Trol. Addition of human 125I-FGF-2 to the extract and immunoprecipitation of the resulting complex by an anti-mPerlecan domain IV antibody have revealed a radioactively labeled band that migrates at approximately 20 kDa, the same position as purified FGF-2. The addition of heparin sulfate to the extract plus 125I-FGF-2 greatly reduces the copurification of the 20-kDa band upon precipitation with the anti-mPerlecan antibody. Little FGF-2 is isolated when the extract plus 125I-FGF-2 is probed with an unrelated mouse antibody (Park, 2003).
The effect of Hedgehog signaling on initiation of neuroblast proliferation was investigated by using hhts2 and hs-hh lines. When larvae were reared at the mildly restrictive temperature of 25°C, all hhts2 animals had fewer dividing neuroblastscompared with sibling controls. In contrast, 77% of homozygous hs-hh larvae raised at 25°C contained excess dividing neuroblasts. On average, the hs-hh samples with extra dividing neuroblasts contained 62% more S-phase stem cells compared with controls (Park, 2003).
Coimmunoprecipitation studies were used to determine whether interactions could be detected between Hedgehog proteins and Trol. Third instar larvae containing a hs-hh construct were placed at 37°C for 30 min to induce increased levels of Hh protein, and protein extracts were prepared. The extracts were size fractionated for ~100-kDa particles and precipitated with an antibody raised against mPerlecan domain IV, a nonspecific antibody, or no antibody at all. Western analysis of the purified complexes using an anti-Hh antibody revealed a band in the sample isolated using the anti-mPerlecan antibody. A much fainter band was observed in the lane prepared without antibody addition, and no band of the expected size was detected in the sample purified using a nonspecific antibody. Addition of heparin sulfate to the extract failed to reduce the intensity of the Hh-immunoreactive band (Park, 2003).
Studies were also carried out to identify possible interactions between mPerlecan and mammalian Hh proteins. Conditioned medium from highly confluent cultures of different primary murine cerebrovascular endothelial (CVE) cell lines derived from different strains of mice was analyzed. Protein complexes from two cell lines were precipitated with the anti-mPerlecan domain IV antibody or a nonspecific antibody, and the resulting samples were analyzed by Western blotting. As observed for Trol, lanes containing samples isolated with the anti-mPerlecan antibody reveal a band of 32 kDa when probed with an anti-Shh antibody; the band is not decreased in the presence of added heparin. Much less signal is observed in lanes containing complexes isolated with a nonspecific antibody (Park, 2003).
Mutations in genes required for Hh signaling also interact with trol alleles. To examine the possibility that trol modulates Hh-mediated neuroblast activation, the extent of stem cell division was examined in trolb22;hh2/+ animals. All trolb22, hh2/+ samples showed decreased levels of neuroblast division compared with normal numbers of S-phase neuroblasts in all control trolb22 and hh2/+ samples. An increase in the severity of the neuroblast proliferation phenotype in trol mutants heterozygous for hh2 was also observed for the independent trol8 allele. Similar results were obtained in trolb22 and trol8 mutants with the protein null allele hhAC. As in the hh2 experiments, 100% of the trolb22; hhAC/+ animals had decreased neuroblast division compared with control hhAC/+ samples. In trol8 mutants, 18% of the samples show a proliferation defect. In contrast, 81% of trol8; hhAC/+ animals had decreased numbers of S-phase neuroblasts compared with controls (Park, 2003).
To verify that decreasing Hh signaling caused decreased activation of neuroblast division in larval stages rather than production of fewer neuroblasts in embryogenesis, a temperature-sensitive hh allele, hhts2, was used to lower Hh activity only in trolb22 first instar larvae. Egg lays and all of embryogenesis were carried out at the permissive temperature of 18°C, and newly hatched larvae were collected in 1-h windows and then moved to 25°C. All of the trolb22; hhts2/+ samples showed decreased neuroblast proliferation, while all trolb22/+; hhts2/+ siblings had normal cell division (Park, 2003).
Tout-velu (Ttv), an acetylglucosaminyltransferase that synthesizes heparan sulfate chains, is required for the reception of the Hh signal in cells of the wing imaginal disc. The neuroblast phenotype of trolb22;ttv00681/+ animals was examined. Every trolb22;ttv00681/+ sample showed supernumerary S-phase neuroblasts, averaging 90% more neuroblasts/brain lobe. In contrast, both trolb22 and ttv00681/+ control samples showed normal neuroblast proliferation. The effect of one copy of ttv in a trol4 mutant background was also examined. In trol4 mutant animals, excess S-phase neuroblasts are never observed; however, in 29% of trol4;ttv00681/+ samples, supernumerary S-phase neuroblasts were detected averaging 21% more S-phase neuroblasts/brain lobe. In addition, 41% of trol4 mutant animals have decreased neuroblast proliferation, while only 5% of trol4,ttv00681/+ animals show a defective proliferation phenotype (Park, 2003).
Hh signaling results in expression of its target gene ptc, and expression of ptc as followed by a ptc-LacZ transgene has been used as a reporter of Hh signaling. Expression of ptc-lacZ was examined in first instar larval brains from trolb22; ttv00681/+, trolb22, and ttv00681/+ animals to determine whether Hh signaling extends further or to more cells in trolb22; ttv00681/+ animals compared with controls. In all trolb22; ttv00681/+ samples examined, stronger lacZ expression as followed by ß-galactosidase activity staining was observed in the posterior lateral portion of the brain lobes than in trolb22 or ttv00681/+ controls (Park, 2003).
Genetic interaction studies with trolb22 and a second hh allele, hh21, also reveal an overproliferation phenotype. One-hundred percent of trolb22; hh21/+ samples contained excess S-phase neuroblasts, with an average of 94% more BrdU-labeled neuroblasts/brain lobe than in sibling trolb22 controls. The overproliferation phenotype was also detected in 18% of animals that are hh21/+. The excess BrdU-labeled neuroblasts are likely to be due to a mutant hh21 protein derived from the hh21 allele as demonstrated by the results from analysis of heterozygosity with hh2 and the protein null allele hhAC (Park, 2003).
Molecular analysis of hh21 revealed a mutant Hh precursor and a processed amino-terminal protein species larger than wildtype. RT-PCR of RNA from hh21/+ and control third instar wing discs using primers for the 3' two-thirds of the hh message produced the expected wildtype band of 730 bp and a larger band approximately 850-900 bp in size in the hh21/+ sample. Use of additional primer pairs demonstrated that the inserted sequence is either 5' or immediately 3' to the DNA encoding the autocleavage site that separates the amino-terminal signaling portion of the Hh protein from the carboxy-terminal autoproteolytic portion of the Hh precursor (Park, 2003).
The hh21/+ genomic DNA revealed no inserted sequence. Use of an anti-HhN antibody against the amino terminal fragment of the Hh protein on a Western blot of extract from stage 9-early stage 10 hh21/+ and wild-type embryos detected a band of the expected size for the wildtype Hh cleavage product with the signal peptide attached in the hh21/+ and control wild-type lanes and larger bands consistent with a mutant aminoterminal hh21 cleavage product with and without signal peptide in the hh21/+ lanes alone (Park, 2003).
bnl06916 and hhP30 lacZ reporter lines were used to assay patterns of bnl and hh mRNA in first instar larval lobes. In larvae at 4 h post-hatch (ph) when the developmentally regulated neuroblasts are still mitotically quiescent, neither bnl- nor hh-driven ß-galactosidase activity is detectable. In contrast, at 16-20 h ph when regulated neuroblasts are beginning to synthesize DNA, both bnl- and hh-driven lacZ expression are present. ß-Galactosidase activity stains reveal low levels of bnl expression in the medial anterior portion of the brain lobe, while hh is more robustly present in the lateral posterior portion of the brain lobe (Park, 2003).
Dystroglycan (Dg) is a widely expressed extracellular matrix (ECM) receptor required for muscle viability, synaptogenesis, basement membrane formation and epithelial development. As an integral component of the Dystrophin-associated glycoprotein complex, Dg plays a central role in linking the ECM and the cytoskeleton. Disruption of this linkage in skeletal muscle leads to various types of muscular dystrophies. In epithelial cells, reduced expression of Dg is associated with increased invasiveness of cancer cells. Dg is required for epithelial cell polarity in Drosophila, but the mechanisms of this polarizing activity and upstream/downstream components are largely unknown. Using the Drosophila follicle-cell epithelium (FCE) as a model system, this study shows that the ECM molecule Perlecan [Pcan; encoded by terribly reduced optical lobes (trol)] is required for maintenance of epithelial-cell polarity. Follicle cells that lack Pcan develop polarity defects similar to those of Dg mutant cells. Furthermore, Dg depends on Pcan but not on Laminin A for its localization in the basal-cell membrane, and the two proteins bind in vitro. Interestingly, the Dg form that interacts with Pcan in the FCE lacks the mucin-like domain, which is thought to be essential for Dg ligand binding activity. Finally, two examples are described of how Dg promotes the differentiation of the basal membrane domain: (1) by recruiting/anchoring the cytoplasmic protein Dystrophin; and (2) by excluding the transmembrane protein Neurexin. It is suggested that the interaction of Pcan and Dg at the basal side of the epithelium promotes basal membrane differentiation and is required for maintenance of cell polarity in the FCE (Schneider, 2006).
In vertebrates, Dg is synthesized as a single polypeptide and post-translationally cleaved into the extracellular glycoprotein αDg and the transmembrane protein ßDg. The two subunits are believed to remain attached to one another through non-covalent interaction of the C-terminal region of αDg with the N-terminal region of ßDg (Sciandra, 2001). αDg shows a dumbbell-like molecular shape in which two less glycosylated globular domains are separated by the mucin-like domain (mucin-domain), a highly glycosylated serine-threonine-proline-rich region (Brancaccio, 1995). Laminin (Lam), Agrin, Perlecan (Pcan) and Neurexin (Nrx) serve as ligands for αDg, and Lam G (LG)-like domains mediate the interaction. The binding site on αDg is not known, but proper glycosylation of αDg is generally considered to be crucial for its ligand-binding activity. Recent studies have demonstrated that Oglycosylation within the mucin-domain is required for Lam (Kanagawa, 2004) and Pcan binding (Kanagawa, 2005), but it is not clear whether the sugar-chains of this domain are directly involved in the interaction or merely play a structural role in supporting the rod-like shape of this region (Schneider, 2006).
The cytoplasmic tail of ßDg interacts with Dystrophin (Dys) in muscle cells, and the Dys-homolog Utrophin (Utr) in epithelial cells. Dys/Utr in turn connect to actin filaments of the cytoskeleton. Dg therefore occupies a central position in an ECM-cytoskeleton link disruption of which leads to various types of muscular dystrophies (Cohn, 2000). In addition, Dg has been suggested to play a key role in the transduction and modulation of various signaling cascades (Schneider, 2006).
In epithelial cells, reduced expression of Dg has been associated with increased invasiveness of cancer cells (Muschler, 2002). In some malignant tumors, e.g. prostate and mammary cancer, the expression of αDg is reduced (Henry, 2001a; Muschler, 2002). Furthermore, the amount of reduction is correlated with the invasiveness of the tumor (Muschler, 2002). Recent results (Sgambato, 2005; Sgambato, 2003) suggest that the loss of αDg might be an early event in carcinogenesis rather than being a consequence of neoplastic transformation (Schneider, 2006).
Some reports have suggested that the major ligand for Dg in non-muscle cells might be Pcan, because the binding of αDg to Pcan LG-domains is five times stronger than that to the most active Lam fragment (Andac, 1999; Talts, 1999). Pcan is the major heparan sulfate proteoglycan in basement membranes (BMs) and connective tissue, and has been implicated in adhesion, proliferation, development and growth-factor binding. The Pcan core protein consists of five domains and binds to a variety of molecules, including FGF-7, Fibronectin, Heparin, Laminin 1, PDGF-B, αDg and Integrins. At the N-terminal domain I and the C-terminal domain V, glucosaminoglycan (GAG) chains are attached that interact with Laminin-1 and Collagen IV and bind to FGF-2, promoting its angiogenic and mitotic activities. Studies in transgenic mice have shown that Pcan is required for the maintenance of the functional and structural integrity of BMs in the heart, but is not needed for BM assembly per se (Schneider, 2006 and references therein).
Not much is known about the function of the interaction between Pcan and Dg. During the development of the neuromuscular junction, binding between Pcan and Dg is required for clustering of acetylcholine esterase at the postsynaptic membrane (Peng, 1999). In addition, cell culture studies with Pcan- and Laminin α2-deficient skin fibroblasts (Herzog, 2004) revealed that shedding of Dg is increased by the lack of Pcan, but not by lack of Laminin α2 (Schneider, 2006 and references therein).
Pcan, Dg and other components of the Dystrophin-glycoprotein complex are conserved in Drosophila and vertebrates. Drosophila Pcan (trol) is required for controlling proliferation of neuronal stem cells in the larval brain (Voigt, 2002). Pcan has been suggested to act in the ECM by binding, storing and sequestering external signals, including FGF and Hedgehog (Voigt, 2002). A role for Pcan in epithelial development has not been reported so far (Schneider, 2006).
Drosophila Dg plays a role in polarizing epithelial cells and the oocyte. In particular, Dg function has been investigated during the development of the follicle-cell epithelium (FCE). The FCE forms through a mesenchymal-epithelial transition and uses mechanisms operating on the apical, lateral and basal side for epithelial differentiation. Contact of follicle cells with the basement membrane and with the germline cells has been suggested to play a role in polarizing the cells. As a result, distinct basal, apical and lateral cell-membrane domains are established by accumulating protein complexes that are actively reinforcing cell-membrane polarity. Loss of Dg leads to an expansion of apical markers to the basal side of the cells and loss of lateral markers. Some Dg mutant cells lose their epithelial appearance, form multiple layers and eventually die (Schneider, 2006).
The finding that Dg is required for epithelial cell polarity is particularly interesting because of its role during the invasive behavior of cancer cells, but little is known about the molecular mechanism behind this polarizing activity. This study investigated the hypothesis that Pcan and Dg constitute a basal polarizing cue required for the differentiation of the basal membrane domain and epithelial cell polarity. The FCE was chosen as a model system for several reasons: (1) all follicle cells are derived from two to three somatic stem cells, making mosaic analysis an excellent tool with which to study gene function in epithelial development; (2) the trol gene is transcribed in follicle cells, and (3) Dg plays a role in follicle-cell polarization (Schneider, 2006).
The phenotypes caused by the loss of Dg or Pcan share many similarities, such loss of cell polarity, formation of multilayers and 'invasion' by mutant follicle cells of the spaces between germ cells. One interesting difference is the behavior of the apical marker Patj, which accumulated at the basal membrane in Dg clones, but was unaffected in trol clones. The reason for this difference is not known, but a possible explanation is that in trol mutant cells, Dg is still present and occasionally even enriched apically (Schneider, 2006).
Patj is a cytoplasmic PDZ domain protein that forms an apical complex with the transmembrane protein Crb. In contrast to Patj, Crb is frequently reduced in trol clones. A similar loss of Crb was observed in embryonic salivary gland after ectopic expression of Dg, suggesting that the apical enrichment of Dg in trol clones might cause the reduction of Crb. Furthermore, the results confirm the existence of a Crb-independent localization and retention mechanism for Patj in the FCE (Schneider, 2006).
Another difference between trol and Dg clones lies is the ability of the cells to survive. Whereas Dg clones eventually die, trol clones can survive until later stages of oogenesis. Studies of embryoid bodies deficient in Dg revealed an accelerated level of apoptosis, which has led to the proposal that Dg has a role in cell survival (Schneider, 2006).
The overall similarity of the trol- and Dg- phenotypes suggests that the two proteins act in the same 'polarity pathway'. In support of this view is the finding that, in trol clones, Dg is frequently lost from the basal-cell membrane. This effect seems to be specific because: (1) Dg is unaffected by the lack of Lam A, and (2) ßPS remains localized in the basal membranes of trol mutant cells that have lost Dg. Pcan could stabilize Dg at the basal cell surface, either by direct binding or indirectly through interaction with other cell-matrix or cell-surface proteins. Recent findings suggested a trimolecular complex of Pcan, Lam and Dg (Kanagawa, 2005). However, a role for Lam in stabilizing Dg in the FCE is unlikely, because Lam is not required for Dg localization. The findings that Pcan domain V can be co-immunoprecipitated with Dg, supports the view that Pcan stabilizes Dg at least in part by direct binding. These results suggest that direct interaction of the ECM molecule Pcan with the transmembrane protein Dg is required for the maintenance of follicle cell polarity (Schneider, 2006).
In this context, it is interesting that mouse Dg is continuously shed from the cell surface of normal cutaneous cells by proteolytic cleavage of ßDg. Cell culture studies with Pcan- and Lam α2-deficient skin fibroblasts further revealed that shedding of Dg is increased by the lack of Pcan, but not by the lack of Lam α2 (Herzog, 2004). Drosophila Dg appears not to be processed into an α and a ß subunit. The antibody used to detect Dg in trol- cells was directed against the cytoplasmic domain (anti-Dgcyto), so clearly at least the intracellular domain of Dg, and probably the whole protein, is lost from the cell membrane in these cells. One might speculate that the loss of Dg in trol clones represents an elevated turnover of Dg, thereby altering the cell-matrix interaction and activity of Dg in the FCE, as shedding of Dg might do in the vertebrate system. In both systems, Pcan, but not Lam, could function to counteract this mechanism and to stabilize Dg at the cell membrane, but the expression pattern of Pcan and Dg makes clear that other mechanisms of stabilizing Dg expression must exist during early stages of oogenesis, when Pcan is not yet present in the ECM (Schneider, 2006).
Glycosylation of Dg is widely accepted to be essential for its function, and recent results suggest an important role for Oglycosylation in the mucin-domain for binding to Lam (Kanagawa, 2004) and Pcan (Kanagawa, 2005). To date, it is unclear whether the sugar-chains in the mucin-domain are directly involved in the interaction or whether they play a primarily structural function required for proper presentation of the ligand-binding domain. The following findings suggest that, in Drosophila, binding of Pcan and Dg does not require the mucin domain: first, the form of Dg that is expressed at the basal side of the FCE and depends on Pcan for its maintained localization does not contain the mucin-like domain; second, ectopic expression of Dg leads to ectopic accumulation of Lam and Pcan independent of the presence of the mucin domain; and third, one single band of ~120 kDa was detected in embryonic protein extracts in overlay binding assays with PcanV. The size of this band corresponds to the size of the two Dg forms Dg-A and Dg-B, which lack the mucin-domain. These results suggest that the mucin-domain plays a structural role that might not be required in the specific surroundings of the FCE. Another possibility is that presence or absence of the mucin-like domain might regulate binding affinity and/or selectivity (Schneider, 2006).
This study is the first demonstrating a function for a Dg splicing variant lacking the mucin-like domain. It will be interesting to find out whether different Dg forms carry out different functions (Schneider, 2006).
Contact with the ECM is important for polarization of several epithelia, including the vertebrate kidney epithelium and the Drosophila midgut, dorsal vessel and follicular epithelia. In Madin-Darby canine kidney (MDCK) cells, contact with the ECM results in the formation of a basal membrane domain and in long-range effects on the differentiation of the non-basal domain. Similar long-range effects of ECM contact during the establishment of polarity have been observed in the Drosophila FCE (Schneider, 2006).
The current results suggest that, after the initial polarization, ECM-cell contact mediated by Pcan and Dg plays a role in the maintenance of cell polarity. The expansion of Arm and the reduction of the lateral marker Dlg in Dg and trol clones might indicate a long-range effect of Dg on cell polarity. It is generally accepted that Dlg functions by preventing invasion of apical proteins and adherens-junction components into the lateral domain, suggesting that the reduction of Dlg in Dg and trol clones is the cause for the expansion of Arm in these clones. The molecular mechanisms underlying the effect of Dg on Dlg remain unknown, but the results show two clear short-range effects of Dg on the differentiation of the basal membrane domain: (1) the recruitment and/or anchoring of the cytoplasmic protein Dystrophin and (2) the exclusion of the basolateral protein NrxIV (Schneider, 2006).
In vertebrates, the cytoplasmic tail of ßDg binds to Dys in muscle cells and its homolog Utr, in epithelial cells. Dys/Utr, in turn, connects to actin filaments of the cytoskeleton. Mutations in Dys cause a reduction of the expression of Dg in the sarcolemma. In Drosophila, Dg and Dys are interdependent for their localization in the basal membrane of the FCE and in wing imaginal discs, suggesting that the interaction between both proteins is conserved. Provided that Drosophila Dys also interacts with actin filaments, this result could explain the defects in basal actin organization that were observed in Dg clones (Schneider, 2006).
In contrast to Dg clones, an abundant cytoplasmic localization of Dys was observed in trol clones. Further experiments are required to understand the precise molecular mechanisms underlying the observed defects in protein localization (Schneider, 2006).
The results raise the issue of whether Dys is also required for cell polarity. In Dys clones, the polarity marker Baz is clearly reduced, indicating a polarity defect in these cells. The difference to Dg clones in which Baz is not affected, and trol clones, in which Baz expression is elevated, indicates that Dys might play a Dg-independent role in cell polarity and that the subcellular localization of Dys could play a role for its function (Schneider, 2006).
Like Pcan and Lam, Neurexins contain several LG-like modules and have been described as putative interaction partners for Dg in the brain. The results suggest that, in the Drosophila FCE, Dg is required to exclude NrxIV from the basal membrane domain. Whether a direct interaction between Dg and NrxIV is involved in this process remains to be seen (Schneider, 2006).
NrxIV is generally regarded as an integral component of pleated SJ. It was surprising to find that NrxIV is located basally to the region where SJ form, in a position that might correspond to the border between the lateral and basal cell membrane domains. The precise function of NrxIV during SJ development in the follicular epithelium remains to be elucidated (Schneider, 2006).
In the embryo, NrxIV forms a complex with Nrg and Cont, and all three proteins are interdependent for SJ localization. The co-localization of NrxIV, Nrg and Cont in dot-like structures, and the fact that Cont co-localizes with ectopic NrxIV in Dg clones, suggest that molecular interactions between NrxIV, Cont and Nrg also occur in the FCE (Schneider, 2006).
On the basis of the current observations, it is proposed that Pcan and Dg provide a basal 'polarizing cue' required for differentiation of the basal membrane and maintenance of epithelial cell polarity in the FCE. Binding of the ECM molecule Pcan to its receptor Dg stabilizes Dg in the basal membrane. Dg is required for stabilizing Dlg at the lateral membrane, which in turn prevents apical markers and ZA components from invading the basolateral membrane domain. In addition, Dg forms a complex with Dys at the basal membrane and exerts a function in excluding NrxIV from the basal membrane. Further investigations will be required to understand the molecular mechanisms underlying the effect of Dg on Dlg localization and the roles of Dys and NrxIV in this process. Hopefully, a better understanding of the function of Dg in epithelial cell polarity will also shed some light on its role in cancer (Schneider, 2006).
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).
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