InteractiveFly: GeneBrief

terribly reduced optic lobes: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - terribly reduced optic lobes

Synonyms - perlecan

Cytological map position - 1A1--3A4

Function - ligand, ECM scaffolding protein

Keywords - brain, regulation of larval neuroblast division, extracellular matrix, FGF pathway

Symbol - trol

FlyBase ID: FBgn0284408

Genetic map position -

Classification - IG domains, concanavalin A-like lectins/glucanases, EGF/Laminin, ligand-binding domain of low-density lipoprotein receptor

Cellular location - secreted

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Crest, J., Diz-Munoz, A., Chen, D. Y., Fletcher, D. A. and Bilder, D. (2017). Organ sculpting by patterned extracellular matrix stiffness. Elife 6. PubMed ID: 28653906
How organ-shaping mechanical imbalances are generated is a central question of morphogenesis, with existing paradigms focusing on asymmetric force generation within cells. This study shows that organs can be sculpted instead by patterning anisotropic resistance within their extracellular matrix (ECM). Using direct biophysical measurements of elongating Drosophila egg chambers, this study documents robust mechanical anisotropy in the ECM-based basement membrane (BM) but not the underlying epithelium. Atomic force microscopy (AFM) on wild-type BM in vivo reveals an A-P symmetric stiffness gradient, which fails to develop in elongation-defective mutants. Genetic manipulation of ECM components Collagen IV, Laminin, and Perlecan showed that the BM is instructive for tissue elongation and the determinant is relative rather than absolute stiffness, creating differential resistance to isotropic tissue expansion. The stiffness gradient requires morphogen-like signaling to regulate BM incorporation, as well as planar-polarized organization to homogenize it circumferentially. These results demonstrate how fine mechanical patterning in the ECM can guide cells to shape an organ.
Ma, M., Cao, X., Dai, J. and Pastor-Pareja, J. C. (2017). Basement membrane manipulation in Drosophila wing discs affects Dpp retention but not growth mechanoregulation. Dev Cell 42(1): 97-106.e104. PubMed ID: 28697337
Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. This study manipulated BM composition to cause dramatic changes in tissue tension. Increased tissue compression when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor beta ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. The results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment.

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF (Branchless) and Hedgehog (Hh) signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system (Park, 2003; Voigt, 2002).

Mutations in trol arrest neuroblasts at G1. The severe partial loss-of-function mutation at trol, trolsd (Datta, 1992) results in a dramatic drop in the number of quiescent neuroblasts that activate cell division at late first instar. Mutations in three alleles result in defective activation of neuroblast division, which can be rescued by expression of cycE, indicating that trol function is likely to be involved in the regulated progression of neuroblasts from mitotic quiescence through G1 to S (Park, 2003).

trol is expressed at all developmental stages, in the larval brain, imaginal discs and fat body, and in the adult gonads. trol message is also detectable in unfertilized eggs, consistent with previous characterization of trol as a maternal effect gene (Garcia-Bellido, 1983). Preliminary results suggest that Trol protein is present either throughout or over the surface of the larval brain at first instar, the latter would be consistent with in situ hybridization showing trol mRNA at the basal surface of the embryonic CNS. This distribution of Trol would allow interaction of Trol with Branchless and Hedgehog either near their sites of expression or near the quiescent neuroblasts, which are located at the cortical surface of the larval brain (Park, 2003).

Trol appears to display functions similar to mammalian Perlecans, which are known to bind FGF-2 and to be required for FGF signaling. Dominant enhancement of the neuroblast proliferation phenotype of two different trol alleles has been observed with mutations in bnl and the Bnl receptor breathless (btl), but not with mutations in the orphan heartless (htl) receptor. The neuroblast proliferation phenotype of trol8 mutant brains was rescued in culture to control levels by addition of human FGF-2. Addition of the MAPK inhibitor PD98059 at 10 hs post-hatching decreased the number of S-phase neuroblasts. Biochemical analysis has shown that FGF-2 can be coimmunoprecipitated with Trol and that the binding of FGF-2 to Trol can be competed by added heparin. This suggests that, like mPerlecan, Trol binds FGF-2 through heparan sulfate residues. These results demonstrate that Trol-mediated FGF signaling is required for initiation of neuroblast proliferation sometime in first larval instar. This similarity to the function of mPerlecans in mammalian FGF signaling and the implications of up-regulation of mPerlecan in tumors strongly imply that trol encodes a functional Drosophila Perlecan homolog (Park, 2003).

Hedgehog has been shown to affect stem cell division of somatic stem cells in the Drosophila ovaries. In addition, blocking Sonic Hedgehog activity decreases the number of neural crest stem cells in the chick, and Sonic hedgehog activity is required for proliferation of precursor cells in avian somites and murine spinal cord. Loss of Hh using a temperature-sensitive mutation results in fewer S-phase neuroblasts at the time these cells begin to proliferate, while production of Hh with an inducible hh gene almost doubles the number of BrdU-labeled neuroblasts. While it is possible that high levels of Hh actually cause apoptosis, which is then repaired by increased neuroblast division, this is found to be unlikely given that induction of the hs-hh transgene rapidly produces excess S-phase neuroblasts. These results indicate that Hh is required to activate stem cell division in quiescent larval neuroblasts and that the level of Hh signaling may limit the number of neuroblasts capable of dividing (Park, 2003).

Genetic interaction studies have demonstrated that the weak lethality of trolb22 is enhanced by mutations in hh or ptc, but not wg. Thus, while trol is required for more than one signaling pathway, there is specificity of trol function. Two independent mutations in hh dominantly enhance the trol proliferation phenotype. Analysis of a temperature-sensitive hh allele shows that, like FGF signaling, Hh signaling is required in first instar for normal activation of neuroblast division. Biochemical studies indicate a potential interaction between Perlecan and Hedgehog proteins in both flies and mammals that is not competed by added heparin. This contrasts with FGF-mPerlecan and FGF-Trol interactions that are competed with heparin (Park, 2003).

Thus, mammalian and Drosophila Perlecan may form complexes with Hedgehog proteins. Future studies will investigate the direct association between Perlecans and Hedgehogs. Genetic analyses and an initial biochemical study suggest that Trol modulates both Hh and FGF signaling, perhaps through formation of a signaling complex (Park, 2003).

Some hh21/+ animals (with normal copies of trol and tout-velu) show extra BrdU-labeled neuroblasts, indicating that the hh21 mutation results in increased Hh signaling. The molecular basis for the hh21 mutation is likely an altered splicing event resulting in an insertion in the portion of the hh mRNA that encodes the signaling activity. Such a mutation could affect the ability of the mutant Hh protein to be processed and released from the cell surface. When placed in a trolb22 background, hh21/+ brains have almost twice as many BrdU-labeled neuroblasts as controls, indicating that a mutation in trol increases the hyperactive phenotype of the hh21 mutation (Park, 2003).

Surprisingly, analysis of trolb22; ttv00681+ brains yielded supernumerary labeled neuroblasts similar to those observed in hs-hh samples. Analysis of expression of the Hh target gene ptc in trolb22; ttv00681/+ brains has revealed a higher level of ptc expression in the portions of the brain expressing hh, when compared with controls. This increase in ptc expression near the source of Hh suggests that the combination of a mutation in trol and decrease of Ttv activity results in higher levels of Hh signaling near hh-expressing cells. A possible model to explain this observation is that when the function of trol is compromised, movement of Hh away from the source of Hh production competes with binding of Hh to nearby responding cells, resulting in stronger signaling nearby and suggesting that trol may function in the diffusion and/or reception of signaling by Hh (Park, 2003).

Taken together with previous studies, these results show that both Drosophila and mPerlecans participate both as structural components of basement membranes and as functional components of the FGF-2 signaling pathway associated with cellular proliferation. In addition, evidence is provided that Perlecans are also involved in signaling by Hedgehog proteins, suggesting an alternative interpretation of perlecan knock-out mouse phenotypes previously attributed to structural defects in extracellular matrix (Costell, 1999). In mPerlecan null mice, chondrocyte division is altered, leading to disorganization of the proliferative zone similar to that seen in the proliferation zones of the optic lobe in third instar trol mutants (Datta, 1992). Altered patterns of chondrocyte proliferation could be due to changes in FGF and Indian hedgehog signaling, correlating with altered neuroblast proliferation in trol mutants with diminished FGF or Hh signaling (Park, 2003).

Perlecan knockout mice have a gap between the proliferative and hypertrophic zones, correlating with the gap between the compound eye and adult optic lobe found in adult trol mosaics (Datta, 1992). These results suggest that Trol/mPerlecan is integral to FGF and Hh signaling and that these signals interface to regulate stem cell proliferation and differentiation. This does not preclude, however, a structural function in assuring the integrity of basement membranes. Given that mPerlecan proteins are in the 450-kDa range with a distinct 'beads on a string' domain structure, it is possible that this versatile protein has multiple developmental roles (Park, 2003).

Several studies on trol and anachronism (ana) have led to the proposal that trol may regulate the reactivation of neuroblast proliferation by suppressing or bypassing the repression by ana, thereby stimulating the G1/S transition through up-regulation of Cyclin E expression (Caldwell, 1998). Other factors required for the activation of mitotically quiescent neuroblasts include the hormone ecdysone and a transacting factor of unknown identity, which is produced in response to the activity of eve (Park, 2001). The homeodomain transcription factor Even-skipped acts in a cell-autonomous manner in areas outside the regulated neuroblasts and is, therefore, likely to control a diffusible signal that impacts neuroblast proliferation in a trol-dependent manner (Park, 2001). In contrast, the activating effect of ecdysone on neuroblast proliferation occurs in a trol-independent manner (Datta, 1999). On the basis of these results, a trol pathway had been proposed in which both ana and eve take part. The finding that trol encodes Perlecan provides a molecular basis toward an understanding of how a trol-dependent pathway could function through the binding and sequestering of proliferative signals (Voigt, 2002).

The study by Voigt (2002) provides slightly different conclusions from that of the Datta lab (Park, 2003) about the function of trol in inducing neuroblast proliferation. A surprising observation was that Perlecan is not expressed in quiescent neuroblasts of the optic lobe area but rather in only a few brain cells outside the area. Although there is no definite proof for the identity of these cells, it appears likely that by analogy to the early expression pattern observed in the embryo, these cells represent a subset of glia cells. Glia cells were shown to produce the secreted ana protein that is necessary to prevent premature optic lobe neuroblast proliferation (Ebens, 1993). These observations suggest that, as in the case of mammalian Perlecan, the Drosophila homolog is able to bind, store, and sequester proliferation-controlling signals that derive from few specialized glial cells to regulate appropriate signal activity to be received and processed by neighboring neuroblasts (Arikawa-Hirasawa, 1999). It is conceivable that Perlecan participates in the control of both activating factors such as those generated in response to eve activity and repressing factors like the secreted ana glycoprotein (Voigt, 2002).

The Perlecan-dependent control of cell proliferation by promoting ligand-receptor interaction for FGF signal, as proposed on the basis of in vitro studies (Aviezer, 1994), would not be consistent with Perlecan function in the fly, because the promotion of any kind of signal-receptor interaction as well as stabilization of the extracellular matrix would require the expression of Perlecan close to or within the target cells. It may, therefore, also not act in a manner analogous to the Drosophila glypicans Dally and Dally-like, shown to bind and stabilize the Wingless signal molecule at the cell surface of target cells. The finding that Perlecan is not expressed in most of its functional target cells, the quiescent optic lobe neuroblasts, is more consistent with the conclusion drawn from the mouse knock-out mutant studies, suggesting that Perlecan binds, stores, and sequesters ligand molecules and thereby modulates the signal activity. This conclusion would also be in agreement with the observation that, in contrast to overexpression of the glypican Dally-like protein, the overexpression of Perlecan has only subtle effects on development and morphogenesis. This result argues further that factors that generate and/or modify heparan sulfate chains of Perlecan, such as the products of the genes sugarless, sulfateless, and fringe connection, are spatially and temporally restricted and are necessary to contribute in a cell-specific manner to proper Perlecan activity. Alternatively, Perlecan expression in glia cells might reflect elevated expression only. It is, thus, possible that low uniform expression might be important, in a cell autonomous manner, for the role of Perlecan in neuroblasts (Voigt, 2002).

The identification of Perlecan as the trol gene product is consistent with a model proposing the ana protein and the unknown eve-dependent factor as putative direct interactors -- these are depleted from the interstitial fluid or become enriched by association with Perlecan. This scenario would suggest that the control that trol exerts on neuronal proliferation is dependent on its timely regulated ability to bind to the growth factors proper, rather than on its place of expression. By analogy to mammalian Perlecan, the Drosophila homolog may also interact with FGF and possibly also other ligand molecules that promote cell proliferation and/or patterning processes during fly development (Voigt, 2002).

Crosstalk between epithelial and mesenchymal tissues in tumorigenesis and imaginal disc development

Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. This study reports a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. Evidence is provided that the secreted proteoglycan Perlecan (Drosophila Trol) can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. This study reports a genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development (Herranz, 2014).

Accumulating evidence indicates that tumor progression results from the interaction between tumor cells and the surrounding normal cells that make up the tumor microenvironment. This study has used the Drosophila wing imaginal disc to dissect the crosstalk between tumor cells and surrounding normal cells, in tumors of epithelial origin. In this model, interaction between the two cell populations is required for tumor growth, neoplastic transformation of the epithelium, and metastasis, even though the genetic modifications were introduced into only one of the two cell populations (Herranz, 2014).

Carcinomas express growth factors involved in the communication between cancer cells and tumor-associated normal cells. The role of TGF-β and Wnt signaling pathways in tumor initiation is well known, but their role as mediators of the interaction between tumor cells and stromal cells has been less well studied. This study observed that EGFR overexpression induced expression of the endogenous Wg and Dpp genes in the epithelial compartment of the tumors. Wg, together with EGFR, is needed in the epithelial cells to drive tumorous growth. The role of the Dpp pathway is different. The findings indicate that Dpp, produced by the epithelial cells, acts on the mesenchymal stromal cells. Dpp signaling activity was not required in the epithelial cells themselves for tumorous growth. Instead, downregulation of the Dpp pathway in mesenchymal cells blocked tumorous growth of the epithelial population. This suggests that Dpp signaling elicits a feedback response from the mesenchymal population. As a consequence, the resulting tumors are composed of a mix of mutant epithelial cells and genetically normal mesenchymal cells, resembling organization observed in human tumors (Herranz, 2014).

EGFR is upregulated in many carcinomas. EGFR is able to promote tissue overgrowth, but additional mutations are required for malignant transformation and invasion. The findings of this study have shown that upregulation of Perlecan is sufficient to cooperate with EGFR to produce neoplastic transformation. Perlecan is a secreted HSPG of the ECM that is overexpressed in many human tumor types. TGF-β ligands have been shown to promote changes in the tumor microenvironment in mammals. Perlecan has also been reported to stabilize Wg and promote Wg activity in Drosophila. Thus, Perlecan production could potentiate the effects of Dpp and Wg produced by the epithelial cells. These findings raise the possibility that Perlecans might have a fundamental role in mediating interactions between epithelial tumor cells and mesenchymal stromal cells, in addition to their known roles in tumor angiogenesis (Herranz, 2014).

The crosstalk between tumor and microenvironment determines the phenotype of the tumor. Signaling from the tumor microenvironment can suppress the malignant tumor phenotype, yet the tumor microenvironment can also promote malignant transformation. The finding that ablation of the mesenchymal cell population reverted the tumor phenotype in this model suggests that signals from the mesenchymal cells are required for tumor progression. This same cell population was required to support growth of the epithelial population in a nontumorous normal tissue context. It is postulated that signals from the adepithelial mesenchymal cells sustain proliferation of the epithelial cells and likewise that signals from the epithelia drive proliferation of the mesenchymal cells. This normal feedback mechanism can be coopted to drive growth of the two tissues, as, for example, when EGFR and Perlecan were overexpressed. A remarkable, unexpected aspect of these findings is that this feedback loop appears to be sufficient to drive the epithelial tissue beyond hyperplasia, through neoplastic transformation, and into metastasis (Herranz, 2014).


Protein Interactions

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).

Genetic interactions between trol and FGF signaling and physical interactions between Trol and FGF

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).

Genetic interactions between trol and Hh signaling and physical interactions between Trol and Hh

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).

Perlecan and Dystroglycan act at the basal side of the Drosophila follicular epithelium to maintain epithelial organization

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).

A functional relationship between SPARC and collagens points to the co-evolution of specialized extracellular matrix macromolecules capable of forming elaborate matrices that provide tissues with their unique biomechanical, biochemical and functional properties

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).

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster

SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. It was previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. This study sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency was found to lead to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell autonomous accumulation of BL components around mutant cells that include Collagen IV (Col IV), Laminin and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. These data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV (Shahab, 2014).

The emergence of multicellular organisms was co-incident with the appearance of genes coding for extracellular matrix (ECM) molecules that gave rise to two major classes of ECMs: interstitial matrices and basal laminae (BL)/basement membranes. In contrast to vertebrate tissues where interstitial matrices predominate, BL are the principal ECMs in animals of lower phyla. Universal components of BLs include network-forming Collagen IV (Col IV), Laminin, Perlecan, and Nidogen, which are assembled into 2D sheet-like networks. In addition to serving as tissue boundaries and an adhesive substratum for cell anchoring and migration, BLs make diverse regulatory contributions to the development of tissues and organs (Hohenester, 2013; Shahab, 2014).

Col IV imparts tensile strength to BL and provides an anchoring substratum for cell adhesion, migration, and secreted signaling molecules. Much of what is known about Col IV is derived from vertebrate studies. Vertebrates express six Col IV α-chains [α1(IV)-α6(IV)] that are assembled in the endoplasmic reticulum into different combinations of heterotrimeric protomers. Upon secretion, the C-terminal globular domain of these trimeric protomers form head-to-head dimers Flexible non-helical interruptions separating collagenous domains of the protomers promote lateral associations during supramolecular assembly of 2D Col IV networks. Further contributing to the stability of these networks, the N-terminal globular domain of the heterotrimers form anti-parallel tetramers. As with fibril-forming collagens, purified Col IV protomers can self-assemble into polymeric networks. In contrast to vertebrates, the Drosophila genome codes for only two Col IV α-chains: Dcg 1/Cg25C and Viking (Vkg). The primary sources of BL components produced within Drosophila embryos and larvae are hemocytes and the fat body (Olofsson, 2005); however, how Col IV and the other BL components are assembled into a stereotypic 2D sheet of precise thickness is unknown (Shahab, 2014).

Previously studies have shown that SPARC (Secreted Protein, Acidic and Rich in Cysteine), a highly conserved matricellular glycoprotein, is a major component of embryonic Drosophila BL (Martinek, 2002; Martinek, 2008). SPARC, also known as osteonectin/ BM40, binds to fibril-forming collagens and Col IV via epitopes located within the C-terminal domain. The absence of interstitial matrices in Drosophila makes it an ideal developmental and genetic model to decipher the role of SPARC in BL assembly and maturation (Shahab, 2014).

Using imprecise P-element excision to generate a mutation/deletion of SPARC in Drosophila, a previous study reported decreased Col IV and BL stability and neural defects resulting in embryonic lethality in the absence of SPARC. However, attempts to rescue embryonic lethality by expressing exogenous SPARC were unsuccessful (Martinek, 2008), raising the possibility that aspects of this phenotype were due to a second site mutation on the 3rd chromosome. The present study, determined that both the neural phenotype and embryonic lethality reported previously, result from a disruption of the neurogenic gene, neutralized. The disruption of SPARC alone leads to larval lethality characterized by compromised fat body homeostasis. The fat body is crucial for development. It acts as the primary source of energy, and fat body together with hemocytes are the principle sources of BL components during larval development. Formed during embryonic development, the larval fat body is a bilateral, multi-lobed organ consisting of a monolayer of about 2,200 polygonal cells called adipocytes. The larval fat body is entirely surrounded by hemolymph, but does not directly interface with it owing to the presence of a BL that covers the entire surface of the fat body. The adipocytes within the fat body have no classical apical-basal polarity. Instead, cell-cell adhesion and shape is mediated by BL surrounding the adipocytes (Pastor-Pareja, 2011). This study reports that a reduction of SPARC leads to defective fat body BL assembly, inducing the resident polygonal adipocytes to round up and accumulate BL components within their microenvironment in a cell-autonomous manner. These findings define a pivotal role for SPARC in the proper assembly of BL surrounding the adipocytes of the Drosophila fat body (Shahab, 2014).

The results of this study demonstrate that loss or knockdown of SPARC expression in Drosophila result in arrest during larval development and disruption of fat body architecture and function. Based upon the SPARC mutation Df(3R)nm136, it was previously reported that loss of SPARC resulted in embryonic lethality associated with severe defects in nervous system development. This study now provide evidence that a second-site mutation present in the neuralized locus, a key regulator of Notch/Delta signalling, is the cause of the Df(3R)nm136 neural phenotype and embryonic lethality. Hence, SPARC is not required for nervous system development (Shahab, 2014).

The new Df(3R)nm136 H2AvD::GFP line, from which the neuralized mutation has been removed, demonstrates that loss of SPARC in Drosophila results in larval lethality and morphological changes of the fat body. The larval fat body is a multifunctional organ essential to fly development. Principle functions of the organ are nutrient storage and regulation of energy availability, functions that may become compromised in SPARC-deficient larvae. SPARC-deficient larvae appear transparent, which is consistent with reduced lipid or energy stores. While it is possible that knockdown of SPARC in hemocytes was responsible for the lethality and fat body morphological defects, knockdown of SPARC selectively within hemocytes using a hemolectin promoter did not result in larval lethality or a fat body phenotype, indicating that the phenotype reported in this study is due to loss of fat body SPARC expression. Moreover, larval lethality and the fat body phenotype of SPARC mutant larvae were rescued by a SPARC transgene that was expressed under the control of either endogenous SPARC or Col IV promoters (Shahab, 2014).

SPARC reduction led to a marked accumulation of BL components in the extracellular microenvironment of affected adipocytes. Temporal expression data from modENCODE indicate that maximum levels of SPARC and Col IV expression occur during the 1st and 2nd instar stages, with expression decreasing during the 3rd larval instar prior to pupariation. Consistent with the idea that SPARC effects are largely mediated prior to the late 3rd instar stage, knockdown of SPARC in 3rd instar had no impact on survival or fat body remodeling (Shahab, 2014).

Pastor-Pareja (2011) showed that knockdown of SPARC results in extracellular assembly of Col IV into thick fibers in the fat body, leading them to speculate that SPARC is required for Col IV secretion and solubility. However, the impact of SPARC knockdown on Col IV secretion, BL integrity, or adipocyte morphology was not addressed in that study. The current study suggests that SPARC deficiency does not prevent Col IV secretion. Consistent with the results of Pastor-Pareja (2011), this study shows extracellular accumulation of Col IV, suggestive of decreased solubility. Moreover, this study shows that Laminin, Perlecan, and Nidogen also accumulate at the surface of SPARC-deficient adipocytes, indicating that all BL components are affected by the loss or knockdown of SPARC (Shahab, 2014).

Biochemical studies have shown that SPARC binds to the triple-helical domains of purified invertebrate and vertebrate Col IV, an interaction that is mediated by two collagen-binding epitopes located in the C-terminal region of SPARC. Col IV is a primary regulator of cell shape and adhesion; thus, alterations in the availability or structure of Col IV fibrils impact cell morphology. Several studies have shown that SPARC has counter-adhesive activity in vitro that causes cells to detach from their substrate and round up. The current data appear paradoxical as loss of SPARC results in cell rounding but does not lead to adipocyte dissociation. However, the impact on cell shape in this instance is likely due to the dysregulation of Col IV polymerization and BL homeostasis, rather than directly to the effect of SPARC on cell-cell or cell-matrix interactions (Shahab, 2014).

A previous studies suggested that SPARC co-localizes with Col IV within secretory vesicles of adipocytes, but it remains to be determined whether SPARC and Col IV directly bind to one another intracellularly. Upon exocytosis, close proximity of SPARC with Col IV enables immediate physical association such that SPARC can regulate Col IV polymerization and sequester Col IV from its cellular receptors. Bradshaw (2009) demonstrated such a relationship between SPARC and Collagen I in mammalian cells. SPARC deficiency does not lead to an increase in intracellular Col IV, demonstrating that the impact of a lack of SPARC on Col IV assembly into BL likely occurs extracellularly. Upon secretion, SPARC may act to maintain solubility of Col IV, preventing it from immediately undergoing polymerization. In the absence of SPARC, Col IV release to the fat body extracellular space occurs; however, without SPARC to delay its polymerization, Col IV may rapidly assemble into a dense meshwork. Other ECM proteins, such as Laminin, Perlecan, and Nidogen, are synthesized and secreted; they encounter polymerized Col IV and are incorporated into the assembled structure as they would in a normal BL. This causes accumulation of multiple BL proteins on the surface of adipocytes. As ECM material accumulates, it promotes the rounding of the cells. The formation of a dense ECM meshwork likely impedes normal adipocyte function and could interfere with a variety of physiological processes such as feeding behavior and energy metabolism (Shahab, 2014).

In light of the diffusible nature of SPARC, the finding of a cell-autonomous phenotype with fat body SPARC knockdown clones was unexpected. The failure of SPARC secreted from adjacent wild-type adipocytes to compensate for the lack of production by SPARC-deficient cell clones indicates that SPARC was not able to diffuse across the BL in sufficient quantities. To date, no study that has addressed the ability of SPARC to diffuse across the BL, but the current data raise the possibility of a charge-dependent barrier that retains SPARC within the microenvironment of a cell. Alternatively, the more immediate interaction of SPARC with Col IV afforded by their intracellular co-localization may be required to effectively prevent premature polymerization of Col IV. Hence, an intracellular interaction between SPARC and Col IV may be required to regulate the kinetics of Col IV polymerization immediately upon its secretion (Shahab, 2014).

SPARC may also regulate BL deposition and remodelling through cell surface receptors. Expression of the cell-matrix adhesion molecules Dg and the βPS integrin subunit was observed on the plasma membrane of wild-type adipocytes. RNAi knockdown of SPARC did not alter the expression or localization of either of these transmembrane receptors in fat body cells indicating that it is unlikely that ECM accumulation around SPARC mutant adipocytes is associated with dysregulation of ECM receptors. However, the possibility that the interaction of BL components with these ECM receptors may have been affected cannot be excluded (Shahab, 2014).

Randomly distributed pits were observed on the surface of adipocytes, which increased in number with the knockdown of SPARC. However, the majority of the pits associated with a SPARC knockdown exhibited thickened circumferential borders underlaid by intracellular lipid-like vesicles. It is conceivable that the pits represent sites of lipid exocytosis. However, preliminary data indicates that the knockdown of SPARC does not affect protein or vesicular endocytosis and exocytosis. Moreover, differences in lipid content between wild-type and SPARC-deficient adipocytes were not observed. Hence the molecular basis of the dramatic difference in the surface topography between wild-type and SPARC-deficient adipocytes remains unknown (Shahab, 2014).

Analysis of the evolutionary history of SPARC revealed a conservation of the collagen-binding epitopes from cnidarians to mammals, which enable SPARC to bind to fibril-forming and network-forming Col IV. While SPARC-null mice develop normally, ultrastructural analysis revealed that interstitial Col IV fibrils are less abundant, smaller and more uniform in size, resulting in fibrils with decreased tensile strength. Biochemical studies indicate that SPARC increases the length of the first stage/lag phase of collagen fibrillogenesis by decreasing the rate of nucleation (Bradshaw, 2009). SPARC is also concentrated in the basal laminae of the nematode C. elegans. RNAi knockdown of SPARC leads to larval lethality for a large percentage of the progeny with a deficiency in gut granules and reduction in body size (Fitzgerald, 1998). It remains to be determined if aberrations in BL lamina assembly is the underlying cause of the phenotype (Shahab, 2014).

Hence, these findings support an emerging concept of SPARC as a critical extracellular collagen chaperone. A detrimental loss of BL homeostasis is evident in the absence of SPARC. The evolutionary conservation of SPARC parallels the advent of BL in multi-cellular organisms, indicating that this chaperone activity of SPARC is important for the maintenance of ECM homeostasis in all metazoans (Shahab, 2014).



A strong expression of the C-terminal domain V of perlecan was found by in situ hybridization and immunohistology at various stages of embryonic development and expression is localized to several basement membrane zones. This indicates, as for mammalian species, a distinct role of perlecan during Drosophila development (Friedrich, 2000).

The spatial expression of Drosophila perlecan was examined using digoxygenin-labelled probes. RNA transcripts are first detected in syncytial blastoderm suggesting maternal expression. During later stages of embryogenesis and until stage 14, only low levels of uniform expression are observed. Only at stage 15 is prominent staining observed in the visceral mesoderm of the gut and in cardiac cells, as well as in the fat body. Similar expression patterns have been found for Drosophila laminin 3/5 chain. No staining is observed in haemocytes, which usually synthesize many proteins of the Drosophila extracellular matrix. At stage 16, staining in visceral mesodermal and cardial cells still persists, although the overall levels of ubiquitous expression increase. During this stage, particularly strong expression is found in cardiac cells, but not on pericardial cells, where the transcripts seems to accumulate more towards the midline. During postembryonic development, transcription of perlecan is also readily detectable in imaginal discs. Particularly strong expression is found in eye and antennal discs, where groups of cells at the morphogenetic furrow, in the presumptive Oc region and in the PalD region show strong staining. Strong staining is also observed in the leg discs in parts of concentric rings (Friedrich, 2000).

To assess the nature and expression of the perlecan protein, the polyclonal antiserum against domain V was used for immunoblotting and on whole mount embryos. Blotting of conditioned medium of Drosophila Kc1 cells shows a single 450 kDa band, while another cell line, Er1, produces a broad smear of lower mobility, suggestive of post-translational modifications. To assess the nature of these modifications, conditioned medium from Er1 cells was subjected to heparinase or heparitinase digestion, followed by immunoblotting. Both treatments change the diffuse band of higher molecular weight into a distinct band of 450 kDa. This indicates that perlecan is substituted with heparan sulfate side chains, although the degree of modification seems not to be as extensive as that for mouse perlecan. A similar broad band could also be detected in SDS extracts of embryonic Drosophila tissue (Friedrich, 2000).

The perlecan protein is first detected in tissues at stage 15 in a quite ubiquitous manner, with particular accumulation around the central nervous system (CNS), the visceral mesoderm and the hindgut. Within these locations, it appears that perlecan accumulates in the basement membrane structures surrounding the tissues. At stage 16, the pattern remains essentially the same, except that on the dorsal side the cardiac cells show an accumulation of perlecan. During stage 16, a particular deposition was also observed in the basement membranes covering the channels of the CNS, on dorsal median cells and on dorsal muscle attachment sites. Since the pattern is reminiscent of the expression of Drosophila laminin, the two patterns were compared and an overlapping localization in the basement membranes surrounding the CNS channel basement membrane and in the heart was found. This suggests that both proteins are integral members of certain basement membrane structures of Drosophila embryos (Friedrich, 2000).

The expression pattern of Drosophila Perlecan during embryogenesis has been analysed with anti-dPCN antibodies directed against domain V of the protein and digoxigenin-labeled antisense RNA probes (Friedrich, 2000). Voigt (2002) monitored the distribution of transcripts by in situ hybridization of whole-mount embryos, using digoxigenin-labeled antisense RNA probes corresponding to the different regions of the 13-kb transcript. The different probes, including probes directed against the 5' most untranslated sequences of trol corresponding to GM02428 cDNA (located close to the insertion sites of the three P-elements) and probes corresponding to all four domains of the open reading frame revealed an identical expression pattern. This finding suggests that the cDNA clones represent different portions of a single, large transcription unit. This conclusion is consistent with the location of the P-element insertions in the 5' region of the trol gene which, together with the 5' untranslated region of transcription units, are the preferred sites for P-element integrations on the X chromosome (Voigt, 2002).

The spatiotemporal in situ expression patterns of the various portions of the trol transcript are identical to those reported by using probes directed against domain V (Friedrich, 2000). The transcripts were strongly expressed during oogenesis. The finding of maternal transcription provides a molecular basis for earlier results showing that the trol15 allele causes maternal-effect embryonic lethality (Robbins, 1990). In addition to the maternal expression, expression of transcripts was noted in a subset of dorsal midline glial cells of the CNS. These cells were found to express Perlecan as detected by anti-Perlecan antibody staining at a later stage of development (Friedrich, 2000; Voigt, 2002).

trol expression was monitored by using a combination of cDNA library screening, in situ hybridization, and RT-PCR. A 2-kb clone representing the 3' end of the trol message was isolated from a 0- to 24-h embryonic cDNA library. RT-PCR studies showed that trol mRNA is expressed in unfertilized eggs and all stages of the life cycle. The trol message is also present in first, second, and third instar brains and the heads, ovaries, and testes of adult flies. In situ hybridization with probes to domain IV- and domain V-encoding regions confirmed trol expression in the developing embryo and imaginal discs. These results correlate with the expression pattern previously observed for the Drosophila perlecan mRNA corresponding to domain V (Friedrich, 2000). In addition, strong staining in the fat body cells is seen adjacent to the salivary glands. These findings have been confirmed by Voigt (2002), who also describe trol expression in small clusters of cells on the medial side of the brain lobes but not the optic lobe region of the late third instar larval brain. It should be noted that the larval brain expression they describe is observed approximately 3 days after the onset of the trol neuroblast proliferation phenotype (Park, 2003).


During larval stages when trol activity is required for the activation of proliferation of the quiescent optic lobe neuroblasts, Perlecan transcripts are expressed in a distinct subpattern of neural cells, which are located outside the optic lobe region. The nature of these cells could not be identified. By analogy to the earlier expression of Perlecan and the transcript in a subset of glial cells of the CNS (Friedrich, 2000), it is anticipated that the Perlecan-expressing cells represent a subset of brain glia. Irrespective of the identity of these cells, however, the remarkable result is that trol-encoded Perlecan is not prominently expressed in the larval optic lobe cells (Voigt, 2002).

Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. This study examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. Two transcriptional regulators were identified that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, the Drosophila heparan sulfate proteoglycan, Trol, was identified as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches (Dragojlovic-Munther, 2013).


Stem cell proliferation is controlled through cell cycle arrest and activation. In the central nervous system of Drosophila, neuroblast quiescence and activation takes place in defined spatial and temporal patterns. Two genes have been identified that regulate the pattern of neuroblast quiescence and proliferation. ana encodes a secreted glial glycoprotein that inhibits premature neuroblast proliferation. trolsd causes a dramatic drop in the number of dividing cells in the larval brain late in development. This study presents evidence that this decrease results from a failure to activate proliferation in the quiescent neuroblast population at the appropriate time. However, trolsd does not affect the maintenance of cell division in already dividing mushroom body neuroblasts. The quiescent optic lobe and thoracic neuroblasts affected by trolsd proliferate in a trol mutant background if they have been activated by a lack of the ana proliferation repressor, demonstrating that trolsd does not affect cellular viability, nor does trol represent a celltype-specific mitotic factor. This also shows that trol acts downstream of ana to activate proliferation of quiescent neuroblasts in an ana-dependent pathway, possibly by inactivating or bypassing the ana repressor. These results suggest that trol and ana are components of a novel developmental pathway for the control of cell cycle activation in quiescent neuroblasts (Datta, 1995).

A culture system has been established in which quiescent neuroblasts in explants of Drosophila larval CNSs initiate cell division in vitro to normal in vivo levels. This activation requires removal of the CNS for culture after a specific developmental stage and the presence of fetal calf serum or a larval extract in the medium. Either supplement is effective when heat-treated. Substitution of the steroid hormone ecdysone or the non-steroidal ecdysone analog RH5992 for either fetal calf serum or larval extract also results in activation of neuroblast proliferation. Culture of trolsd CNSs with wildtype larval extract or ecdysone results in the defective neuroblast proliferation phenotype observed in trol mutants in vivo, while culture of wildtype CNSs with trolsd extract produces normal neuroblast proliferation (Datta, 1999).

Several genes have been identified that control the pattern of neuroblast quiescence and proliferation in the central nervous system (CNS), including anachronism (ana), even skipped (eve) and terribly reduced optic lobes (trol). eve acts in a non-cell-autonomous manner to produce a transacting factor in the larval body that stimulates cell division in the population of quiescent optic lobe neuroblasts. ana encodes a secreted glial glycoprotein proposed to repress premature proliferation of optic lobe and thoracic neuroblasts. trol acts downstream of ana to activate proliferation of quiescent neuroblasts either by inactivating or bypassing ana-dependent repression. trol codes for Drosophila Perlecan, a large multidomain heparan sulfate proteoglycan originally identified in the extracellular matrix structures of mammals. The results suggest that trol acts in the extracellular matrix and binds, stores, and sequesters external signals and, thereby, participates in the stage- and region-specific control of neuroblast proliferation (Voigt, 2002).

The trol locus of Drosophila is localized in the chromosomal band 3A4 on the X-chromosome and is characterized by 134 mutant alleles (Datta, 1992; Flybase). Several of the in-depth analyzed trol-mutant alleles such as trolsd and trol15 show a severe size reduction of the larval optical lobe area, attributed to the loss of reactivation of neuroblast proliferation from mitotic quiescence (Datta; 1995). To molecularly identify the trol transcription unit, a X-chromosomal collection of lethal P-element insertion lines was screened. Three P-element insertions, l(1)G0023, l(1)G0271, and l(1)G0374, failed to complement the trol alleles trol13 and trol15 (Judd, 1972), suggesting that the corresponding P-element insertions have generated trol alleles. To show that the P-element insertion is indeed the cause of the trol mutation, remobilization experiments were performed. Each of the insertion lines could be reverted to viability, indicating that each of the three P-element insertions had hit the trol locus (Voigt, 2002).

It was next asked whether the newly identified trol alleles show the reduced optic lobe phenotype (Datta, 1992). Hemizygous l(1)G0271 mutant larvae develop smaller optic lobes than their heterozygous siblings, as observed earlier with mutant trol alleles (Datta, 1992). Other parts of the brain show overgrowth defects, which indicate that proliferation control in the brain is strongly impaired. Thus trol mutations do not only specifically interfere with the reinitiation of optic lobe neuroblast proliferation but have severe effects on larval brain growth in general. This conclusion is consistent with the finding that the imaginal discs of such larvae are extensively folded compared with wild-type discs at the corresponding developmental stages. In addition, it was noted that mutant discs are either significantly enlarged or, in other instances, smaller or not detectable. These diverse and even opposing observations with wing discs suggest that trol participates not only in a cell-specific manner in the control of proliferation as shown in the case of the optic lobe neuroblasts (Datta, 1995) but contributes also in a more general manner to larval development. This conclusion is also consistent with the finding that the development of hemizygous l(1)G0271 larvae is slowed down, meaning that it takes the mutant larvae at least 1 day longer to reach the third larval stage where they eventually die (Voigt, 2002).

The strong hypomorphic trol15 allele causes polyphasic lethality during first and second larval instar (Datta, 1992), whereas the three newly identified P-element insertion mutations survived until third instar larval stage. Only few escapers develop into pupae to die as pharate adults. These observations indicate that the P-element-associated trol mutations are generally weaker alleles than the strong trol15 hypomorphic allele (Voigt, 2002).

To correlate, by molecular means, the P-element insertion sites with a transcription unit, 'plasmid rescue' experiments were performed to identify P-element-adjoining genomic DNA sequences within the Drosophila genome. The three P-element insertions are located within a DNA segment of less than 1 kb, followed by several large and a multitude of small exons that could be conceptually combined to a single transcription unit spanning a genomic region of more than 40 kb. Sequences of the transcription unit are represented by several expressed sequence tags (ESTs), that cover approximately 55% of the coding region of a 13-kb mRNA. Conceptional translation of the open reading frame indicates that the transcript encodes the Drosophila homolog of mammalian Perlecan, parts of which had been described previously (Friedrich, 2000; Voigt, 2002).

To obtain a trol null mutation, a deletion spanning the genomic region of the transcription unit was generated. The deletion was the result of the simultaneous mobilization of the P-elements l(1)G0271 and EP(1)1619, which are located immediately 5' of the putative transcription start site and approximately 4 kb 3' to the transcription unit, respectively. Excision of the two P-elements was monitored by the reappearance of the white-eye phenotype, indicating that both P-element-associated marker gene copies were lost. Heterozygous female individuals were collected and examined by PCR analysis by using primers directed to 5' adjacent sequences of the l(1)G0271 insertion site and to 3' adjacent sequences of the EP(1)1619 insertion site, respectively, followed by sequencing of the amplified DNA. The sequence data showed that the excision of the P-elements in combination with a recombination event had caused a 47,908bp deficiency, removing the entire Perlecan transcription unit exclusively. The deficiency mutation causes lethality without resulting in an obvious and morphologically distinct larval cuticle phenotype as has been observed with the hypomorphic trol mutations. Questions concerning the maternal effect of this new trol allele, which represents a trolnull mutation, can now be addressed (Voigt, 2002).

EP(1)1160 is inserted within the region of the trol gene where the three above-described P-elements are located. It was asked, therefore, whether this line could be used to drive the expression of trol by the GAL4/UAS system. The homozygous EP(1)1160 females were with males homozygous for the engrailed-GAL4 driver. Embryo in situ hybridization with trol anti-sense RNA probes and anti-Perlecan domain V-specific antibodies revealed an engrailed-like expression pattern, indicating that endogenous trol can be activated in response to transgene-dependent GAL4 activity (Voigt, 2002).

It was next asked whether ectopic trol expression interferes with normal embryonic and larval development. GAL4 driver lines were crossed with EP(1)1160 to overexpress trol in response to (1) the maternal and ubiquitous V3-GAL4 driver, (2) the sca-GAL4 driver in neurons, (3) the en-GAL4 driver in a series of stripes along the longitudinal axis, and (4) the Actin5C-GAL4 driver (in order to obtain constitutive ubiquitous expression). Lethal and phenotypic consequences of ectopic trol expression were examined in each case. Surprisingly, only minor effects of the ectopic trol overexpression could be observed in low penetrance. These include defects in the arrangement of macrochaete in response to neurospecific trol expression and extra wing veins in response to ectopic trol activity. These observations indicate that ectopic expression of trol under the experimental procedures described has only subtle effects on development as compared to, for example, the overexpression of heparan sulfate proteoglycans encoded by dally-like and dally (Voigt, 2002).

A chemical mutagenesis with diepoxybutane (DEB) was used to generate new alleles of trol. Approximately 2000 mutagenized F1 males produced three independent trol alleles: trol4, trol5, and trol6. All are homozygous lethal. Screening of an additional 150 X chromosome lethal lines from a previous DEB mutagenesis resulted in the identification of two more trol alleles: trol7 and trol8 (Park, 2003).

The activation of neuroblast proliferation in animals mutant for four trol alleles was assayed by counting the number of neuroblasts labeled by BrdU from 16-20 h post-hatching. trol4, trol5, trol6, and trolS1 all produced a defective neuroblast proliferation phenotype at late first instar. The ability of induced cyclin E expression to rescue the proliferation phenotype of trol4, trol5, trol6, and trolS1 was assayed by BrdU incorporation. In all trol alleles examined, cyclin E expression is able to rescue the mutant proliferation phenotype. Proliferation was never observed in the ventral nerve cord, nor was overproliferation observed in the lobes (Park, 2003).

RFLP, PCR, and sequence analysis of the existing trol alleles were carried out to identify lesions in the predicted Drosophila perlecan gene. Sequence analysis of genomic DNA from the X-ray-induced allele trolb22 revealed a single base pair deletion in domain V. The deletion results in a predicted frame shift and truncation of the protein from 450 to 400 kDa. The single base deletion is also observed in RT-PCR fragments derived from larval RNA, indicating that the trolb22 lesion lies within the transcribed sequence. Sequence comparison of a second independent allele, trol7, and the parental chromosome in domain IV revealed a 25-base pair deletion in trol7 from 127431 to 127456 in the genomic sequence (7966-7991 in the predicted mRNA sequence) resulting in a frame shift and truncation of the predicted protein from 450 to 267 kDa. Analysis of two additional independent trol alleles, trol6 and trol8, also led to the identification of molecular lesions within the GC7981 coding sequence (Park, 2003).

The Perlecan protein in third instar trol mutant larvae was analyzed by Western blot of a native gel using either an anti-mPerlecan domain IV antibody or 10E4, an antibody specific for heparan chains. A total of 100 micrograms of size-fractionated larval protein was isolated from animals mutant for each of four independent trol alleles, and the parental strain for two of the alleles was analyzed. Third instar trolb22 extracts produced multiple bands compared with control extracts when probed with either antibody (Park, 2003).

Extracts from trolsd, trol7, and trol8 had greatly decreased anti-mPerlecan immunoreactivity. Consonant with the relative severity of the mutations, extract from trol8 animals showed more staining than did extracts from either trolsd or trol7. The predicted protein truncations for Trolb22 and Trol7 proteins are not observed, presumably due to separation based on shape and charge as well as by size, and perhaps additionally due to instability of the Trol7 protein (Park, 2003).

Due to the large size of the predicted trol open reading frame, RNAi assays were carried out to further verify that Drosophila Perlecan protein is the product of the trol locus. Double-stranded RNA was made from an ~1-kb section at the 3' end of the putative trol cDNA, covering multiple exons. Double-stranded RNA, single-stranded sense RNA, and buffer were used to inject wildtype embryos, and the proliferation phenotype of the resulting larvae was assayed. When injected with doublestranded RNA from the putative trol cDNA, 45% of larvae had a proliferation defect compared with only 5% of the single-stranded or buffer-injected animals, further evidence that trol is the Drosophila perlecan gene (Park, 2003).

Genetic interaction between even-skipped and trol

The regulation of stem cell division by developmental cues is critical for the assembly and function of multicellular organisms. Stem cell division in the Drosophila brain is controlled by trol, which is required for activation of proliferation by quiescent neuroblasts at the appropriate stage of larval development. The transcriptional regulator eve has been shown to be part of the trol activation pathway by the identification of eve as a dominant enhancer of a weak trol allele, trolb22. Known eve mutations are capable of enhancing the lethality of trolb22 and uncovering a defective neuroblast proliferation phenotype. Additionally, genetic and molecular analysis has revealed that an independent mutation that acts as a dominant enhancer of trol is also an allele of eve. The enhancement of trolb22 lethality can be suppressed by the presence of an eve transgene. Interestingly, extra copies of eve supplied by the eve transgene also enhance trolb22 lethality, suggesting that the level of Eve protein may be critical for neuroblast activation. Finally, activation of neuroblast proliferation is normal in eve4 heterozygotes, suggesting that the proliferation defect observed in trolb22;eve/+ animals is due to a synergistic interaction (Park, 1998).

Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. Genetic interaction and phenotypic analysis is presented showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain (Park, 2001).

Several genes have been identified that affect neuroblast proliferation, including anachronism (ana), terribly reduced optic lobes (trol) and eve. tr ol was originally identified in a genetic screen for abnormal larval brain morphology due to defective patterns of neuroblast proliferation in the larval brain. Mutations in trol cause a dramatic decrease in the reactivation of proliferation from mitotic quiescence. Recent studies suggest that trol may regulate this reactivation of neuroblast proliferation by stimulating the G1/S transition through upregulation of Cyclin E (CycE) expression. Several studies on trol and ana have led to the hypothesis that trol is required to overcome the repression of neuroblast cell division imposed by ana. eve was identified in a screen for enhancers of a hypomorphic allele trol. Mutations in eve enhanced both the trol proliferation phenotype and the associated lethality, indicating that eve may regulate transcription of cell cycle genes in the trol pathway (Park, 2001).

Analysis of explants has shown that ecdysone enables activation of neuroblast division and can substitute for larval extract. Furthermore, addition of ecdysone does not rescue the proliferation phenotype of cultured trol mutant brains, implying that ecdysone acts upstream of trol. Thus, ecdysone can overcome the lack of eve-induced activity in extracts of mutant flies. Interestingly, almost complete rescue is obtained when animals are fed ecdysone from 16-20 hours posthatching, indicating that the time between ecdysone action and S phase entry is at most four hours (Park, 2001).

The genetic interaction between eve and trol has all the characteristics expected for two components of a common pathway: (1) the eve;trol interaction is not allele specific and the known functional domains of Eve are implicated in the interaction; (2) the strength of the interaction mirrors the strength of the eve allele in segmentation; (3) eve mutants themselves have the predicted proliferation phenotype, and (4) neuroblasts arrested in trol;eve double heterozygotes can be rescued by expression of CycE, as can the neuroblasts arrested in a strong trol mutant. The latter is especially revealing, as induction of CycE expression in trol mutants results in the activation of cell division only in the number of neuroblasts appropriate to the developmental stage of the induction. That is, not all mitotically quiescent neuroblasts are arrested at the same cell cycle phase, and the extent to which CycE is a limiting factor is developmentally controlled. Therefore, as in embryonic segmentation and determination of neuronal identity, eve appears to function in a specific genetic pathway to affect the behavior of specific cells at specific times (Park, 2001).

However, Eve is not detectable in regulated neuroblasts at any time during first instar. Furthermore, eve function is not required within the larval CNS, but is required within the larval body from which extracts are prepared. Moreover, low levels (10%-20%) of extract made from eve plus animals will not support activation of neuroblast division while higher concentrations will. This concentration dependence indicates that eve does not inhibit production of a trans-acting proliferation repressor that is produced at higher levels in a eve mutant, since dilution of such a repressor would allow neuroblast division at lower rather than higher extract concentrations. These results strongly suggest that eve function is required for the production of a trans-acting factor that stimulates neuroblast division (Park, 2001).

Is ecdysone the trans-acting factor produced in response to eve? Ecdysone can rescue eve-dependent proliferation defects both in vivo and in vitro, but not the proliferation defect of trol mutants in vitro. This suggests that ecdysone acts upstream of trol, as would be expected if it is the eve-dependent trans-acting signal, and trol acts within the receiving cells. However, while the ecdysone receptor has been detected in a few neurosecretory cells of the first instar CNS, it has not been detected in neuroblasts. This may indicate that only a few high-affinity receptors are required to transduce the ecdysone signal, or that ecdysone acts indirectly through the products of the neurosecretory cells. However, since Eve is not detectable in the neurosecretory cells in wild-type brain lobes, it is unlikely that the added ecdysone rescues mutant animals by compensating for a loss of Eve activity in those cells. In each of these cases, eve could be acting through ecdysone production. Alternatively, ecdysone may act through a pathway parallel to the one stimulated by an (unknown) eve-dependent signal. While the relationship between eve and ecdysone is not yet clear, it seems likely that eve is required for the production of an organismal-level trans-acting signal that is specifically required to stimulate larval neuroblast proliferation (Park, 2001).

The Drosophila Perlecan gene trol regulates multiple signaling pathways in different developmental contexts

Heparan sulfate proteoglycans modulate signaling by a variety of growth factors. The mammalian proteoglycan Perlecan binds and regulates signaling by Sonic Hedgehog, Fibroblast Growth Factors (FGFs), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), among others, in contexts ranging from angiogenesis and cardiovascular development to cancer progression. The Drosophila Perlecan homolog trol has been shown to regulate the activity of Hedgehog and Branchless (an FGF homolog) to control the onset of stem cell proliferation in the developing brain during first instar. This study has extended the analysis of trol mutant phenotypes to show that trol is required for a variety of developmental events and modulates signaling by multiple growth factors in different situations. Different mutations in trol allow developmental progression to varying extents, suggesting that trol is involved in multiple cell-fate and patterning decisions. Analysis of the initiation of neuroblast proliferation at second instar demonstrated that trol regulates this event by modulating signaling by Hedgehog and Branchless, as it does during first instar. Trol protein is distributed over the surface of the larval brain, near the regulated neuroblasts that reside on the cortical surface. Mutations in trol also decrease the number of circulating plasmatocytes. This is likely to be due to decreased expression of pointed, the response gene for VEGF/PDGF signaling that is required for plasmatocyte proliferation. Trol is found on plasmatocytes, where it could regulate VEGF/PDGF signaling. Finally, it was shown that in second instar brains but not third instar brain lobes and eye discs, mutations in trol affect signaling by Decapentaplegic, Wingless and Hedgehog. These studies extend the known functions of the Drosophila Perlecan homolog trol in both developmental and signaling contexts. These studies also highlight the fact that Trol function is not dedicated to a single molecular mechanism, but is capable of regulating different growth factor pathways depending on the cell-type and event underway (Lindner, 2007).

Mutations in trol prevent the onset of neuroblast division in the first instar brain, and most trol mutations are lethal. Mutations in a second gene, anachronism, also affect the onset of neuroblast proliferation but in the opposite manner: in anachronism mutants, mitotically regulated neuroblasts begin cell division too early. However, when a lethal trol mutation was combined with a viable allele of anachronism, the lack of neuroblast division was rescued (double mutants exhibited the anachronism phenotype of premature neuroblast division) but lethality was not. This outcome suggested that trol function is required for other developmental events necessary for survival. Further analyses revealed that trol modulates Hh and Bnl signaling in the first instar brain. This study has demonstrated that trol function is required for developmental progression to third instar and for pupariation. Analogous to its function in the first instar brain, trol is required to initiate the division of a second, independent and spatially distinct population of neuroblasts in the second instar brain. This initiation of division is also dependent on Bnl and Hh signaling. It has also been demonstrated that the Trol protein is localized to the surface of the brain at all larval stages, which places it in close proximity to the regulated neuroblasts. This localization is consistent with the model where Trol regulates Bnl and Hh signaling to cells adjacent to the regulated neuroblasts by binding the growth factors directly. Trol protein localization to the basal lamina is not limited to the larval brain, as Trol-GFP studies also showed Trol protein in the basal lamina surrounding the salivary glands. trol function is not limited to the nervous system, as mutations in trol also diminish the number of circulating plasmatocytes by decreasing expression of pnt, a PVR response gene in plasmatocytes. It is speculated that trol may be necessary for signaling by the Drosophila PDGF and/or VEGF growth factor, just as mammalian Perlecan has been shown to function during angiogenesis. These studies of Dpp and Wg indicate a positive feedback between dpp expression and Dpp signaling and wg expression and Wg signaling in the second instar ventral ganglion. Signaling by Dpp and Wg is also dependent on trol in the second instar brain, but not (or very little) in the third instar brain lobes and eye discs, despite the fact that Dpp and Wg signaling are taking place in those tissues. In fact, even Hh signaling appears to be independent of trol in this context. These results highlight an important concept in trol, and indeed, in proteoglycan function: that the Trol protein will be used at different times and places to regulate the signaling of different growth factors. Deciphering the role of trol in different developmental decisions will require that each event individually be examined individually, as trol will not necessarily mediate the same molecular mechanism each time (Lindner, 2007).

The requirement for heparan sulfate proteoglycans in signaling by different families of growth factors is well established, but what is not yet clear is why different organs and tissue types use different HSPGs to modulate these signaling pathways. One possibility is that the specific mechanism(s) through which these molecules modulate signaling activity allows for site-specific variations in the regulation of signaling activity. HSPGs with varied amino acid sequence can act in the same signaling pathway, such as Syndecan-4 and Perlecan for FGF2 or Glypicans, Syndecan-3 and Perlecan for Hh. Mutations that affect heparan sulfate synthesis or modification strongly affect FGF2 and Hh signaling. Furthermore, Perlecan isolated from various endothelial cell sources has different binding affinities for FGF2. These data initially suggested that the protein core of the HSPG might have little to do with signaling specificity and that the main functional domain of HSPGs is concentrated in the sequence of the heparan sulfate chains (Lindner, 2007).

The carbohydrate-centric view is being challenged by studies that indicate a role for the protein-protein interactions of HSPGs with growth factors and other signaling molecules. For example, expression of chimeric molecules has shown that the cytoplasmic tail of Syndecan is specifically required for FGF2 signaling in addition to its heparan sulfate chains. Perlecan protein-protein interactions include the ability of Perlecan to bind growth factors and extracellular matrix molecules at various sites on its protein core. Further mechanisms that allow for differential regulation include processing of HSPGs. These studies suggest a reason for the use of a particular HSPG during an individual developmental decision – the flexibility of combining both carbohydrate-based regulation and protein-based regulation of cell-cell signaling may make a specific HSPG uniquely suited for a given situation (Lindner, 2007).

In the context of combined carbohydrate and protein inputs into HSPG function, it becomes clear that a given HSPG may be expressed and function in very specific contexts that take advantage of its unique regulatory abilities. It is interesting to note that Perlecan has been connnected with FGF and Hh signaling in the developing fly brain while mouse studies have shown that Perlecan knock-out mice have cerebral cortex abnormalities. trol mutant larvae have decreased numbers of circulating hemocytes that are likely due to decreased Ras-MAPK signaling by VEGF/PDGF. Perlecan knock-out mice also have defects in chondrogenesis and cardiovascular development and mammalian studies have demonstrated a role for Perlecan in angiogenesis driven by FGFs, VEGF and PDGF. Finally, this study has shown that Perlecan is required for SHH signaling during human prostate cancer growth, which reveals a new system for the investigation of the mechanism of Perlecan action. Further analysis of the ability of HSPGs to substitute for each other in cell fate decisions and the means by which they individually regulate cell-cell communication will lead to a clearer understanding of the inputs necessary for cells to carry out a developmental or disease progression (Lindner, 2007).

The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland

The heparin sulfate proteoglycan Terribly Reduced Optic Lobes is the Drosophila melanogaster homolog of the vertebrate protein Perlecan. Trol is expressed as part of the extracellular matrix (ECM) found in the hematopoietic organ, called the lymph gland. In the normal lymph gland, the ECM forms thin basement membranes around individual or small groups of blood progenitors. The pattern of basement membranes, reported by Trol expression, is spatio-temporally correlated to hematopoiesis. The central, medullary zone which contain undifferentiated hematopoietic progenitors has many, closely spaced membranes. Fewer basement membranes are present in the outer, cortical zone, where differentiation of blood cells takes place. Loss of trol causes a dramatic change of the ECM into a three-dimensional, spongy mass that fills wide spaces scattered throughout the lymph gland. At the same time proliferation is reduced, leading to a significantly smaller lymph gland. Interestingly, differentiation of blood progenitors in trol mutants is precocious, resulting in the break-down of the usual zonation of the lymph gland. which normally consists of an immature center (medullary zone) where cells remain undifferentiated, and an outer cortical zone, where differentiation sets in. Evidence is presented that the effect of Trol on blood cell differentiation is mediated by Hedgehog (Hh) signaling, which is known to be required to maintain an immature medullary zone. Overexpression of hh in the background of a trol mutation is able to rescue the premature differentiation phenotype. These data provide novel insight into the role of the ECM component Perlecan during Drosophila hematopoiesis (Grigorian, 2013).

Basement membrane manipulation in Drosophila wing discs affects Dpp retention but not growth mechanoregulation

Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. This study manipulated BM composition to cause dramatic changes in tissue tension. Increased tissue compression was found when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor beta ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. These results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment (Ma, 2017).

Basement membranes (BMs) are laminar polymers of extracellular matrix proteins which underlie epithelia and surround organs in all animals. The main components of BMs are collagen IV, nidogen, laminin, and perlecan, all conserved from insects to humans. Despite long-known conservation, ubiquity in animal tissues, and extensive biochemical knowledge, understanding of the developmental roles of BMs is comparatively poor. Nonetheless, significant progress has been made in recent years with the help of model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, thanks to limited genetic redundancy of BM components in these systems. In this way, it has been shown in the fruit fly Drosophila that collagen IV is required for full Dpp activity in dorsal cells of the embryo and for the response to Dpp of renal tubules. In addition, BMs are now known to play an essential role in mechanically shaping tissues: in the absence of a BM, tissues such as the egg follicleand the larval imaginal discs uffer profound deformations (Ma, 2017).

Drosophila adult wings develop from the pouch region of the wing imaginal disc, a widely studied model for tissue growth regulation. The wing pouch of the third instar larva (L3 stage) is a highly columnar monolayered epithelium where each cell attaches to the BM . Recently, the hypothesis that mechanical factors contribute to the regulation of wing growth has gathered considerable momentum. The observations that cell compression is higher at the center of the pouch and that compression increases during larval development have led to several models postulating a negative effect of compression on growth. This negative effect of compression on growth is invoked to solve the apparent paradox that combined concentration of growth promoters Dpp and Wingless (Wg) is higher at the center of the pouch, yet the distribution of cell proliferation is roughly homogeneous throughout the disc. In this context, the Hippo signaling pathway, known to respond to cell contact, cell crowding, and cytoskeletal tension has been postulated as a mediator of mechanical inputs into wing growth. However, the difficulty of experimentally changing tissue constriction in an internally developing organ has precluded definitive testing of this hypothesis (Ma, 2017).

To investigate the developmental role of the BM and explore the influence of mechanical factors on wing growth, this study subjected wing discs to different BM manipulations changing tissue constriction in order to assess their effect on disc development and adult wing size. The results show a lack of effect of mechanical constriction on Hippo signaling and wing growth. In contrast, BM was foudn to contribute to tissue growth by enhancing tissular retention of Dpp (Ma, 2017).

The results of the experiments changing tissue constriction through BM manipulation are difficult to reconcile with a physiological role of cell compression in regulation of normal wing growth, a central tenet of wing growth mechanoregulation models. Increase in compression when perlecan was knocked down, and decreased compression when the BM was degraded, both failed to produce the predicted effects: smaller and larger wings, respectively. In contrast to the results in the larval wing, tissue size regulation by cell crowding and apoptosis has been shown to occur in the notum during metamorphosis. Since both the wing and the notum derive from the same imaginal disc, it follows that mechanical effects on size must be highly dependent on the specific developmental context (Ma, 2017).

The failure to observe changes in Hippo activity after dramatic changes in tissue shape also challenges the role of Hippo signaling in regulating wing growth in response to compression. Nonetheless, several manipulations of cytoskeletal components clearly influence Hippo signaling in the wing, affecting growth. Because the actin-rich zonula adherens is the physical locus where Hippo signaling complexes assemble, Hippo signaling may act as a critical sensor of cell polarity or cell contact. According to the current results, however, it does not act in the wing as a tension-growth feedback regulator slowing growth in response to cell crowding (Ma, 2017).

Discs made of larger, fewer cells have long been known to give rise to normally sized adult wings, indicating that some parameter different from cell numbers contributes to defining final wing size, for instance some physical dimension of the tissue such as planar area or tissue volume. BM manipulations dramatically changed apical area and height of individual cells and of the tissue as a whole, but they may not have changed cell size, as suggested by the fact that cell density in the adult wing did not change. These findings, therefore, would be consistent with a model in which tissue mass or volume contributes to determination of final wing size. Normally sized discs and adult wings made of larger, fewer cells, in addition, offer a further argument against mechanical regulation of wing growth, as these larger cells would display very different physical properties in terms of their apical areas and the tensions supported by their membranes and cytoskeletons (Ma, 2017).

Even though no mechanical effects on Hippo signaling or wing growth were detected following profound tissue deformations, it cannot be completely rule out that BM manipulations caused secondary effects that negated putative effects of mechanical signals. Such is the case, it is arguee, of the discs flattened by BM elimination. These discs gave rise to smaller adult wings, an effect that further experiments indicate is a result of the specific requirement of the BM in Dpp signaling. Nonetheless, this study also failed to detect changes in cell proliferation or adult wing size when discs were flattened in vivo through direct application of force. Importantly, a contribution of the directionality of compression is also a possibility that cannot be rule out, as cells in the periphery of both act > troli and rn > Mmp2 discs change their apical discs change their apical area, but maintain the tendency of the wild-type to align their major axis tangentially to the center of the disc. Therefore, if the vector of the compression rather than its magnitude is readable by a cell or its neighbors, the results cannot rule out a role for this in regulating wing growth. This pattern of cell orientation has been attributed to a slightly higher proliferation rate in the center of the wing pouch, a fact overlooked in the past and possibly responsible in the first place for the higher cell compression in the center of the wing. BM modifications, therefore, would not affect this intrinsically different proliferation rate in the central and peripheral wing regions. The results, finally, do not rule out the possibility that more extreme mechanical inputs could impact wing growth, for instance in wound healing or damage-stimulated growth (Ma, 2017).

Despite the lack of influence on Hippo signaling in the BM manipulations, the data show that the BM itself is required to preserve a growth-promoting environment by hindering diffusion of Dpp out of the disc. Collagen IV, the main component of BMs, physically interacts with Dpp through the C-terminal NC1 domains of both collagen IV chains. The effects of collagen IV loss on Dpp signaling in the wing, the dorsal blastoderm and germarium, and renal tubules are all consistent with a role of collagen IV in Dpp concentration. Elimination of the BM, however, did not seem to affect signaling by the other diffusible ligands Wg and Hh, which are, unlike Dpp, quite hydrophobic and may not require a mechanism preventing their escape from the tissue. The role of the BM in maintaining the concentration of extracellular ligands, therefore, may not be general, but ligand specific or specific to Dpp (Ma, 2017).

A role has been attributed to Dpp signaling in modulating cell height in the wing epithelium. Even though the current experiments eliminating the BM caused both a Dpp deficit and decreased cell height, it is unlikely that the effects on cell height in this experiment are caused by the Dpp deficit. First, the effects of collagenase treatment on disc morphology are immediate, which is difficult to explain as a deficit in Dpp signaling, specially a transcriptionally mediated effect. Second, discs in which the BM was simultaneously degraded and Dpp signaling was activated were still flattened, supporting the idea that effects on tissue shape elicited by BM degradation are not due to a Dpp deficit (Ma, 2017).

Since Dpp does not seem to accumulate basally in the wing disc, it is hypothesized that transient binding of Dpp allows the wing BM to act as a semipermeable barrier hindering Dpp diffusion, although not completely preventing it. This is a function that other BMs are long known to serve in the vertebrate kidney or the blood-brain barrier. Indeed, the results showing homogeneously high levels of Dpp signaling in the disc when Dpp was expressed in the fat body demonstrate an ability of Dpp to cross the BM. This result has also implications for understanding of Dpp signaling in the wing, as it shows that Dpp presentation by apical cytonemes is not absolutely required for signaling. A function of the BM in limiting basal escape of Dpp is, in addition, highly consistent with recent findings showing that a Dpp.GFP fusion could be immobilized at the BM, with effects on patterning and growth similar to the ones observed when the BM was eliminated. The findings support a critical role for basolaterally diffusing Dpp against a competing hypothesis stating that the functional Dpp gradient forms apically. It must be noted, however, that the role of the medial Dpp stripe in regulating growth has been called into question during the third larval instar, when a non-stripe source in the anterior compartment would serve this growth-promoting function instead. Because BM elimination reduces not just medial spalt and pMad, but also growth, it follows that the BM is required to maintain the concentration of Dpp from both sources: the medial stripe and the unknown anterior non-stripe source (Ma, 2017).

Given the conservation of BM components and Dpp, BM degradation and epithelial-to-mesenchymal transitions may enhance BMP/TGF-β signaling across tissue layers in development. The results also suggest a way in which tumoral BM degradation could contribute to tissue signaling misregulation in cancer by allowing escape of these diffusible signals. Finally, the visualization of an apico-basal gradient of Dpp in this highly columnar epithelium calls for the inclusion of the apico-basal dimension in future quantitative studies of Dpp gradient formation (Ma, 2017).


UNC-52 is a C. elegans perlecan homolog

Mammalian integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen for proteins binding the integrin beta(1) subunit cytoplasmic domain. ILK has been implicated in integrin-mediated signaling and is also an adaptor within integrin-associated cytoskeletal complexes. The C. elegans pat-4 gene has been identified in genetic screens for mutants unable to assemble integrin-mediated muscle cell attachments. pat-4 encodes the sole C. elegans homolog of ILK. In pat-4 null mutants, embryonic muscle cells form integrin foci, but the subsequent recruitment of vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked. Conversely, PAT-4/ILK requires the ECM component UNC-52/perlecan, the transmembrane protein integrin, and the novel cytoplasmic attachment protein UNC-112 to be properly recruited to nascent attachments. Transgenically expressed 'kinase-dead' ILK fully rescues pat-4 loss-of-function mutants. UNC-112 has been identified as a new binding partner for ILK. These data strengthen the emerging view that ILK functions primarily as an adaptor protein within integrin adhesion complexes and identifies UNC-112 as a new ILK binding partner (Mackinnon, 2002).

The unc-52 gene of C. elegans encodes a homolog of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. It is proposed that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways (Merz, 2003).

Mutations in the smu-1 gene of C. elegans suppress mutations in the genes mec-8 and unc-52. mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes. unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. A presumptive null mutation in smu-1 suppresses nonsense mutations in exon 17 but not exon 18 of unc-52 and enhances the phenotype conferred by an unc-52 splice site mutation in intron 16. Reverse transcription-PCR and RNase protection were used to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. smu-1 has been identified molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. It is proposed that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and other genes (Spike, 2001).

C. elegans MEC-8 is a putative RNA-binding protein that promotes specific alternative splices of unc-52 transcripts. MEC-8 is a nuclear protein found in hypodermis at most stages of development and not in most late embryonic or larval body-wall muscle. Overexpression of MEC-8 in hypodermis but not muscle can suppress certain unc-52 mutant phenotypes. These are unexpected results because it has been proposed that UNC-52 is produced exclusively by muscle. Various tissue-specific unc-52 minigenes were constructed and fused to a gene for green fluorescent protein. These minigenes have allowed monitoring of tissue-specific mec-8-dependent alternative splicing; mec-8 must be expressed in the same cell type as the unc-52 minigene in order to regulate the minigene's expression, supporting the view that MEC-8 acts directly on unc-52 transcripts and that UNC-52 must be synthesized primarily by the hypodermis. Indeed, this analysis of unc-52 genetic mosaics has shown that the focus of unc-52 action is not in body-wall muscle but most likely is in hypodermis (Spike, 2002).

Perlecan is involved in the cellular uptake of bFGF

A survey of defined species of cell surface and extracellular matrix heparan sulfate proteoglycans (HSPG) was performed in a search for cellular proteoglycans that can promote bFGF receptor binding and biological activity. Of the various affinity-purified HSPGs tested, perlecan, the large basement membrane HSPG, is found to induce high affinity binding of bFGF both to cells deficient in HS and to soluble FGF receptors. Heparin-dependent mitogenic activity of bFGF is strongly augmented by perlecan. Monoclonal antibodies to perlecan extract the receptor-binding promoting activity from active HSPG preparations. In a rabbit ear model for in vivo angiogenesis, perlecan is a potent inducer of bFGF-mediated neovascularization. These results identify perlecan as a major candidate for a bFGF low affinity, accessory receptor and an angiogenic modulator (Aviezer, 1994).

The internalization mechanism of basic fibroblast growth factor (bFGF) at the blood-brain barrier (BBB) has been investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin-resistant binding of [125I]bFGF to TM-BBB4 cells was found to be time-, temperature-, osmolarity- and concentration-dependent. Kinetic analysis of the cell-surface binding of [125I]bFGF to TM-BBB4 cells has revealed saturable binding with a half-saturation constant of 76 nm and a maximal binding capacity of 183 pmol/mg protein. The heparin-resistant binding of [125I]bFGF to TM-BBB4 is significantly inhibited by a cationic polypeptide poly-L-lysine, and compounds that contain a sulfate moiety, e.g. heparin and chondroitin sulfate-B. Moreover, the heparin-resistant binding of [125I]bFGF in TM-BBB4 cells is significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG-mediated endocytosis. RT-PCR analysis has revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine-kinase receptors for bFGF) mRNA are expressed in TM-BBB4 cells. Moreover, immunohistochemical analysis demonstrates that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes (Deguchi, 2002).

Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bind similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulate the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulates those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation is similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that is capable of binding FGF-2 is incapable of activating any BaF3 cell line unless the HS is removed from the protein core. The HS chains also exhibit greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity (Knox, 2002).

Other Perlecan protein interactions

Domain IV of mouse perlecan, which consists of 14 immunoglobulin superfamily (IG) modules, was prepared from recombinant human cell culture medium in the form of two fragments, IV-1 (IG2-9, 100 kDa) and IV-2 (IG10-15, 66 kDa). Both fragments bind to a heparin column, being eluted at ionic strengths either below (IV-2) or above (IV-1) physiological level, and can thus be readily purified. Electron microscopy demonstrated an elongated shape (20-25 nm), and folding into a native structure was indicated by immunological assay and CD spectroscopy. Solid-phase and surface plasmon resonance assays demonstrated strong binding of fragment IV-1 to fibronectin, nidogen-1, nidogen-2 and the laminin-1-nidogen-1 complex, with Kd values in the range 4-17 nM. The latter binding apparently occurs through nidogen-1, as shown by the formation of ternary complexes. Only moderate binding was observed for fibulin-2 and collagen IV and none for fibulin-1 and BM-40. Fragment IV-2 showed a more restricted pattern of binding, with only weaker binding to fibronectin and fibulin-2. None of these activities could be demonstrated for recombinant fragments corresponding to the N-terminal perlecan domains I to III. This indicates a special role for domain IV in the integration of perlecan into basement membranes and other extracellular structures via protein-protein interactions (Hopf, 1999).

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. The crystal structure of the mouse nidogen-1 G2 fragment has been solved; this fragment contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding (Hopf, 2001a).

Perlecan, a major basement membrane proteoglycan, has a complex modular structure designed for the binding of many cellular and extracellular ligands. Its domain IV, which consists of a tandem of immunoglobulin-like modules (IG2-IG15), is rich in such binding sites, which have been mapped to different modules obtained by recombinant production. Heparin/sulfatide binding is restricted to IG5 and depends on four arginine residues that are close in space in beta strands B and E of the C-type IG fold. The nidogen-1 and nidogen-2 isoforms bind to IG3 with high affinity [K(d) approximately 10 nM]. This interaction depends on the globular nidogen domain G2 and is crucial for the formation of ternary complexes with laminins. Two loops of IG3 located between beta strands B/C and F/G, which are spatially close, make a major contribution to binding. Fibronectin binding is localized to IG4-5, and fibulin-2 binds to IG2 and IG13-15 with different affinities. This implicates a complex cluster of heterotypic interaction sites apparently important for the supramolecular organization of perlecan in tissues (Hopf, 2001b).

Perlecan, a widespread heparan sulfate proteoglycan, functions as a bioactive reservoir for growth factors by stabilizing them against misfolding or proteolysis. These factors, chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high affinity binding and receptor activation. However, rather little is known about biological partners of the protein core. The major goal of this study was to identify novel proteins that interact with the protein core of perlecan. Using the yeast two-hybrid system and domain III of perlecan as bait, approximately 0.5 106 cDNA clones were screened from a keratinocyte library and a strongly interactive clone was identified. This cDNA corresponded to FGF-binding protein (FGF-BP), a secreted protein previously shown to bind acidic and basic FGF and to modulate their activities. Using a panel of deletion mutants, FGF-BP binding was localized to the second EGF repeat of domain III, a region very close to the binding site for FGF7. FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein. Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of various squamous cell carcinomas suggesting a potential in vivo interaction. Thus, FGF-BP should be considered a novel biological ligand for perlecan, an interaction that could influence cancer growth and tissue remodeling (Mongiat, 2001).

PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, it was found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bind PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibits the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I does not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, does not bind perlecan. Confocal immunohistochemistry shows that PRELP outlines basement membranes with a location adjacent to perlecan. PRELP also binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualizes a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, a function is proposed for PRELP as a molecule anchoring basement membranes to the underlying connective tissue (Bengtsson, 2002).

The collagen-tailed form of acetylcholinesterase (AChE) is concentrated at the vertebrate neuromuscular junction (NMJ), where it is responsible for rapidly terminating neurotransmission. This unique oligomeric form of AChE, consisting of three tetramers covalently attached to a collagen-like tail, is more highly expressed in innervated regions of skeletal muscle fibers, where it is externalized and attached to the synaptic basal lamina interposed between the nerve terminal and the receptor-rich postsynaptic membrane. Although it is clear that the enzyme is preferentially synthesized in regions of muscle contacted by the motoneuron, the molecular events underlying its localization to the NMJ are not known. Perlecan, a multifunctional heparan sulfate proteoglycan concentrated at the NMJ, is shown to be the unique acceptor molecule for collagen-tailed AChE at sites of nerve-muscle contact and is the principal mechanism for localizing AChE to the synaptic basal lamina (Arikawa-Hirasawa, 2002)

The yeast two-hybrid system was used to find interactive partners of perlecan; perlecan domain V was used as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, a ~1.6-kb cDNA insert was isolated that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a novel splice variant that was named ECM1c. The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants. The C-terminus of ECM1 interacts specifically with the EGF-like modules flanking the LG2 subdomain of perlecan domain V. Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia. ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells and induce angiogenesis. Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro- and anti-angiogenic activities. Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression (Mongiat, 2003b).

Perlecan mutation

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme causes invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes develop a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation (Costell, 1999).

The gene encoding perlecan (Hspg2) was disrupted in mice. Approximately 40% of Hspg2-/- mice died at embryonic day (E) 10.5 with defective cephalic development. The remaining Hspg2-/- mice died just after birth with skeletal dysplasia characterized by micromelia with broad and bowed long bones, narrow thorax and craniofacial abnormalities. Only 6% of Hspg2-/- mice developed both exencephaly and chondrodysplasia. Hspg2-/- cartilage shows severe disorganization of the columnar structures of chondrocytes and defective endochondral ossification. Hspg2-/- cartilage matrix contained reduced and disorganized collagen fibrils and glycosaminoglycans, suggesting that perlecan has an important role in matrix structure. In Hspg2-/- cartilage, proliferation of chondrocytes is reduced and the prehypertrophic zone is diminished. The abnormal phenotypes of the Hspg2-/- skeleton are similar to those of thanatophoric dysplasia (TD) type I, which is caused by activating mutations in FGFR3, and to those of Fgfr3 gain-of-function mice. These findings suggest that these molecules affect similar signalling pathways (Arikawa-Hirasawa, 1999).

Schwartz-Jampel syndrome (SJS1) is a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses. Electromyographic investigations reveal repetitive muscle discharges, which may originate from both neurogenic and myogenic alterations. The SJS1 locus has been localized to chromosome 1p34-p36.1; no evidence was found of genetic heterogeneity. Mutations, including missense and splicing mutations, of the gene encoding perlecan (HSPG2) are described in three SJS1 families. This study identifies the first human mutations in HSPG2, underscoring the importance of perlecan not only in maintaining cartilage integrity but also in regulating muscle excitability (Nicole, 2000).

Mice lacking the perlecan gene (Hspg2) have a severe chondrodysplasia with dyssegmental ossification of the spine and show radiographic, clinical and chondro-osseous morphology similar to a lethal autosomal recessive disorder in humans termed dyssegmental dysplasia, Silverman-Handmaker type. This study reports a homozygous, 89-bp duplication in exon 34 of HSPG2 in a pair of siblings with DDSH born to consanguineous parents, and heterozygous point mutations in the 5' donor site of intron 52 and in the middle of exon 73 in a third, unrelated patient, causing skipping of the entire exons 52 and 73 of the HSPG2 transcript, respectively. These mutations are predicted to cause a frameshift, resulting in a truncated protein core. The cartilage matrix from these patients stained poorly with antibody specific for perlecan, but there was staining of intracellular inclusion bodies. Biochemically, truncated perlecan was not secreted by the patient fibroblasts, but was degraded to smaller fragments within the cells. Thus, DDSH is caused by a functional null mutation of HSPG2. These findings demonstrate the critical role of perlecan in cartilage development (Arikawa-Hirasawa, 2001).

Mice lacking exon 3 of perlecan (Hspg2) gene were generated by gene targeting. Exon deletion does not alter the expression or the reading frame but causes loss of attachment sites for three heparan sulfate (HS) side chains. Hspg2Delta 3/Delta 3 mice are viable and fertile but have small eyes. Apoptosis and leakage of cellular material through the lens capsule are both observed in neonatal lenses, and lenses degenerate within 3 weeks of birth. Electron microscopy revealed altered structure of the lens capsule through which cells had formed extensions. No kidney malfunction, such as protein uria, was detected in Hspg2Delta 3/Delta 3 mutant mice, nor were ultrastructural changes observed in the glomerular basement membranes (BMs). To achieve further depletion in the HS content of the BMs, Hspg2Delta 3/Delta 3 mice were bred with collagen XVIII null mice. Lens defects were more severe in the newborn Col18a1-/- x Hspg2Delta 3/Delta 3 mice and degeneration proceeded faster than in Hspg2Delta 3/Delta 3 mice. The results suggest that in the lens capsule, HS chains have a structural function and are essential in the insulation of the lens from its environment and in regulation of incoming signals (Rossi, 2003).

The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin

Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding (Hohenester, 1999).

Perlecan is required for laminin matrix assembly processes

Dystroglycan (DG) function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, roles for other molecules in this process have been defined. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell (Henry, 2001b).

Perlecan inhibits angiogenesis

Perlecan plays key roles in blood vessel growth and structural integrity. The C terminus of perlecan potently inhibits four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan is active at nanomolar concentrations and blocks endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth it has been named 'endorepellin.' Endothelial cells possess a significant number of high affinity [K(d) of 11 nm] binding sites for endorepellin and endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo (Mongiat, 2003a).


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Biological Overview

date revised: 25 March 2015

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