BIOLOGICAL OVERVIEW

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, trol^sd (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 trol^b22 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 trol^b22 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 trol^b22; ttv⁰⁰⁶⁸¹+ brains yielded supernumerary labeled neuroblasts similar to those observed in hs-hh samples. Analysis of expression of the Hh target gene ptc in trol^b22; 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).

GENE STRUCTURE

cDNA clone length - 13041 bp (AJ487018)

Bases in 5' UTR - 207

Exons - 39

Bases in 3' UTR - 161

PROTEIN STRUCTURE

Amino Acids - 4117, 4179. 4223, and 4228 amino acids

Structural Domains

Perlecan is a posttranslationally modified extracellular matrix protein that has heparan/chondroitin sulfate side chains (Hassell, 1980; Noonan, 1991; Danielson, 1992). It is subdivided into five distinct regions that harbor domains structurally related either to LDL receptors (domain II), the N-terminal region of both laminin A and B short arms (domain III), the N-CAMs (domain IV), or the globular C-terminus of the laminin A chain (domain V) (Noonan, 1991; Kallunki, 1992; Noonan, 1993). Sequence analysis of three overlapping cDNA clones (SD04592, GM03359, and GM01116), which cover approximately 7 kb, has revealed coding regions corresponding to the regions IV and V, and a major part of domain III of mammalian Perlecan (Voigt, 2002).

The most striking difference to mammalian Perlecan is the absence in Drosophila of an equivalent to domain I, which consists mainly of one SEA domain. Sequences corresponding to this domain have not been found in either cDNA clones or in the genomic DNA sequences. This domain is also absent from C. elegans Perlecan (Noonan, 1991; Kallunki, 1992; Rogalski, 2001). Another major difference is that domain II, which is characterized by LDL-R motifs, is extended in Drosophila Perlecan. Whereas mammalian homologs exhibit four such motifs (Noonan, 1991; Cohen, 1993), Drosophila Perlecan has a total of at least 22 (Voigt, 2002).

The C-terminal domain V of the basement membrane proteoglycan perlecan plays a major role in extracellular matrix and cell interactions. A homologous sequence of 708 amino-acid residues from Drosophila (the complete protein has 4223 amino acids) has now been shown to be 33% identical to mouse perlecan domain V. It consists of three laminin G-type (LG) and epidermal growth factor-like (EG) modules but lacks the EG3 module and a link region found in mammalian perlecans. Recombinant production of Drosophila perlecan domain V in mammalian cells yields a 100-kDa protein which folds into a linear array of three globular LG domains (Friedrich, 2000).

Like the mouse sequence, Drosophila domain V consists of three LG modules (LG1 to LG3), but has only three analogs of EG modules (EG1, EG2 and EG4) interrupting the LG tandems. Module EG3 is replaced in Drosophila by a 30-residue sequence containing seven prolines but no cysteines. A further difference is the lack of a 19-residue link region between EG4 and LG3, that in the mouse contains a single glycosaminoglycan attachment site and a protease-sensitive site. Yet Drosophila domain V has three potential SGE or SGD sequences suitable for glycosaminoglycan attachment and seven NXT/S sites for N-linked glycosylation. It also has a potential cell-adhesive RGD sequence at the beginning of the LG3 module, which is not conserved in mouse perlecan. The 5' end of the sequenced clone (GM01490) encodes an additional 50 residues with 50% sequence identity to an Ig module from the adjacent domain IV of mouse perlecan (Friedrich, 2000).

Genetic analyses indicate that trol maps to 3A3-5 on the X chromosome (Datta, 1992; Shannon, 1972). Genetic mapping and sequence information from the Drosophila genome project have suggested that trol might correspond to GC7981, which comprises 44 exons that add up to a 13.5-kb open reading frame. The predicted GC7981 protein is about 450 kDa in size and has significant sequence identity to human Perlecan (Murdoch, 1992) in domains III (34%), IV (24%), and V (30%), whereas no significant similarity is found to domains I and II (Park, 2003).

date revised: 21 May 2003

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