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).
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).
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).
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date revised: 25 June 2007
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