See the embryonic expression pattern of bun at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
bunched is expressed in a hedgehog-dependent stripe in the undifferentiated cells just anterior to the morphological furrow of the eye imaginal disc. It appears to be involved in the transmission of the differentiation-inducing signal; a reduction in bunched function leads to a delay in differentiation and to a loss of photoreceptors in the adult. bunched is also required for a morphogenetic movement in the brain that reorients the second optic lobe relative to the first. Input from the eye is required for this rotation. Additional functions of shs include a role in wing margin development and a requirement for both male and female fertility (Treisman, 1995).
The mutant is lethal, but a few surviving adults have slightly small, rough eyes. The first observable phenotype evinces a delay in neural differentiation. The mutation is enhanced by a single copy of hedgehog or decapentaplegic. The mutant phenotype is suppressed by the loss of one copy of wingless (Treisman, 1995).
In the follicle cell (FC) epithelium that surrounds the Drosophila egg, a complex set of cell signals specifies two cell fates that pattern the eggshell: the anterior centripetal FC that produce the operculum and the posterior columnar FC that produce the main body eggshell structure. The long-range morphogen DPP represses the expression of the bunched (bun) gene in the anterior-most centripetal FC. bun, which encodes a homolog of vertebrate TSC-22/GILZ, in turn represses anterior gene expression and antagonizes Notch signaling to restrict centripetal FC fates in posterior cells. From a screen for novel targets of bun repression, the C/EBP homolog slow border cells (slbo) has been identified. At stage 10A, slbo expression overlaps bun in anterior FC; by stage 10B they repress each other's expression to establish a sharp slbo/bun expression boundary. The precise position of the slbo/bun expression boundary is sensitive to Notch signaling, which is required for both slbo activation and bun repression. As centripetal migration proceeds from stages 10B-14, slbo represses its own expression and both slbo loss-of-function mutations and overexpression approaches reveal that slbo is required to coordinate centripetal migration with nurse cell dumping. It is proposed that in anterior FC exposed to a Dpp morphogen gradient, high and low levels of slbo and bun, respectively, are established by modulation of Notch signaling to direct threshold cell fates. Interactions among Notch, slbo and bun resemble a conserved signaling cassette that regulates mammalian adipocyte differentiation (Levine, 2007).
bunched refines a DPP activity gradient by antagonizing Notch signaling to establish the posterior edge of the operculum-forming centripetal FC. This study reveals that bunched is part of an intricate switch reliant on Notch activation of slbo to direct alternate FC fates. These observations contribute to a model in which bunched connects long-range morphogen cues to short range, cell contact-dependent signaling. Together with recent work on the bunched homologue GILZ in mammalian cell culture, these data suggest that this family of proteins is part of a conserved signaling cassette regulating cell fate decisions, as detailed below (Levine, 2007).
In different contexts cells migrate either as integrated sheets, such as during convergent extension, or as small groups of cells, such as during neural crest migration. During border cell migration from stages 8-10, a subset of anterior FC transiently loses epithelial polarity, delaminates and rounds into a small semi-polarized cell cluster that migrates through the nurse cell complex. In contrast, during centripetal migration from stages 10-14 a ring of anterior follicle cells changes shape and squeezes through the oocyte/nurse cell complex in a process coordinated with rapid nurse cell dumping. Marker gene expression indicates that the centripetal FC stretch to cover the anterior of the oocyte and retain epithelial contacts with the anterior and posterior nurse cell FC and columnar FC groups, respectively, throughout this mass cell ingression. While unique genetic pathways likely regulate these distinct cell migrations, because both the border cells and the centripetal FC coordinately migrate through the germ line cyst and arrive in the same vicinity at the anterior of the egg, it is unsurprising that common components are involved in both processes. Non-muscle myosin (zipper) and DE-cadherin (shotgun) are expressed and required for migration in both cell types. As well, it has been shown that slbo itself is required for DE-cadherin accumulation during both border cell and centripetal FC migrations, an observation consistent with the role for slbo function in the centripetal FC that are demonstrated in this study. Recently, screens for border cell-specific gene expression have identified many transcripts expressed in both tissues (Levine, 2007).
Comparing the role and regulation of slbo during the centripetal FC sheet and border cell cluster migrations reveals both shared and unique requirements. Weak slbo mutations, which completely block border cell migration, have no discernable effect on centripetal FC migration, which is disrupted only in stronger allelic combinations. While early slbo mutant clones reduced DE-cadherin accumulation in the dorsal anterior FC and in the border cells, late slbo mutant clones in the nurse cell FC and centripetal FC are difficult to recover and properly stage. These clones result in several effects on late stage egg chambers. First, these resulted in increased levels of DE-cadherin and decreased levels of DLG consistent with changes in epithelial polarity and adhesion. Second, large anterior slbo mutant clones are associated with a failure of centripetal FC ingression to coordinate with nurse cell dumping. It is noted that slbo mutant phenotypes are distinct from DE-cadherin shotgun (shg) mutants, which result in ectopic centripetal migration between posterior nurse cells. slbo mutants do resemble dlg mutant phenotypes associated with defects in FC shape and epithelial invasiveness. And third, ectopic slbo-lacZ expression associated with disintegration of the follicular epithelia and egg chamber collapse which are likely connected to defects in epithelial maintenance. Thus previous reports that the strong slbo allele has no effects on centripetal FC migration may result from difficulties recovering and staging these highly aberrant and friable late stage mutant egg chambers (Levine, 2007).
The mechanism of slbo regulation in the border cells and centripetal FC is also distinct. It has been shown that post-transcriptional regulation of slbo protein levels is critical to proper border cell migration but does not occur in the centripetal FC. This study shows that in both cell groups, Notch initiates slbo expression and slbo is necessary and sufficient to repress its own expression as centripetal migration proceeds. SLBO protein can bind to a DNA sequence element located near the start site of its own promoter, and several matches to the canonical C/EBP binding site occur as well in the sequence of the slbo2.6 element that is sufficient to mediate autorepression, so this regulation is likely direct. Thus slbo adopts two strategies to fine-tune its levels: post-transcriptional regulation specifically in the border cell and transcriptional autoregulation in the both cell groups, as shown in this study (Levine, 2007).
It has been shown that DPP establishes the position of the bun expression boundary in the anterior FC and this boundary coincides with the posterior edge of the operculum eggshell structure. This study shows that as this boundary forms, slbo and bun expression patterns initially overlap and subsequently slbo and bun repress each other's expression to resolve respective expression patterns into two distinct cell groups. Notch signaling plays a central role in these interactions: Notch activates slbo expression in the centripetal FC and bun is required to antagonize Notch activation in posterior cells adjacent to the boundary (Levine, 2007).
The position of the boundary is highly sensitive to Notch activity so that increased Notch signaling leads to increased slbo2.6 expression both in the centripetal FC and, surprisingly, in adjacent columnar FC. Ectopic slbo expression in Nintra-expressing columnar FC at stage 10B is not associated with changes in FC proliferation and thus the spread of Notch activity likely relies on cell–cell signaling. This may arise either from (1) Notch activation of slbo expression in a large group of centripetal FC precursors that is not subsequently downregulated to a more narrow domain or (2) a Nintra-dependent activation of Notch signaling in adjacent columnar FC leading to cell contact-dependent posterior spread of slbo expression. The latter explanation is preferred because slbo2.6GAL4 expression expanded to almost all columnar FC in many egg chambers. In this way the position of the DPP-dependent cell fate boundary that defines the operculum is quite flexible but always drawn sharply by Notch activation (Levine, 2007).
While several canonical bun and Suppressor of Hairy [Su(H)] binding sites are located in the slbo2.6 element indicating slbo regulation by bun1 and Notch signaling, respectively, might be direct, several observations indicate slbo regulation at the boundary by bun is likely more complex. It has been noted previously that: (1) high levels of Notch and Notch target gene expression occur in anterior FC, with slightly reduced levels in centripetal FC in contact with bun-expressing cells and (2) increased levels of Notch targets occur in all cells of bun mutant clones at the centripetal FC boundary except those that contact bun+ cells. A parallel relationship is observed between bun and the Notch target slbo: (1) reduced levels of slbo occur in cells adjacent to bun-expressing cells in WT egg chambers, and (2) slbo expression occurs in bun mutant clones located at the centripetal FC boundary, with lower slbo levels in bun− cells in contact with bun+ cells. Thus while bun may repress slbo directly, bun also antagonizes Notch activation of slbo in a non-cell autonomous manner. Consistent with this, bun clones removed from the centripetal FC do not lead to increased slbo expression and bun1 is not sufficient to block Nintra activation of slbo2.6 in the centripetal FC (Levine, 2007).
Notch modulation of slbo expression may be indirect as well. Because the Nts; slbo01310/slbo01310 double mutant egg chambers retain strong slbo-lacZ expression throughout the FC compared to Nts; slbo01310/+ egg chambers stained in parallel, it is hypothesized that Notch blocks SLBO protein's ability to repress its own expression. In this scenario, which must be further tested, the rapid reduction in slbo expression as centripetal migration proceeds results from both (1) decreasing Notch activation of slbo via Su(Hw) sites in the slbo promoter and (2) relief of a block on slbo autorepression. Consistent with rapid changes in Notch levels in the migrating centripetal FC, as slbo levels decrease a corresponding increase is seen in the levels of Cut protein, a key target of Notch repression in these cells. Because reduced dorsal appendages and opercula are seen in Nintra-expressing egg chambers, it is likely that rapid reduction in Notch levels is critical to permit the further patterning of anterior structures (Levine, 2007).
Dynamic interactions among bun, slbo and Notch signaling tightly regulate DE-cadherin levels in the centripetal FC. bun mutant clones lead to increased Notch signaling and DE-cadherin accumulation and Nintra is sufficient to increase DE-cadherin levels in the FC. slbo mutant clones lead to loss of DE-cadherin expression early and ectopic DE-cadherin levels late. Thus a recurring theme is that tight modulation of DE-cadherin levels is required in the FC at late oogenesis for epithelial transitions including border cell migration, centripetal FC migration and dorsal appendage elongation (Levine, 2007).
Recently, it has been shown that the bun homolog GILZ antagonizes the ability of C/EBP to activate expression of the key fat cell master regulator gene PPARγ2 (Peroxisome Proliferator Activator γ2) in adipogenic mesenchymal stem cells (Shi, 2003). GILZ binds a promoter element required for C/EBP-mediated activation and recruits HDAC1 (Histone Deacetylase 1) to repress PPARγ2 expression and promote the osteogenic cell fate. GILZ can also directly bind to C/EBP in vitro. Shi (2003) proposes that a balance of GILZ repressor and C/EBP activator in precursor mesenchymal cells regulates levels of PPARγ2, the master fat cell regulator. The similarities between these pathways are striking and it is proposed they constitute a conserved signaling cassette required for cell fate commitment. In support of a role for Notch in both, it has been shown that Notch signaling promotes adipogenesis in tissue culture , although the specific role of Notch in adipogenesis has been questioned. Targets may be conserved as well: expression of a gene homologous to PPARγ2 in the centripetal FC has been noted. While a connection between border cell specification and adipogenesis has been noted, slbo has no role in fly fat body formatio. However, bun expression hduring fat body formation has been detected suggesting that portions of this fly signaling cassette may operate in a general pathway required for storage cell differentiation (Levine, 2007).
The bunched (bun) gene encodes the Drosophila member of the TSC-22/GILZ family of leucine zipper transcriptional regulators. The bun locus encodes multiple BUN protein isoforms and has diverse roles during patterning of the eye, wing margin, dorsal notum and eggshell. This study reports the construction and activity of a dominant negative allele (BunDN) of the BUN-B isoform. In the ovary, BunDN expression in the follicle cells (FC) results in epithelial defects including aberrant accumulation of DE-cadherin and failure to rearrange into columnar FC cell shapes. BunDN expression in the posterior FC leads to loss of epithelial integrity associated with extensive apoptosis. BunDN FC phenotypes collectively resemble loss-of-function bun mutant phenotypes. BunDN expression using tissue-specific imaginal disk drivers results in characteristic cuticular patterning defects that are enhanced by bun mutations and suppressed by co-expression of the BUN-B protein isoform. These data indicate that BunDN has dominant negative activity useful to identify bun functions and genetic interactions that occur during tissue patterning (Ash, 2007).
The bunched (bun) gene encodes the fly member of the TSC-22/GILZ/BUN family of proteins whose structures share conserved leucine zipper and DNA binding motifs. Several lines of evidence indicate that these proteins act as transcriptional regulators: (1) GILZ has been show to be a sequence-specific DNA binding protein with histone deacetylase-dependent transcriptional repressor activity in tissue culture cells (Shi, 2003); (2) TSC-22 has activator and repressor functions, depending on the method of assay; and (3) bun is a potent repressor of gene expression in migrating ovarian cells (Ash, 2007).
TSC-22 and GILZ both function in sundry developmental processes linked to cell differentiation, cell growth and migration. GILZ mediates glucocorticoid (GC)-stimulated tissue differentiation including T-cell maturation (Asselin-Labat, 2004; Ayroldi, 2002; Berrebi, 2003; Cohen, 2006; Mittelstadt, 2001; Riccardi, 2001), stem cell maintenance (Kolbus, 2003), and adipogenesis (Shi, 2003). TSC-22 is widely expressed in the early mouse embryo (Dohrmann, 1999, Kester, 1999) and in adult tissues including the mouse hair follicle, chick feather bud tract, and human colon and erythyroid cell lineages (Choi, 2005; Dohrmann, 2002; Gupta, 2003; Soma, 2003). Misexpression of TSC-22 in cell culture leads to cell type-specific effects on growth and apoptosis (Hino, 2002; Ohta, 1997; Shostak, 2003; Xu, 2003). Conversely, RNAi knockdown of Xenopus TSC-22 increases cell division and delays embryonic blastopore closure (Hashiguchi, 2004). These outcomes support the notion that TSC-22 links cell proliferation and tissue morphogenesis and consistent with this, TSC-22 has tumor suppressor properties in several cancer cell types (Iida, 2005; Rentsch, 2006; Shostak, 2005; Ash, 2007 and references therein).
Deletion of the N- and C-terminal domains of TSC-22 required for transcriptional regulation generates a mutant version (DN-TSC-22) that exhibits dominant negative properties in tissue culture (Gupta, 2003). This study demonstrates that a corresponding derivative of the BUN-B protein has dominant negative properties during the patterning of several adult tissues (Ash, 2007).
A requirement for bun has been demonstrated in cells that contact the centripetally migrating FC in the ovary: in bun mutant clones that contact the boundary of the centripetal migrating FC increased expression of Notch target genes is observed and accumulation of several cell junction proteins including DE-CAD, DLG and ARM (Dobens, 2005; Levine, 2007). This study shows that similarly, Flp-out clones expressing BunDN in anterior FC that contact the centripetal FC resulted in cell autonomous increases in accumulation of DE-cadherin (DE-CAD). The similarity between BunDN-expressing FC clones and bun loss-of-function clones argues strongly that BunDN blocks bun activity in these cells. Effects of BunDN expression in the FC suggest other unreported roles for bun in FC patterning. In large anterior BunDN clones, cells fail to rearrange and flatten to form stretch FC at stage 10 apparently leading to a collapsed egg chamber phenotype. In posterior FC, BunDN clones result in a striking loss of epithelialization of the follicle cell layer. This latter phenotype resembles that of bun mutant clones that lose adhesion to the posterior of mutant egg chambers. The loss of epithelial polarity in posterior FC found in both bun loss-of-function clones and in clones expressing BunDN correlates with increased levels of DE-cadherin, DLG and ARM throughout the clone and increased TUNEL staining in some cells of the clone. A role for BUN in apoptosis parallels the requirement for GILZ in apoptotic protection of IL-2 starved T-lymphocytes and suggests a conserved role for these genes in hindering cell death (Ash, 2007).
From a screen of imaginal disk GAL4 drivers it was shown BunDN expression interferes with cuticle patterning in the eye and notum. In the notum, a pnr-dependent notum cleft phenotype was enhanced by both BunDN expression and bun loss-of-function alleles; conversely the cleft phenotype was suppressed by BUN-B. BunDN effectively blocked the latter BUN-B suppression phenotype. In stronger mutant combinations – such as bun-pnr- double mutants and the PnrGAL4>3xUAS-BunDNHA genotype – a significant increase was observed in the abundance of notum bristles and defects in their polarity. The opposing effects of gain- and loss-of-bun activity on pnr phenotypes suggest that bun functions normally in the pnr pathway to (1) promote the spreading and fusion of the dorsal wing disc epithelium required for proper notum formation and (2) limit bristle formation. The interaction between bun and pnr suggests that a balance of these factors is required to pattern this tissue, and consistent with this, pnr and bun reporter genes have overlapping expression in the dorsal notum of the wing disk primordia (Ash, 2007).
In a second key assay of BunDN activity, the OmbGAL4 driver was used to test BUN-B and BunDN interactions at the forming wing margin. BunGAL4 expression occurs in cells flanking the wing margin indicating that bun normally restricts Notch activity to the margin and in support of this, both weak bun mutations and expression of BunDN resulted in wing notch phenotypes. OmbGAL4 expression of BUN-B in distal wing margin cells resulted in strong wing notch phenotypes that correlated with repression of the Notch target WG at the distal margin of the wing pouch. BunDN coexpression with BUN-B at the margin effectively reduced BUN-B wing notches and increased WG levels in the wing pouch. BunDN overexpression leads to increased Notch activity at the margin and is associated with wing overgrowth, which has been cited as a consequence of Delta overexpression in the wing. The previous observation that bun antagonizes Notch signaling in the FC and the observation here that BunDN blocks BUN-B repression of Notch signaling at the forming margin points to a common mechanism by which bun regulates Notch signaling during tissue patterning. This notion fits a speculative model that the TSC-22/GILZ/BUN family of genes has a conserved role for in regulating Notch signaling (Ash, 2007).
Because other drivers that are expressed at the distal margin, such as DppGAL4 or PtcGAL4, gave no phenotype in combination with BunDN or BUN-B (not shown), it was surmised either that the OmbGAL4 driver is significantly stronger than those drivers, or that the OmbGAL4 insertion sensitizes distal cells to changes in BUN levels. Consistent with the latter possibility, the Omb insertion results in a wing margin phenotypes that can be suppressed by the bun alleles bun6903 and bun4230 (data not shown), indicating that bun and Omb have opposing activities during wing margin patterning. bun interactions with Omb are likely complex: Optomotor blind (Omb) gene, which encodes a T-box sequence, is regulated by Dpp and WG signals, and Omb mutants show a loss of Dpp signaling, increased Notch expression, and both apoptosis of the central wing blade cells and cell proliferation in lateral cells (Ash, 2007).
While both increases and decreases in bun levels have opposing effects on BunDN phenotypes in the wing, notum and eye, in some cases BunDN mutant phenotypes resulted only from expression of multiple copies of the transgene. It is notable that in Westerns of ovarian protein extracts expressing BunDN, increased level of several high molecular weight species were detected that cross-reacted with BUN antisera. Some of these species correspond in size to BUN-B and BUN-A proteins. Such an outcome suggests that BUN might repress its own expression normally and may explain why driving BunDN expression by BunGAL4 and hsGAL4 led to only mild cuticular phenotypes. The BUN-B homolog GILZ is thought to repress its own expression by transcriptional repression of its activator FoxO3 indicating that bun autoregulation may be conserved (Asselin-Labat, 2005). Previously bun was shown to repress Serrate levels in the FC, so the observation that SerrateGAL4 driving expression of BUN-B or BunDN resulted in no wing patterning defects (not shown) may be explained by feed back modulation of the expression of this driver. Thus interpreting the effect of BunDN on patterning is subject to complexities such as (1) bun autoregulation, (2) cross-regulation of transcription of the driver promoter and (3) genetic interactions between reduced bun activity and the driver insertion (Ash, 2007).
Recent sequencing efforts have identified three new bun splice isoforms that, in addition to BUN-A, -B and -C, represent a set of at least six BUN proteins that can interact to form heterodimers via a common leucine zipper structure encoded by a shared 3'exon. Preliminary data indicates that complex, overlapping expression patterns of the six Bun RNA isoforms occurs in the FC. Thus resolving the developmental contribution of specific BUN isoforms will be aided by a tool like BunDN, which can be used to compare tissue-specific 'targeted blockade' of bun activity and with the effects of isoform add-back (Ash, 2007).
The role of the transmembrane receptor Notch in the adult brain is poorly understood. This study provides evidence that bunched, a negative regulator of Notch, is involved in sleep homeostasis. Genetic evidence indicates that interfering with bunched activity in the mushroom bodies (MBs) abolishes sleep homeostasis. Combining bunched and Delta loss-of-function mutations rescues normal homeostasis, suggesting that Notch signaling may be involved in regulating sensitivity to sleep loss. Preventing the downregulation of Delta by overexpressing a wild-type transgene in MBs reduces sleep homeostasis and, importantly, prevents learning impairments induced by sleep deprivation. Similar resistance to sleep loss is observed with Notchspl-1 gain-of-function mutants. Immunohistochemistry reveals that the Notch receptor is expressed in glia, whereas Delta is localized in neurons. Importantly, the expression in glia of the intracellular domain of Notch, a dominant activated form of the receptor, is sufficient to prevent learning deficits after sleep deprivation. Together, these results identify a novel neuron-glia signaling pathway dependent on Notch and regulated by bunched. These data highlight the emerging role of neuron-glia interactions in regulating both sleep and learning impairments associated with sleep loss (Seugnet, 2011).
The evidence presented suggests that Notch signaling controls factors that reduce the negative consequences of waking as measured by an attenuated sleep rebound and intact learning following 12 hr of sleep deprivation. Although it is tempting to speculate that the intact learning seen following sleep loss is simply due to the flies not being sleepy, previous studies have shown that sleepiness does not result in performance impairments in aversive phototaxic suppression (APS; Seugnet, 2008). Thus, Notch activation may preserve learning by preventing neuronal overstimulation during extended waking. Reducing neuronal stimulation may also prevent the buildup of sleep debt and thus explain the lack of sleep rebound. Canonical Notch signaling leads to Su(H)-dependent changes in transcription, but several other downstream pathways have been identified; thus, further work is required to determine which pathway downstream of the receptor is effectively involved in this context. The results suggest that Notch is mediating a neuron-glia signaling mechanism. These data provide additional support to recent work showing an involvement of glia in sleep homeostasis and cognitive impairments. In mammals, adenosine released by glia appears to play a critical role (Halassa, 2009). Given that mutants for the only known Drosophila adenosine receptor have normal sleep homeostasis (Wu, 2009), other factors are likely to be involved. It is interesting to note in this context that expression of the cell adhesion molecule Klingon, required for long-term memory and controlled by Notch in the adult brain, has been reported to be expressed in the glia. It should be noted that Notch localization and activation in glia may seem at odds with reports showing a requirement for Notch as well as the downstream effector Su(H) in MB neurons for memory consolidation. The data do not exclude a low level of Notch expression in neurons. In fact, it would not be surprising if Notch is expressed in both cell types and mediates two-way signaling between adjacent cells, given that it occurs commonly during developmental processes (Seugnet, 2011).
To maintain tissue homeostasis, some organs are able to replace dying cells with additional proliferation of surviving cells. Such proliferation can be localized (e.g., a regeneration blastema) or diffuse (compensatory growth). The relationship between such growth and the growth that occurs during development has not been characterized in detail. Drosophila melanogaster larval imaginal discs can recover from extensive damage, producing normally sized adult organs. This study describes a system using genetic mosaics to screen for recessive mutations that impair compensatory growth. By generating clones of cells that carry a temperature-sensitive cell-lethal mutation, patches of tissue in the imaginal disc were conditionally ablated and the ability of the surviving sister clones to replace the lost tissue was assessed. This system was used together with a modified whole-genome resequencing (WGS) strategy to identify several mutations that selectively compromise compensatory growth. Specific alleles of bunched (bun) and Ribonucleoside diphosphate reductase large subunit (RnrL) were found to reduce compensatory growth in the imaginal disc. Other genes identified in the screen, including two alleles of Topoisomerase 3-alpha (Top3α), while also required for developmental growth, appear to have an enhanced requirement during compensatory growth. Compensatory growth occurs at a higher rate than normal growth and may therefore have features in common with some types of overgrowth. Indeed, the RnrL allele identified compromises both these types of altered growth and mammalian ribonucleotide reductase and topoisomerases are targets of anticancer drugs. Finally, the approach described is applicable to the study of compensatory growth in diverse tissues in Drosophila (Gerhold, 2011).
Ablation of the recessive lethal mutation sec5ts did not induce blastema formation. Rather imaginal disc recovery occurred by compensatory proliferation in two stages. At early stages when levels of apoptosis were highest, ablating discs had a greater proportion of mitotic cells than control discs, suggesting that damage may directly promote additional proliferation. If surviving cells were responding directly to dying cells, it would suggest the existence of a regulatory mechanism distinct from those that are employed during normal disc development. However, unlike the nonautonomous proliferation induced by undead cells, no clear evidence was found that the increased proliferation is localized adjacent to the deleted tissue. In addition, at late stages, when normal growth in control discs slowed, ablated discs continued to show levels of proliferation comparable to slightly younger discs. As the amount of apoptosis in ablated discs was relatively low at this time, this late growth is unlikely to occur in direct response to damaged tissue. Thus compensatory growth may be a broader tissue-wide response. As such, it may be regulated by the same patterning and size-sensing mechanisms that function during normal development (Gerhold, 2011).
A screen was conducted for recessive mutations that block compensatory growth. Three of the mutations that were identified as being most likely to specifically affect compensatory growth were found to disrupt the genes bun, Gmd, and >RnrL. The data indicate that bun is required to produce a normal amount of compensatory growth. The normal biological function of bun and its mammalian ortholog TSC-22 are still poorly understood. In Drosophila, overexpression of BunA alone has no effect on imaginal disc growth. However, coexpression with Madm, a protein with a catalytically inactive kinase-like domain that can bind to BunA, results in increased growth of imaginal discs. Mutant forms of bun have been shown to have a subtle effect on growth under normal conditions. Clones of cells lacking the long isoform of bun (bunA) display, at best, mild growth defects in imaginal discs. However, eye imaginal discs that are composed almost entirely of mutant bun cells, develop into adult eyes composed of both fewer and smaller cells. These discs were generated by mitotic recombination with a chromosome bearing a cell-lethal mutation and therefore produce a situation not all that dissimilar to our sec5ts ablation system. Additionally, bun has been shown to be upregulated in imaginal discs undergoing regeneration after either surgical or genetic ablation of a portion of the disc. Taken together with the current findings, these observations implicate bun in the regulation of both regenerative and compensatory growth (Gerhold, 2011).
The mechanisms by which bun regulates growth are not known. Since bun encodes a protein that has a leucine zipper as well as a TSC box that appears capable of binding DNA in vitro, it was initially assumed that it functions as a transcriptional regulator. More recent studies have shown that Bun and Madm colocalize to the Golgi in S2 cells (Gluderer, 2010) and that knockdown of Madm by RNAi interferes with protein secretion. Since both the expression and requirement for Bun seem to be increased when regenerative or compensatory growth are required, future of studies of Bun in this biological context may provide more mechanistic insights into Bun function (Gerhold, 2011).
Imaginal discs in which the compensating cells were GmdB3-1 recovered in size following ablation but were still unable to produce a morphologically normal adult structure. This suggests that Gmd may be more important for tissue patterning or morphogenesis following ablation than for compensatory growth per se. In Drosophila, Gmd has been studied primarily in the context of Notch signaling, where loss of Gmd blocks expression of Notch target genes and results in reduced growth of the wing disc (Okajima, 2005). However, when this observation was tested by clonal analysis, rather than in homozygous mutant animals, Gmd clones did not have an obvious phenotype. Only when very large Gmd clones were generated was loss of the Notch target Wingless observed and only at sites distant from any wild-type cells, suggesting that the requirement for Gmd may not be cell autonomous (Okajima, 2008). No defects were observed in the expression of the Notch target Wingless even when the majority of the posterior compartment was composed of GmdB3-1 mutant cells, suggesting that the defects that were observed in ablated adult structures were not a consequence of reduced Notch activity in the larval disc. Fucosylation is also necessary for the formation of many different glycan structures that modulate the activity of a variety of proteins, including signaling and cell adhesion molecules, one or many of which may participate in disc patterning and morphogenesis (Gerhold, 2011).
Reducing RnrL function did not reduce compensatory growth as mutant cells were able to generate a normally sized posterior compartment; rather, mutant cells engaged in compensatory growth appeared to be predisposed to apoptosis. In Drosophila, no mutant phenotype has been characterized at the cellular level for either RnrL or RnrS. However, RnrL transcription is induced at the G1-to-S transition by E2F and is presumably required for DNA synthesis. Thus compromising RnrL function may render cells incapable of increasing their rate of cell-cycle progression to keep pace with the increased rate of tissue growth required following tissue ablation. Consistent with this possibility is the observation that RnrLA4B5 restricts the growth of clones that overexpress an activated form of Yki and that the growth of these cells appears to have outpaced their rate of division. Interestingly increased RR activity is associated with many malignant cancers, indicating that an increase in RR activity may be required to sustain increased levels of cell proliferation. Consistent with this, RRs are the targets of several common chemotherapeutic agents used in cancer treatment. Together, these observations suggest that some similarities may exist between compensatory growth and hyperplastic overgrowth (Gerhold, 2011).
The findings also indicate that some of the differences between compensatory growth and normal growth are likely to be quantitative rather than qualitative. The RnrL allele that was identified may be hypomorphic and more severe alleles may perturb normal growth. When the causative mutations were identified in three of the complementation groups found in the screen, each disrupted a gene that likely functions during cell growth or proliferation under normal conditions, yet the requirement for these genes appears to be enhanced when additional growth is required. Interestingly, mutations were identified in Top3α as strongly disrupting both sec5ts- and IR-induced compensatory growth. Topoisomerases are chemotherapeutic targets and topoisomerase IIα was recently shown to be haploinsufficient for liver regeneration in zebrafish, suggesting that abnormally proliferating cells may be particularly sensitive to the loss of topoisomerase activity. The screen may therefore have enriched for a set of mutations that reduce gene function to a level that is near the threshold required for developmental growth and is insufficient for compensatory growth, which requires either additional or more rapid cell divisions. These findings reinforce the idea that compensatory growth is intimately connected to developmental growth, but might place an increased demand on certain pathways that sustain normal cellular requirements for cell-cycle progression and growth (Gerhold, 2011).
The results also validate the approach of identifying EMS-induced mutations by whole-genome resequencing. In the one previous case where this strategy has been applied successfully in Drosophila, it was possible to distinguish genuine mutations from those that arose during library construction or sequencing by looking for those that had a high allele frequency. Unfortunately the approach is not suitable for situations where mutations are homozygous lethal. Therefore a method was developed of library construction and computational analysis that is generally applicable to mutations that are homozygous lethal, and it was used successfully to identify three of the mutations generated in the screen. Moreover, these programs have also been used more recently to identify chemically induced mutations in C. elegans (Gerhold, 2011).
Finally, it was shown that the system that was developed for studying compensatory growth is indeed capable of generating and characterizing mutations that, at least to some extent, impair compensatory growth more than normal growth. The issue of whether there are genes that are required exclusively for compensatory growth is still unresolved. However, using this system and others like it, it will be possible to conduct more extensive genetic screens for mutations that influence compensatory growth or regeneration. The system can be extended in two ways. First, by engineering fly lines in which transposon insertions on each of the chromosome arms carry a wild-type copy of the sec5 gene in a sec5ts background, it should be possible to screen most of the genome. Second, by targeting FLP to a variety of tissues, it should be possible to investigate the capacity of each of those tissues for compensatory growth (Gerhold, 2011).
Search PubMed for articles about bunched
Ash, D. M., Hackney, J. F., Jean-Francois, M., Burton, N. C. and Dobens, L. L. (2007). A dominant negative allele of the Drosophila leucine zipper protein Bunched blocks bunched function during tissue patterning. Mech. Dev. 124(7-8): 559-69. PubMed ID: 17600691
Asselin-Labat, M. K,m et ak, (2004), GILZ, a new target for the transcription factor FoxO3, protects T lymphocytes from interleukin-2 withdrawal-induced apoptosis. Blood 104: 215-223. PubMed ID: 15031210
Ayroldi, E., et al. (2002). Glucocorticoid-induced leucine zipper inhibits the Raf-extracellular signal-regulated kinase pathway by binding to Raf-1. Molec. and Cell. Biol. 22: 7929-7941. PubMed ID: 12391160
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date revised: 10 February 2012
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