1 University Museum of Zoology, Department of Zoology, University of Cambridge, Downing Street, Cambridge, UK, CB2 3EJ
2 Institut de Biologia Molecular de Barcelona (CSIC). C/ Jordi Girona 18-26, 08034, Barcelona, Spain
Present address: Centre de Biologie du Développement, Université Paul Sabatier, Bât 4R3, 118 Route de Narbonne, 31062, Toulouse, France
*Author for correspondence (e-mail: roch{at}cict.fr)
Accepted 22 November 2001
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SUMMARY |
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Key words: Capicua, Bullwinkle, Ras/Raf pathway, Transcriptional repression, Vein development, Drosophila
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INTRODUCTION |
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A well-established model of RTK signalling concerns the formation of veins in the Drosophila wing. The insect wings consist of a bilayer of epidermal cells closely apposed by their basal sides that secrete a thin and transparent cuticle. In Drosophila, wings display a stereotyped pattern of five longitudinal veins and two transverse crossveins. The veins are specialised epithelial cells present in both wing surfaces. During late differentiation, dorsal and ventral veins match accurately providing a hollow space for the passage of axons and trachea (García-Bellido and de Celis, 1992).
Vein tissue specification occurs during larval and pupal stages within the wing imaginal discs, and involves a complex network of gene regulation. Central to this network is the Epidermal Growth Factor Receptor (EGFR) RTK (Díaz-Benjumea and Hafen, 1994), which, in Drosophila, functions in a wide spectrum of developmental processes such as oogenesis, patterning of embryonic structures such as the ventral epidermis and trachea, and specification of photoreceptor cells in the compound eye (Schweitzer and Shilo, 1997
). In wing discs, activation of EGFR in rows of cells causes their determination as prospective wing veins. Consistent with this function, loss-of-function mutations in components of the EGFR pathway cause loss of veins, whereas gain-of-function alleles and/or overexpression of those components result in ectopic vein development (Díaz-Benjumea and Hafen, 1994
).
The role of EGFR signalling in vein patterning involves complex molecular interactions that (1) restrict its activity to appropriate cells, and (2) impose cell fate changes in response to signalling. Activation of EGFR in precise rows of cells depends on several signalling pathways that pattern the wing disc (de Celis and Barrio, 2000), and relies ultimately on the localised activity in the veins of different EGFR ligands and the Rhomboid (Rho) transmembrane protein, a mediator of EGFR activation (Schnepp et al., 1996
; Sturtevant et al., 1993
). Regulated EGFR activation specifies vein fates by both activation and repression of several downstream target genes. These encode transcription factors and also signalling molecules, such as the Decapentaplegic (Dpp) TGFß fly homologue, that have to be activated in the veins for their proper differentiation (de Celis, 1997
).
Although a broad picture of how veins are specified is emerging, little is known about how the EGFR cascade regulates target gene expression in the nucleus of developing vein cells. In this work, we present evidence that the EGFR pathway functions, at least in part, by promoting destabilisation of the Capicua (Cic) protein in vein nuclei. Cic is an HMG-box transcription factor that controls early embryonic patterning in response to Torso RTK signalling. Cic functions as a repressor of two genes expressed in the terminal regions of the embryo, tailless (tll) and huckebein (hkb). Cic accumulates in central regions of the embryo, but is under negative post-transcriptional control by Torso signalling at the termini, thus permitting expression of tll and hkb at the embryonic poles (Jiménez et al., 2000).
Our results support a similar regulation of the Cic protein by EGFR signalling in the wing. We have found that Cic activity prevents differentiation of vein tissue in the wing pouch. In turn, restricted activation of the Ras/Raf pathway in the presumptive veins antagonises Cic repressor activity in this tissue, thus allowing localised expression of genes mediating vein differentiation. We discuss the role of Cic as a general transcriptional repressor regulated by the Ras/Raf pathway.
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MATERIALS AND METHODS |
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Clonal analysis
Adult cic mutant FRT/FLP (Xu and Rubin, 1993) M+ clones marked with the forked mutation were induced in males of the following genotypes: yhsFLPf36a; FRT82B UbqGFP83 f+87D M(3)95A/FRT82B bwk
11 e and yhsFLPf36a; FRT82B UbqGFP83 f+87D M(3)95A/FRT82B sr cic2 e. Clones mutant for Ras-1 and the double mutant clones Ras-1/cic were induced, respectively, in males of the genotype yhsFLPf36a; FRT82B UbqGFP83 f+87D M(3)95A/FRT82B Ras
C40b and yhsFLPf36a; FRT82B UbqGFP83 f+87D M(3)95A/FRT82B Ras
C40bsr cic2 e. The expression of the argos lacZ reporter was monitored in cic mutant clones generated in flies with the genotype yhsFLPf36a; FRT82B UbqGFP83 f+87D M(3)95A/aosw11 FRT82B sr cic2 e. Clones were generated at 60±12 hours AEL with a 5 minutes heat shock pulse at 37°C.
Staining procedures
Immunostaining was performed according to standard protocols. For staining of Cic protein we used a polyclonal antibody from rat (1/300). Other primary antibodies were: mouse anti-Bs (1/600) (kindly provided by E. Martín-Blanco), rat anti-Vvl (1/300) (Llimargas and Casanova, 1997), mouse anti-ß-gal (1/100) (Promega) and rabbit anti-ß-gal (1/1000) (Promega). Secondary antibodies include anti-rat and anti-mouse conjugated to fluorescein and Texas Red and anti-rabbit and anti-rat conjugated to Cy5, all of them diluted 1/200 (Jackson Laboratories). Fluorescence was visualised in a Leica TCS confocal microscope. In situ hybridisation was carried out according to Sturtevant et al. (Sturtevant et al., 1993
) with DIG-labelled antisense riboprobes made with the full cDNAs of the dpp, rho and cic genes.
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RESULTS |
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The original bwk allele, bwk8482, is a P-element-induced female sterile mutation that affects the patterning of the ovary follicle cells and the early embryo, giving rise to embryos with abnormal eggshells that also show a bicaudal phenotype (Rittenhouse and Berg, 1995). bwk8482 also has zygotic effects: homozygous adult flies display ectopic vein tissue in their wings (Rittenhouse and Berg, 1995
). In contrast, the original cic1 allele is a female sterile mutation caused by the insertion of a hobo transposon 300 bp away from the bwk8482 insertion (Fig. 1). Homozygous cic1 females produce embryos with strongly suppressed trunk regions that are replaced by terminal structures (Jiménez et al., 2000
).
The genetic relationship between cic and bwk mutations is unclear. Although cic1 and bwk8482 complement each other, both mutations are uncovered by specific small deficiencies (less than 500 bp) in the region (Jiménez et al., 2000). To help clarify this issue, we analysed the phenotypes observed in heteroallelic combinations of several cic- and bwk-related alleles. This analysis shows that both cic1 and bwk8482 mutations are not complemented by cic2 and specific P-element excision alleles obtained by mobilisation of bwk8482 (e.g. bwk
11 and bwk
14) (Fig. 1), resulting in cic- and bwk-associated phenotypes, respectively (Table 1). Thus, cic2, bwk
11 and bwk
14 behave as strong hypomorphs or nulls for both cic and bwk genetic functions. These observations, together with the proximity of the bwk8482 and cic1 transposon insertions, raise the possibility that cic and bwk mutations disrupt different functions of a single gene. Alternatively, bwk and cic may be adjacent genes that are simultaneously affected by cic2 and some bwk8482 derived excisions.
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Cic is required during wing development for intervein specification
To study the role of cic in wing development, we examined the effects of a complete loss of function of the gene in an heteroallelic combination of the cic2 allele with the deficiency bwk11, which removes the entire cic-coding region (Fig. 1). This combination is generally lethal, but adult scapers can be obtained with a low frequency. The most striking feature of these flies is their abnormal wings, which are reduced in size and often show blisters that separate the ventral and dorsal surfaces. The mutant wings display abundant extravein tissue, preferentially close to the normal veins, which appear thickened (Fig. 2A-D). This phenotype is a consequence of intervein cells acquiring morphological features of vein cells, which are typically smaller, more pigmented and with shorter and thicker trichomes than wild-type intervein cells. However, close examination reveals that the transformation to vein tissue is not complete and many intervein cells acquire intermediate morphologies between those shown by typical vein and intervein cells (Fig. 2C,D). In addition, some regions such as the area between veins L3 and L4 never differentiate as vein tissue in mutant wings, indicating that specification of intervein fate in this territory is independent of cic (see Discussion). Other structures, such as the wing margin bristles and the campaniformia sensilla located along the L3 vein, appear in their normal positions in the mutant wings, indicating that cic absence only affects the process of vein/intervein specification.
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To analyse the effects of cic in vein development further, we generated clones homozygous for either cic2 or bwk11 mutations with the Minute technique, which allows the generation of clones that cover large territories (Morata and Ripoll, 1975
) (see Materials and Methods). Both sets of experiments gave similar results; we describe below those corresponding to cic2. In our analysis, we studied 45 clones, covering the complete wing surface. The mutant clones are consistently smaller than their respective controls, suggesting that proliferation of cic2 mutant cells is impaired. Mutant cells often differentiate as vein tissue in ectopic positions, particularly if they are close to normal veins (Fig. 2E-G). However, these changes in cell fate are not strictly cell autonomous, as many intervein cells within the mutant clones differentiate normally. This could be due in part to a rescue of the phenotype by wild-type cells situated in the other surface of the wing, because mutant clones that occupy only one wing surface display always less extravein tissue than clones occupying both dorsal and ventral surfaces (Fig. 2G). In addition, mutant clones can also induce neighbouring wild-type cells to differentiate as vein tissue, either in the same wing surface or in the opposite one (Fig. 2F,H). We conclude that cic functions as a negative regulator of vein differentiation in intervein territories, probably as part of a network involving cell interactions.
Post-transcriptional regulation of Cic protein in the wing disc
We next examined the expression pattern of cic in wild-type wing discs. In situ hybridisation of third instar discs showed uniform distribution of cic transcripts (Fig. 3A). By contrast, staining of similar discs with a specific Cic antibody revealed a complex pattern of protein accumulation: Cic accumulates at high levels in the wing pouch and in the primordial hinge region, but not in the notum region (Fig. 3B). At this stage, Cic levels begin to drop in the presumptive third longitudinal vein and in two rows of cells running along the D/V boundary, which correspond to the future wing margin (Fig. 3B,C). Moreover, the remaining Cic protein in those cells is cytoplasmic, whereas in other regions of the wing pouch (and in the adjacent peripodial cells), Cic is clearly nuclear (Fig. 3C,D). During pupariation [from 6 to 34 hours after puparium formation (APF)], Cic levels also decline in all presumptive longitudinal wing veins and crossveins (Fig. 3E-G). This specific accumulation of Cic in intervein sectors is consistent with its role as a negative regulator of vein differentiation.
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If this hypothesis is correct, Ras/Raf signalling should be dispensable, at least in part, for vein differentiation in the absence of cic activity. We tested this idea in two different ways. In one case, we eliminated Ras/Raf signalling in the wing by mutating factors required for EGFR activation. The hypomorphic viable combination rhove vn1 affects the activity in the wing of both the vein (vn) locus, which encodes a putative EGFR ligand (Schnepp et al., 1996), and rho, which encodes a transmembrane protein involved in EGFR activation (Sturtevant et al., 1993
). In flies mutant for this combination, the activation of MAPK in the presumptive vein cells is prevented (Martín-Blanco et al., 1999
) and as a consequence, veins fail to differentiate (Fig. 4A). By contrast, flies with the triple mutant combination rhove vn1 cic2/rhove vn1 bwk
11 display the same extravein phenotypes as cic2/bwk
11 mutant wings (Fig. 4B,C), suggesting that Cic functions downstream of EGFR activation. We generated large M+ clones homozygous for the Ras
C40b and cic2 null alleles. Mutant clones lacking only the Ras-1 (Ras85D FlyBase) function show the expected loss of vein tissue (Fig. 4D), but this effect is suppressed in clones lacking both Ras-1 and cic activities, which show ectopic vein development (Fig. 4E,F). These results support the notion that Ras/Raf signalling inhibits cic function during vein differentiation.
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Cic acts as a repressor of wing vein-specific genes
The above results suggest that EGFR signalling activates the expression of genes involved in vein patterning by antagonising cic repressor activity in presumptive vein cells. To test this hypothesis, we examined the role of cic in regulating expression of genes induced by the EGFR pathway. One such gene is aos, which encodes a secreted factor that mediates negative feedback modulation of EGFR signalling in many developmental processes (Golembo et al., 1996; Wasserman and Freeman, 1998
). In the wing, aos is expressed specifically in presumptive vein cells, where EGFR activity is maximal (Gabay et al., 1997
) (Fig. 5A). We analysed expression of an aos-lacZ reporter gene in third larval instar and pupal cic2/bwk
11 mutant discs and found a dramatic derepression compared with the wild-type pattern. Ectopic aos-lacZ expression is observed throughout the wing pouch, except in the presumptive wing margin (Fig. 5B). This ectopic expression is maintained in intervein cells until at least 30 hours APF (Fig. 5D). However, we note that in cic mutant discs, aos is expressed at higher levels in presumptive vein cells than in intervein territories, suggesting that cic is not the only factor involved in aos regulation (Fig. 5B). We have also analysed whether aos expression is repressed autonomously by cic. We find that in clones homozygous for the cic2 mutation, the aos-lacZ reporter is expressed by all the cells within the clones (Fig. 5E,F). Thus, cic behaves in a cell autonomous way as a repressor of aos transcription in intervein cells.
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The differentiation of vein cells during pupal stages also requires the function of the secreted Dpp factor, that is implicated in the refinement of vein patterning during pupal development (de Celis, 1997). In wild-type pupal wings, dpp transcripts accumulate in precise stripes corresponding to the developing veins (Fig. 6I). By contrast, such transcripts are clearly observed throughout the wings of cic2/bwk
11 mutants by 24-30 hours APF (Fig. 6J). Dpp levels are still higher in regions that will give rise to vein tissue in the mutant, but there is ectopic dpp expression in most of the future intervein cells, indicating that cic affects dpp expression in late pupal wings. This effect on a secreted factor that diffuses from its source may explain, at least in part, the non autonomous phenotypes of cic mutant clones (see Discussion).
The above results indicate that Cic is required during larval and pupal stages for repression in intervein cells of several vein-specific genes. We also checked if the expression of intervein genes is affected in the absence of Cic. One such gene is blistered (bs), which encodes a homologue of the mammalian SRF transcription factor (Montagne et al., 1996) and is autonomously required for intervein differentiation (Fristrom et al., 1994
). In wild-type discs, Bs and the Cic repressor are co-expressed in all intervein cells throughout late larval and early pupal stages (not shown). We find that Bs expression is normal in third larval instar and 6 hours APF pupal discs of cic2/bwk
11 mutant flies (not shown), indicating that cic is not required for bs regulation at these stages. However, Bs levels are clearly reduced in intervein cells at 24-30 hours APF (Fig. 6D). As Cic appears to be a dedicated repressor, it seems unlikely that it could contribute directly to the maintenance of Bs expression in the same cells; rather, we interpret this late repression of bs as an indirect effect caused by ectopic expression of vein-specific genes in intervein territories of these mutant discs. Indeed, it has been shown that ectopic expression of rho in intervein cells causes downregulation of bs during pupal stages (Roch et al., 1998
).
The analysis of Bs and Vvl expression led also to the interesting observation that both proteins are co-expressed in many cells throughout the wing pouch of cic2/bwk11 mutants (Fig. 6D,F), whereas they show almost perfect complementary expression patterns in wild-type discs (Fig. 6C,E). The co-expression of vein and intervein markers, such as Dpp, Vvl and Bs could explain the combination of vein and intervein cell morphologies observed in cic2/bwk
11 adult wings.
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DISCUSSION |
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Role of Cic in wing vein development
There are two key aspects of Cic function as a developmental regulator: its ability to repress specific target genes in defined territories, and its inhibition by the Ras/Raf pathway to allow expression of those targets in complementary positions. In the blastoderm embryo, Cic is required for development of trunk body regions and represses genes mediating differentiation of terminal structures. Torso RTK activation at each pole of the embryo alleviates Cic-dependent repression and initiates the terminal gene expression program (Jiménez et al., 2000). Our results support a similar model for cic function during specification of vein versus intervein fate in the wing. Loss of cic function in the wing causes formation of ectopic vein tissue, implying that Cic mediates intervein specification by restricting vein formation to appropriate regions. In intervein territories, Cic behaves as a repressor of vein-specific genes such as aos and vvl but does not seem to affect directly the expression of bs, which is required for the specification of intervein fates. Finally, EGFR signalling leads to downregulation of Cic protein levels in vein nuclei, thus relieving Cic-mediated repression and promoting vein development (Fig. 7).
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Moreover, vein differentiation is not a mere result of EGFR activation but depends on other signals such as Dpp and Notch (de Celis, 1997; de Celis et al., 1997
), and on the distribution of additional transcription factors that contribute to wing patterning. For example, the Collier/Knot nuclear factor has been shown to induce high levels of Bs expression between veins L3 and L4, promoting intervein development in this region (Vervoort et al., 1999
). All these inputs are linked in a complex circuit of intercellular signalling and gene regulation that progressively refines vein determination during late larval and pupal development. This signalling network could provide an explanation for the observed non-autonomy of cic phenotypes during vein specification. Thus, although cic represses aos expression in a cell-autonomous manner, this and other cic targets are likely to participate in signalling mechanisms that affect adjacent cells. Consistent with this idea, we find that cic mutant cells express ectopic Dpp product, a diffusible factor that promotes vein differentiation (de Celis, 1997
).
We have shown that in cic mutant wings, many cells differentiate, acquiring morphological features that are intermediate between those observed in either vein and intervein cells. In these wings, most cells co-express Bs and Vvl proteins, which are normally restricted to vein and intervein cells, respectively, suggesting that vein/intervein fate specification may result from a balance of these factors rather than on a simple binary switch. In this context, the concerted activities of signalling cascades such as Dpp, Notch and Ras/Raf pathways may regulate cell differentiation by modulating the balance of nuclear factors that act in a dose-dependent way. This hypothesis provides a mechanism that could explain the enormous variability observed in the cell morphologies of different insect wings.
Role of Cic as a general repressor downstream of RTK signalling
In this work, we have shown that Cic acts in wing development in a similar way to that previously described in the early embryo. Moreover, the fact that mutant clones for the Groucho repressor display extraveins, similarly to cic clones (de Celis and Ruíz-Gómez, 1995), indicates that these two proteins could interact as partners during wing development, as it is the case during embryonic development (Jiménez et al., 2000
). Indeed, we have observed weak genetic interactions between different cic and gro alleles during wing development (F. R., unpublished). Thus, Cic and Gro could be part of a conserved repressor complex downregulated by the Ras/Raf molecular cassette in different cellular contexts. In this regard, the phenotype of bwk mutations suggests that Cic may also function as a target of other RTK signals during patterning of the eggshell in the ovary (Rittenhouse and Berg, 1995
). However, it should be noted that cic does not seem to act in all developmental processes mediated by Ras/Raf signalling in Drosophila. For example, the eyes of cic mutant flies appear normal (F. R., unpublished), even though the Ras/Raf pathway controls several aspects of cell fate specification and patterning in this tissue (Freeman, 1997
). These observations support the idea that the Ras/Raf pathway can regulate cell specification in a cic-independent way depending on the cell context.
Previous work has shown that during patterning of ovary follicle cells, the expression of rho is controlled by the Ras/Raf pathway via another transcriptional repressor, the CF2 protein (Hsu et al., 1996). CF2 is tagged for cytoplasmic retention and degradation after direct phosphorylation by MAPK (Mantrova and Hsu, 1998
). The Cic protein has also consensus sites for phosphorylation by MAPK (Jiménez et al., 2000
), suggesting that Cic levels could be regulated post-transcriptionally in a similar way to CF2. This indicates that localised downregulation of specific repressors is a common mechanism for the activation of target genes by the Ras/Raf pathway. The identification of highly conserved cic homologues in mice and humans suggests that regulation of gene expression by RTK signalling in vertebrates may also involve relief of Cic-dependent repression.
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ACKNOWLEDGMENTS |
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