Transcriptional Regulation

Rhomboid and Ras activities prevent net transcription. Ectopic expression of rho under the control of a heat-inducible promoter during late third instar or early pupal development causes broadening of veins and ectopic vein formation, whereas absence or reduction of rho activity, or impaired Egfr-dependent signaling, generates the complementary phenotype, i.e. loss of vein structures. To test if the development of ectopic veins is linked to the repression of net, net transcription was examined in wing discs after ectopic activation of UAS-rho by MS1096. Indeed, in such discs, which express rho strongly in almost the entire disc (the pattern is identical to that of net transcripts), no net transcripts are detectable, which demonstrates that ectopic rho expression represses net transcription. It appears that in these wing discs all intervein regions develop as veins, since the resulting adult wings have a tube-like appearance and consist entirely of vein-like tissue. This phenotype is much stronger than that of net null mutants. Ectopic Rho protein suppresses intervein fate in regions where such a fate is independent of net expression. Hence, it is assumed that in these regions rho represses intervein-promoting and vein-suppression genes that are different from net, and thus activates vein-promoting genes. These genes may show partial redundancy with net functions in regions in which their expression overlaps that of net (Brentrup, 2000).

To investigate further whether rho mediates its repression of net through activated components of the Egfr signaling pathway, constitutively active Ras protein was expressed in MS1096/+;UAS-Dras1V12 /+ wing discs. Constitutive Ras activity produces overgrown wing discs that fail to express net. Although the adult wing phenotype could not be observed in these flies because they die as early pupae, ectopic activation of the Ras/mitogen-activated protein kinase (MAPK) signaling pathway in wing discs has been shown to give rise to ectopic veins. These results illustrate that ectopic expression of rho or activated components of the Egfr signaling pathway represses the transcription of net and presumably of additional vein-suppression genes, and suggest that the repression of these genes is a prerequisite for vein formation (Brentrup, 2000).

Wing discs in which Ras is constitutively active accumulate high levels of rho transcripts in intervein regions. In contrast, rho expression is disrupted in wing discs and pupal wings that are homozygous for rhove. In such discs, net transcription expands into the vein primordia of third instar wing discs and remains expressed ectopically in the distal portions of L3 to L5 of pupal wings, where vein development is suppressed. This result is consistent with the observation that ectopic expression of net is able to repress vein formation only in regions where vein formation depends on rho expression. These findings show that net and rho transcripts accumulate in mutually exclusive patterns in developing wings, and specify intervein and vein precursor cells, respectively. Thus, net expression is negatively regulated by Egfr signaling and, in turn, represses rho expression (Brentrup, 2000).

Finally, UAS-net and UAS-rho were co-expressed ubiquitously under the control of MS1096. Surprisingly, while ubiquitous expression of either net or rho in developing wings generates curved wings with suppressed distal L5 or tube-like wings composed of vein-like tissue, ubiquitous co-expression of net and rho results in a nearly wild-type wing blade. Since the ubiquitous expression of Rho, which is under the control of MS1096, is independent of Net, this result indicates that Net represses, in addition to rho, other vein-promoting genes downstream of Rho in Egfr signaling. If this were not the case and the only function of Net was to repress the endogenous rho gene, the same phenotype would be expected after ubiquitous co-expression of Net and Rho as after ubiquitous expression of Rho, rather than the nearly wild-type wing observed. Therefore, Net suppresses vein development in interveins through a control that consists of at least two tiers, one repressing rho, the other interfering with the activation of vein-promoting genes downstream of Rho-enhanced Egfr signaling. Curiously, although ubiquitous expression of Net is able to suppress the effect of ubiquitously co-expressed Rho in intervein regions, it is unable to repress vein development in veins. It follows that differences between future vein and intervein regions exist that are sufficient to correctly determine vein versus intervein fates in the presence of an excess of both Net and Rho. These findings further suggest a considerable redundancy in the gene networks participating in the specification of vein versus intervein fates. This conclusion is also borne out by the observation that if both Rho and Net are reduced or lost in rhove:net1 double mutants, veins are less severely truncated than in wings of rhove single mutants (Brentrup, 2000).

Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators involved in the maintenance of developmental decisions. Although their function as transcriptional regulators of homeotic genes has been well documented, little is known about their effect on other target genes or their role in other developmental processes. The patterning of veins and interveins in the wing has been used as a model with which to understand the function of the trxG gene ash2 (absent, small or homeotic discs 2). ash2 is required to sustain the activation of the intervein-promoting genes net and blistered (bs) and to repress rhomboid (rho), a component of the EGF receptor (Egfr) pathway. Moreover, loss-of-function phenotypes of the Egfr pathway are suppressed by ash2 mutants, while gain-of-function phenotypes are enhanced. These results also show that ash2 acts as a repressor of the vein L2-organising gene knirps (kni), whose expression is upregulated throughout the whole wing imaginal disc in ash2 mutants and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes, and (2) keeping kni in an off state in tissues beyond the L2 vein. It is proposed that the Ash2 complex provides a molecular framework for a mechanism required to maintain cellular identities in the wing development (Angulo, 2004).

Loss of ash2 function causes differentiation of ectopic vein tissue, indicating that ash2 is required for intervein development, where it functions as an activator of the intervein-promoting genes net and bs, restricting rho expression to vein regions. In addition, the loss-of-function phenotypes of Egfr alleles are rescued in ash2 mutants, while the gain-of-function phenotypes are enhanced. Furthermore, rho mRNA exhibits an expanded expression pattern in ash2 mutant tissues. Thus, ash2 promotes the maintenance of intervein fate, either by activation of net and bs or by repression of the Egfr pathway. Since rho and bs/net expression is mutually exclusive, it cannot be determined whether the Ash2 complex interacts directly with one or all of them. However, since bs expression is inhibited by the loss-of-function of ash2 during larval and pupal stages, it can be proposed that ash2 acts as a long-term chromatin imprint of bs that is stable throughout development (Angulo, 2004).

Targets of Activity

In homozygous net1 wing discs, rhomboid is ectopically expressed in broad domains, alternating with areas devoid of rho expression. To determine if this pattern of rho expression in wing discs reflects the null phenotype of net mutants, rho expression was examined in net deficiency wing discs, which fail to express net in the wing pouch. These discs display the same rho expression during larval and pupal stages as described for net1 mutants, i.e. rho expands into all intervein regions with the exception of the sector between veins L3 and L4, but the effect is most pronounced in the distal intervein regions B (between L2 and L3) and D (between L4 and L5). Hence, it appears that net represses rho in intervein regions of the wild-type wing, except in the region between L3 and L4. These effects of net mutations on rho expression are consistent with the null phenotype in the wing blade of net deficiency flies, which show ectopic veins most frequently in the distal intervein sectors B and D. This net null phenotype is indistinguishable from that of net1 mutants, which confirms the assumption that the bHLH domain of Net is indispensable for its function (Brentrup, 2000).

To test if Net acts as a repressor of rho transcription, net was expressed ectopically in wing discs by means of the GAL4/UAS system. When activated by the GAL4 driver line MS1096, a GAL4 P-element insertion at the Beadex locus, UAS-net is expressed nearly ubiquitously in the wing pouch and widely represses rho transcription, which displays a pattern similar to that in homozygous rhove discs. Consistent with this effect on rho expression, the wing phenotype of MS1096/Y;UAS-net flies is almost identical to that of homozygous rhove flies: the most distal part of L3 and the distal half of L5 are missing, while the distal part of L4 is also absent or interrupted by gaps. In contrast, L2 is wild type except in occasional flies, in which its most distal part is missing, although rho expression is clearly absent from L2 proveins. In addition, wings in which net is ectopically expressed during development are smaller (Brentrup, 2000).

Interestingly, when the dosage of MS1096 and UAS-net is reduced to one copy in net1 females, the net1 phenotype is rescued. The suppression of distal L5 formation is independent of net1, while L4 is no longer suppressed at the reduced dosage. The absence of the anterior crossvein is not caused by the ectopic net expression, but induced by the MS1096 driver in the absence of its UAS-net target (Brentrup, 2000).

It is concluded that Net is able to repress the transcription of rho in the wing pouch, but net activity is crucial for the repression of rho in wild-type discs, mainly in the distal intervein sectors A, B, D and E. Repression of rho by ectopic Net has no effect on the proximal portions of wing veins and, in most flies, on L2, which is consistent with the phenotype of rho;ve mutants, demonstrating that vein formation completely depends on rho activity only in the distal portions of longitudinal veins L3 to L5 (Brentrup, 2000).


Northern blot analysis shows that the 2.2 kb net transcript is expressed during all stages of the Drosophila life cycle. In situ hybridization of a net cDNA probe to whole-mount embryos and imaginal discs reveals complex expression patterns. Since, during development, the putative net lack-of-function alleles cause discernible defects only in the wing, the specific expression of net in the embryo may serve a redundant function. Therefore, the net function has been analysed only during wing development. In wing discs, net expression is first observed in early third instar larvae. net transcripts are confined throughout the third instar to prospective intervein sectors and to a narrow region straddling the wing margin, but are excluded from primordial wing veins expressing rho. The complementary expression patterns of net and rho are maintained until the pupal wings have developed and expression of rho is restricted from seven- to eight-cell wide stripes of vein-competent provein cells to the narrow two to three cell wide stripes of future vein cells in P1 pupae. Shortly before this P1 pupal stage, net expression is enhanced along the wing margin and in regions flanking proveins (Brentrup, 2000).

During P1 and the subsequent P2 pupal stage, when crossveins are recognizable, net is expressed in the intervein regions, but continues to be repressed in the broad provein stripes. After 30 hours APF (after puparium formation) at 22°C, net remains repressed in proveins while its expression in intervein regions is considerably reduced. Thus, net and rho expression appear to be mutually exclusive during the entire development of the wing, although net expression does not expand when rho expression is reduced to the narrow stripes of vein cells in P1 pupae (Brentrup, 2000).

Effects of Mutation or Deletion

The Extramacrochaetae gene encodes a transcription factor with an HLH domain without the basic region involved in interaction with DNA present in other proteins that have this domain. Emc forms heterodimers with bHLH proteins preventing their binding to DNA, acting as a negative regulator. The function of emc is required in many developmental processes during the development of Drosophila, including wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc indicate that during wing morphogenesis emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation. The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signaling pathway (egfr, vein, veinlet and Gap), blistered, plexus and net, in both adult wing phenotypes and cell behavior in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behavior of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signaling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically, during cell differentiation, repressing Emc expression (Baonza, 1997).


Angulo, M., Corominas, M. and Serras, F. (2004). Activation and repression activities of ash2 in Drosophila wing imaginal discs. Development 131(20): 4943-53. 15371308

Baonza, A. and Garcia-Bellido, A. (1997). Dual role of extramacrochaetae in cell proliferation and cell differentiation during wing morphogenesis in Drosophila. Mech. Dev. 80(2): 133-46.

Brentrup, D., Lerch, H.-P., Jackle, H. and Noll, M. (2000). Regulation of Drosophila wing vein patterning: net encodes a bHLH protein repressing rhomboid and is repressed by rhomboid-dependent Egfr signaling. Development 127: 4729-4741.

Cook, O., Biehs, B. and Bier, E. (2004). brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development 131: 2113-2124. 15073155

Diaz-Benjumea, F. J. and García-Bellido, A. (1990). Genetic analysis of the wing vein pattern of Drosophila. Roux's Arch. Dev. Biol. 198: 336-354

Matakatsu, H., Tadokoro, R., Gamo, S. and Hayashi, S. (1999). Repression of the wing vein development in Drosophila by the nuclear matrix protein Plexus. Development 126: 5207-5216. A>

net: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 9 December 2004

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