BIOLOGICAL OVERVIEW

The combgap (cg) locus, first described by C. B. Bridges in 1925, is a gene required for proper anteroposterior pattern formation in the limbs of Drosophila. combgap mutation has pleiotropic effects on bristle number (most notably for the male sex comb), wing venation , and oogenesis. Based on genetic interactions and phenotypic analysis, cg was assigned to the 'vein' group of loci that function in wing vein patterning (Song, 2000 and references therein). combgap encodes a chromosomal protein with 11 C2H2 zinc fingers. Limb defects found in combgap mutants consist of either loss or duplication of pattern elements in the anteroposterior axis and can be explained through the inappropriate expression of cubitus interruptus (ci) and its downstream target genes (Campbell, 2000; Svendsen, 2000; Song, 2000). A genetic interaction between cg and some ci mutants was identified many years ago (House, 1953, 1961; Waddington, 1953). In cg mutants, ci is ectopically expressed in the posterior compartments of wing imaginal discs and is downregulated in the anterior compartment of legs, wings and antennae. Combgap protein binds to polytene chromosomes at many sites including the ci locus, suggesting that it could be a direct regulator of ci transcription. The adult viable cg mutants are sterile and also exhibit defects such as eye roughening and ectopic thoracic bristles. Several deficiencies from the Bloomington Deficiency Collection have dominant phenotypes (i.e., wing notching, ectopic veins) when in compound heterozygotes with cg². While some of these phenotypes may be due to effects on ci regulation, others are not easily ascribed to changes in ci expression. Thus, cg may be required for the regulation of many genes but ci may be particularly sensitive to reduction in cg function (Svendsen, 2000).

Transcriptional control of the ci gene and post-translational regulation of the Ci protein are essential for the placement and function of the A/P organizer in limb development. The action of the Hedgehog-signaling pathway results in a relatively high ratio of Ci-155 (the activator form of Ci) to Ci-75 (the repressive form of Ci) at the A/P boundary, which ensures the localized expression of Hh-responsive genes necessary for the formation of the A/P organizer. Hh signaling influences the post-translational modification of the Ci protein at multiple levels including proteolytic cleavage, phosphorylation, subcellular location and nuclear import. In contrast to the details of post-translational modification of Ci-155 by elements of the Hh-signaling pathway, relatively little is known about the transcriptional control of ci. Decreased levels of both Ci protein and expression from ci-lacZ reporter constructs are observed in the anterior compartments of cg mutant imaginal discs, as well as ectopic expression in posterior cells of wings. Thus, Cg affects both the activation and repression of ci transcription (Svendsen, 2000).

A dominant interaction between cg and engrailed/invected mutations that gives rise to a gap in vein L4 strongly suggests that Cg and En/Inv act together to repress posterior ci transcription. Posterior expression of En represses the transcription of ci resulting in anterior specific expression. En has been shown to interact directly with the ci regulatory elements. In cg mutant wing imaginal discs, weak ectopic expression of ci-lacZ reporter constructs are found in posterior cells, thus Cg may act in concert with En to repress posterior ci. Hypomorphic mutants in either cg or en/inv can give rise to the reduction in vein L4 that is characteristic of ectopic ci expression (Svendsen, 2000).

Many proteins with multiple C2H2 zinc finger motifs like those found in cg have been shown to be transcription factors, DNA-binding proteins or chromatin proteins. The widespread localization of Cg on salivary gland chromosomes is consistent with all of these activities. While the data have not yet established direct action of Cg on the ci regulatory elements, binding of Cg to the ci region of polytene chromosomes suggests that Cg could be a direct regulator of ci transcription. Direct binding of Cg (produced in E. coli) to DNA from the ci regulatory region has not been detected. However, given that the transcriptional regulation of ci is likely to be complex, Cg may not act at the level of direct DNA binding. The involvement of the Pc-group genes in the repression of ci suggests that intricate regulatory modes are necessary to maintain the correct levels and spatial patterns of ci transcription during imaginal disc development. Furthermore, the ci-regulatory regions have been shown to be subject to transvection effects, indicating that interchromosomal interactions also govern ci regulation. Thus Cg may act at any level, from generally influencing the chromosome pairing through to direct binding of ci enhancer elements. Finally, the positive and negative effects of cg mutants on ci transcription and the genetic interaction with en/inv suggest that Cg may be required in conjunction with other transcription factors for the function of ci enhancers and that Cg may not specify activation or repression itself (Svendsen, 2000).

The changes in the A/P pattern observed in cg mutant limbs are caused by the mis-regulation of Hh-responsive genes regulated by the Ci-155 and Ci-75 transcription factors. In cg mutant wing imaginal discs, Ci-155 is ectopically expressed in the posterior compartment and is associated with posterior compartment defects and posterior misexpression of genes such as patched and knot. Ectopic expression of kn is sufficient to suppress vein fate. Thus, the misexpression in the posterior compartment of kn and other genes regulated by high levels of Ci-155 probably leads to the vein defects described in this study. The occurrence of both higher levels of posterior Ci and Kn expression, and higher frequency of posterior compartment defects in cg¹/cg¹ mutant wings supports this explanation. Stronger allelic combinations of cg have lower levels of ectopic Ci and Kn, and lower incidence of posterior compartment wing defects, but they result in a greater reduction in Ci in the anterior compartment and more anterior vein defects. The posterior and anterior vein defects, as well as occasional anterior wing margin bifurcations, resemble the effects of regulatory mutants of ci that cause the ectopic expression of ci in posterior cells and the reduction of ci expression in the anterior compartment (Svendsen, 2000 and references therein).

In legs and antennae, overall Ci levels are decreased in the anterior compartment, resulting in circumferential overgrowth of the anterior compartment and ectopic anterior expression of the morphogens wg and dpp. Similar effects on leg morphology have been previously reported when wg and dpp were ectopically activated in anterior cells. The rescue of cg mutant leg defects by additional expression of ci in the anterior compartment using the Gal4/UAS system indicates that the phenotypes result from a reduction of Ci-75 leading to the derepression of wg and dpp. Thus, it is concluded that the Cg protein is critical for the proper levels and spatial patterns of Ci and that the A/P limb patterning defects in cg mutants are due largely, if not completely, to mis-regulation of ci (Svendsen, 2000).

The effects of cg mutants on ci expression are seen only in the anterior compartment of legs but in both anterior and posterior compartments in wings. What is the basis for this difference? While anterior Ci is reduced in both limbs, ectopic posterior Ci is only seen in wings. One possibility is that the alleles that have been studied may have different effects on cg expression in anterior versus posterior compartments and/or legs versus wings. However, little Cg imaginal disc staining was seen in cg²/cg², suggesting little or no Cg protein is produced, and so the phenotype may be near null (there are no deficiencies uncovering the cg locus, so this could not be tested genetically). Cg may not be required for repression of ci in the posterior compartment of leg discs, or alternatively, there may be a much lower threshold for Cg function in legs. The different effects on ci expression in cg mutant leg and wing imaginal discs suggest that while the broad framework is similar, there may be unique aspects to A/P patterning in dorsal versus ventral limbs. The predominance of wing phenotypes in ci^W and similar ci regulatory mutations also suggests a difference in the way ci is regulated in wings versus legs. Another difference was seen in the effects of reduced Ci levels on the expression of dpp. A greater reduction of Ci staining is seen in the anterior compartments of wings compared with leg imaginal discs; paradoxically, ectopic expression of dpp is seen in all cg mutant leg imaginal discs but none is seen in cg²/cg² wing imaginal discs. Although there are limits to how accurately real levels of Ci may be inferred from histochemical staining in different tissues, the simple conclusion is that dpp responds to different thresholds of Ci in legs and wings, and that the effect of cg is indirect (Svendsen, 2000).

Similar conclusions about the role of cg in ci regulation have been reached in the Campbell (2000) study. The posterior wing venation defect in cg hypomorphs is very similar to that found in ci mutants and this phenotype is enhanced in cg/+;ci/+ transheterozygotes. These ci mutants, however, are gain-of-function mutants; they show ectopic expression of ci in the posterior. In fact, direct misexpression of ci in the posterior using the UAS/Gal4 system can also produce the same vein defects as seen in these mutants and in cg mutants. Analysis of cg mutant discs reveals ectopic ci expression in the posterior, indicating that the cg posterior phenotype is almost certainly the direct result of deregulation of ci expression in this compartment (Campbell, 2000).

Ci expression is also abnormal in the anterior of cg mutant discs, being found at much lower levels than in wild-type discs. Loss of ci expression in the wing results in hedgehog gain-of-function phenotypes, including overgrowth and misexpression of dpp. Reduced Ci levels in the leg also result in the characteristic overgrowth phenotype, with ectopic expression of wg and dpp, found following ubiquitous expression of Hh -- i.e., the same phenotype as that found in cg mutant leg discs. Support for the proposal that the anterior combgap phenotype in the leg is also the direct result of deregulation of ci expression, in this case lowered levels of expression, comes from the observation that raising ci levels in cg mutant leg discs using the UAS/Gal4 system can suppress the overgrowth and ectopic dpp expression (Campbell, 2000).

One difference between ci and cg mutants is that wing discs from the former have a hedgehog gain-of-function phenotype with overgrowth and ectopic dpp in the anterior, while the latter do not show overgrowth and only very weak ectopic dpp. It is possible that the leg and wing are differentially sensitive to Ci levels and the Ci levels are still high enough in the wing in cg mutants to repress most dpp expression. Protein levels detected with antibody staining in ci hypomorphs and cg mutants are too low to detect significant differences with confidence, so the reason for the difference between ci and cg wings remains to be determined. Ci is also required during embryogenesis, but the putative null cg mutant survives to the early pupal stage. This suggests either that lower levels of Ci are sufficient for embryonic but not larval development or that cg RNA is maternally supplied. The first possibility is supported by the observation that hypomorphic ci mutants are not embryonic lethal and survive to the early pupal stage. However, in situ analysis reveals that CG RNA is maternally supplied so that the question of whether cg is required during embryogenesis will require the generation of germline clones (Campbell, 2000).

Full-length Ci acts as a transcriptional activator and there is evidence that the lowered levels of Ci in cg mutants also compromises Ci function as an activator. Although, dpp is misexpressed in cg discs, the level of expression, even at the compartment border, is lower than that found in wild-type discs. A similar phenomenon has been demonstrated for loss of ci in the wing and it appears that the high levels of dpp in wild-type discs require activation by Ci-155, as well as the absence of Ci-75. Thus, the lower levels of dpp in cg discs are presumably due to lower levels of Ci-155. Another gene directly activated by Ci is en in late third instar wing discs. Ci-dependent en activation in the anterior compartment does not occur in cg mutant cells, again presumably because the level of the Ci-155 activator form is too low (Campbell, 2000).

Thus Cg is required to activate ci expression to its normal levels in the anterior compartment and to repress ci expression in the posterior. The Cg protein contains multiple zinc fingers and is most probably a DNA-binding protein that would be expected to bind to elements at the ci locus. However, understanding the mechanism by which it regulates ci expression requires further studies. It is possible that Cg functions as a standard transcription factor and activates ci transcription in the anterior and represses it in the posterior. If this is the case, its activity must be modified in either the anterior or posterior compartments. Analysis of the Cg protein outside of the zinc fingers does not reveal any classical activator or repressor domains, but as these are often not well defined it is impossible to determine whether the protein has these activities without more-detailed studies (Campbell, 2000).

An argument against such a direct involvement of Cg in transcription is the well-documented role of En in regulating ci expression. En is a transcription factor that represses expression of several genes including ci, dpp and wg, and has been shown to bind to elements at the ci locus. It would appear likely that En is the primary factor that represses transcription of ci in the posterior. If this is the case, the function of Cg in regulating transcription may be indirect and may be to assist the binding of other transcription factors to the ci gene. If so, the misexpression of Ci in the posterior of cg mutant discs would be due to a lowered ability of En to bind in the absence of Cg protein, while the lowered Ci levels in the anterior would be due to a lowered ability to bind a currently unidentified transcriptional activator of ci. There are several possible mechanisms by which Cg might affect the binding of other factors. For example, there may be direct physical interactions between Cg and these other factors. Alternatively, Cg action could be more indirect, for example, it could modify chromatin structure at the ci locus producing a more open conformation. Further studies are required to test these possibilities (Campbell, 2000).

The morphogenesis of specialized structures within the CNS relies on the nonautonomous activity of cell populations that play the role of organizers. In the Drosophila visual system, cells on the dorsal and ventral margins of the developing visual cortex express the Wnt family member Wingless (Wg) and the TGF-beta Decapentaplegic (Dpp). The activity of these morphogens in establishing cortical cell fates sets the stage for the guidance of photoreceptor axons to their retinotopic destinations in the Drosophila brain. One role for Wg in cortical development is to induce and maintain the expression of Dpp, a key step in the assignment of dorsoventral cell identities. Dpp is induced early in cortical development, shortly after the onset of Wg expression in a few dorsal and ventral margin cells, and is maintained by Wg activity until at least the time of retinal axon pathfinding. Wg is a developmental signal in many different tissues, and acts by regulating different target gene sets to elicit a constellation of different cell fates. Wingless-controlled targets include distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, labial in the gut, and sloppy-paired in the embryonic CNS. Conversely, Dpp belongs to a Hedgehog-controlled circuit in the wing (Song, 2000 and references therein).

A regulatory mechanism is described that relays Wg signal reception to the tissue-specific expression of target genes in the visual cortex. In a screen for mutants in which photoreceptor axons project aberrantly to their destinations in the brain, a mutation in combgap was discovered. Retinal axon navigation defects in combgap animals are due to the role of cg in the establishment of cortical cell identity. cg represses the expression of Wg target genes in a positionally restricted manner in the visual cortex. wg+ induction of its cortical cell targets occurs via the downregulation of cg. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination of cell fate in the visual cortex (Song, 2000).

The translation of extracellular signals into cell-type identities is an important step in the elaboration of complex patterns of neuronal connectivity. In the visual system of Drosophila, a population of about 40 cells set aside in embryogenesis gives rise to thousands of cortical neurons that form three precisely interconnected visual ganglia. Hundreds of neuronal and glial cell types are generated, with more than 120 distinct neuronal cell types identified in the medulla cortex alone. Wingless plays an important role in the determination of visual system cortical cell fates, acting from cell populations at the dorsal and ventral margins to induce expression of the morphogen Dpp and the transcriptional regulators Distal-less. combgap is a key regulator in this pathway for cortical cell fate determination (Song, 2000).

A combgap mutation was recovered in a screen for mutants with aberrations in retinal axon projections. On the basis of its effects on target region gene expression and the outcome of mosaic analysis, it is evident that a role for combgap in the specification of cortical cell identity underlies its requirement for the establishment of retinotopic connectivity in the visual system. In cg loss of function animals, three markers under wg⁺ control are expressed in expanded dorsal and ventral portions of the retinal axon target field. The requirement for cg to repress the markers within these domains is autonomous. The lamina midline region, however, appears phenotypically normal in homozygous or mosaic cg animals. This positionally restricted requirement for cg⁺ activity is correlated with the pattern of cg expression, since cg is not expressed in the midline region where it is not required. Since wg⁺ misexpression is sufficient to induce wg⁺-dependent markers in the midline region, another regulatory system must control these markers there. Hence, the consequences of wg signal reception at different dorsoventral positions within the cortical precursor field would appear to involve a set of regulatory molecules that divide the cortex into specific domains for pattern formation (Song, 2000).

As in the wing and leg, cg clones that include the cortical lamina are deficient in Ci expression. These effects on Ci expression could account for the reduction of the Hh-controlled expression of Engrailed and Dpp at the anterior-posterior compartment border of the developing wing. However, Ci loss and gain of function experiments have not revealed a role for Ci in the regulation of Wg target gene expression in visual system cortical cells where Ci functions as in other imaginal tissues as an effector of Hedgehog signal reception. Moreover, the cortical markers under Wg control do not respond to Hh (Song, 2000).

The constellation of genes under Wingless control displays considerable tissue specificity. Wingless-controlled targets include Distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, and sloppy-paired in the embryonic CNS. Though Dpp and Omb belong to a Hedgehog-controlled circuit in the wing, they are under Wg control in the visual cortices of the brain. With respect to the control of cell fate, Wg signal transduction apparently follows a canonical pathway from a pair of redundant receptors at the cell surface to the cytoplasmic control of Armadillo stability and nuclear translocation. This raises the question of how the tissue specificity of wg target gene expression is achieved (Song, 2000).

The observations that cg regulates dpp, optimotor blind and aristaless in the visual cortex place cg in a second tier of regulation, as a component of a tissue-specific relay mechanism between the Wg signal transduction pathway and the target genes that are wg dependent in visual system cortical cells. The evidence in support of this hypothesis is as follows: (1) epistasis analysis with the wg pathway negative regulator Axn places the requirement for cg downstream of the cytoplasmic complex that includes APC, GSK-beta, and Armadillo; (2) the induction of at least three downstream effectors of wg⁺ activity is mediated by negative regulation of cg expression -- cg expression is reduced in the dorsal and ventral domains of the cortical lamina where these wg target genes are expressed and ectopic cg expression blocks wg target gene expression within these domains; (3) ectopic wg⁺ clones repress cg expression, yielding Cg-negative domains in which wg target genes are ectopically expressed. The presence of consensus Pangolin binding sites in the first intron of cg suggests cg may be a direct target of Wg signal transduction. How the Armadillo/Pangolin complex might participate in the negative regulation of cg is unclear. Cg might act by binding directly to wg target gene regulatory elements as a transcriptional repressor (Song, 2000).

Additional molecules have been identified as tissue- and/or stage-specific modulators of wg signal transduction. The lines gene product is specifically required for late stage Wg signaling in the dorsal epidermis of the embryo. Wg regulates the nuclear accumulation of Lines rather than its expression. Teashirt, a zinc finger protein, modulates Wg signaling specifically in the ventral epidermis by binding to the C terminus of Arm. In addition, Smad transcriptional effectors of TGF-beta signaling can bind the Arm/dTCF complex to mediate tissue-specific cross-regulatory interactions between the TGF-beta and Wnt pathways. Along with the mode of action of Cg in the visual cortex, the limited number of examples so far point to a diversity of mechanisms for achieving the tissue-specific consequences of Wnt signaling (Song, 2000 and references therein).

GENE STRUCTURE

Genomic length - 7 kb

Exons - 9

PROTEIN STRUCTURE

Amino Acids - 671

Structural Domains

The predicted open reading frame of LD05357 contains 11 canonical C₂H₂ zinc-finger sequences (C-X_1-2-C-X₃-F-X₅-L-X₂-H-X_3-4-H) typical of many DNA- and chromatin-binding proteins. A glutamine-rich region (19/24 residues) spans residues 569 to 592. No significant matches were found to other proteins outside the zinc-finger and glutamine-rich motifs (Svendsen, 2000 and Campbell, 2000).

combgap encodes a protein with ten putative Krüppel-like C2H2 zinc finger domains. The amino-terminal region of ~150 amino acids bears little homology to known proteins. The first eight zinc fingers follow in a tandem array, each separated by 6-7 amino acids. Similar clustering of multiple zinc fingers, as well as certain conserved amino acids between and within the zinc fingers, are found in several predicted human, mouse, and Xenopus multi zinc finger proteins. The presence of a glutamine-rich region flanking the ninth and tenth zinc fingers suggests that Combgap may act as a DNA binding transcription factor. Consistent with this role, Cg displays nuclear localization (Song, 2000).

date revised: 5 December 2000

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