Immunolocalization of the Cg protein using three independent anti-sera has shown uniform Cg expression in all imaginal discs and in many larval tissues, as well as uniform expression in embryos. As was most clearly seen in the peripodial membrane of imaginal discs and in larval tissues, such as the fat body, the protein is enriched in the cell nuclei in all tissues (Svendsen, 2000 and Campbell, 2000).
The effects of cg mutations on limb development can be largely if not completely explained through misregulation of ci. Does the zinc-finger protein encoded by cg directly regulate ci expression? Recombinant Cg protein produced in E. coli does not show specific binding to the 5' region of the ci gene using gel mobility-shift assays. However, the distribution of Cg protein was examined in the polytene chromosomes of salivary glands and dozens of strong, discrete binding sites were found on each of the five major chromosome arms, and many more weak sites of Cg binding. This staining is not observed in the chromosomes of cg2/cg2 mutants. Of particular interest, a band of Cg binding was found on chromosome 4 at approximately 101F, the cytological location of the ci gene. This suggests that the Cg protein may be a direct regulator of ci transcription. Attempts to show that Cg localizes to ci regulatory elements in transgenes inserted at ectopic chromosomal locations have been confounded by the large number of endogenous Cg-positive bands (Svendsen, 2000).
In order to understand how the ubiquitously expressed Cg protein acts in A/P limb patterning, the effect of cg mutations on both morphological and molecular markers of limb development was investigated. Wings from the three cg genotypes producing viable adults (cg1/cg1, cg1/cg2 and cg1/cg3) have defects in some combination of the posterior compartment longitudinal veins 4 and 5 (L4 and L5) and the anterior compartment longitudinal vein 2 (L2). Defects in vein L4, ranging from a slight thinning of the vein to gaps of increasing size, are seen in 100% of wings from all hetero-allelic combinations. There is a higher penetrance of posterior compartment (L4 and L5) defects in cg1/cg1 flies, which is predicted to be the weakest mutant combination, based on both viability and leg phenotypes. Wings from the stronger cg1/cg2 mutants have a higher incidence of anterior compartment defects. More rarely (<1%), all three genotypes display outgrowths in the costal region of the anterior wing margin. These outgrowths are reminiscent of the pattern triplications caused by mutations in ci and other genes affecting Hh signaling (Svendsen, 2000).
The L4 gap phenotype is also seen in loss-of-function en alleles and in gain-of-function ci regulatory alleles, both of which result in ectopic posterior ci expression. Vein L4 gaps have also been reported in flies heterozygous for both cg1 and either en or ci alleles, as well as compound heterozygotes of en and ci. Compound heterozygotes of all three cg alleles with Df(2R)en 11 -- a small deficiency removing engrailed and invected -- have the L4 gap phenotype. Flies heterozygous for cg2 or cg3 and ciW have an enhancement of the vein L4 defects observed in ciW/+. Many of these lack vein L4 completely and thus resemble ciW/ciW homozygous wings. The dominant interactions between ci, en and cg suggest that the ectopic ci expression may be due to the disruption of En-mediated repression of ci in posterior cells, and that cg plays a role in this regulation of ci by En (Svendsen, 2000).
To clarify the relationship between cg, ci and en further, the expressions of ci and en were examined in cg mutant wing imaginal discs. Using the 4D9 antibody that recognizes both En and Inv, it was found that the expression of En/Inv is normal in cg mutant discs. To assess the effects of cg on ci expression, ci-lacZ reporter constructs were crossed into cg mutant backgrounds. Weak ectopic expression of ci-lacZ was found in the posterior compartment and reduced anterior ci-lacZ was found, compared with wild-type. To confirm these results and exclude the possibility of an artefact that was due to the reporter construct, cg imaginal discs were stained using antibodies to Ci proteins. The different forms of Ci could be distinguished using either an N-terminal-directed antibody that recognizes both Ci-155 and Ci-75, or an antibody directed against the C terminus that is specific for Ci-155. The levels of Ci-155 are reduced in the anterior compartments of cg mutant discs and Ci-155 is ectopically expressed in the posterior. This is also true of overall Ci levels detected with the N-terminal-directed antibody. The effects are more pronounced in stronger allelic combinations. For example, in cg1/cg1 homozygotes, there is less of a reduction of Ci in the anterior compartment but more ectopic expression of Ci in the posterior compartment, when compared with the strongest allelic combination, cg2/cg2. Thus, effects of cg mutants on the expression of ci are very similar to those described for gain-of-function ci alleles that are disrupted in a regulatory element 5' of the ci gene. These mutations also result in the ectopic expression of ci in the posterior compartment and diminished expression of ci in cells of the anterior wing compartment. Given these results and the similarity of the phenotypes of the cg and ci gain-of-function alleles, it seems most likely that the effects of cg on wing patterning are due to the misregulation of ci. The wild-type function of cg in wing development appears to be different in the two compartments (Svendsen, 2000).
How does the misexpression of Ci in the cg mutants affect the regulation of hh and Hh-responsive genes ptc, dpp, and kn? In even the strongest cg mutant backgrounds, hh-lacZ and dpp-lacZ are expressed in the wild-type pattern. The transcription of both hh and dpp are normally repressed in the anterior compartment by Ci-75 and thus sufficient levels of Ci-75 must be present in cg mutants for the repression of both hh and dpp. The occasional anterior bifurcations seen in cg mutant wings may be due to rare (<1%) ectopic expression of either dpp or hh and may thus have been missed in the sampling. Unlike dpp and hh, two Hh-responsive genes activated by Ci-155 in the intervein region between veins L3 and L4 are altered in cg mutants. The ptc gene is normally expressed weakly throughout the anterior compartment of wing discs, except for an elevated level of expression in the stripe of anterior organizer cells adjacent to the A/P compartment boundary. In cg mutants, Ptc is ectopically expressed in the posterior cells, consistent with observed ectopic Ci expression in cg mutants. The kn locus encodes a transcription factor required for the suppression of vein formation in the 3/4 inter-vein. kn expression is usually restricted to the stripe of anterior cells adjacent to the A/P boundary in the wing pouch. Using an anti-Kn antibody, it was found that, like ptc, this protein is expressed ectopically in posterior cells of cg mutant imaginal wing discs. In general, the levels of both ectopic and endogenous Kn decreases with increasing strength of the cg allelic combination used, which in turn probably reflects altered ci expression. In cg1/cg1 homozygotes, the levels of staining in the endogenous domain of Kn expression is similar to wild type, with weaker, ectopic staining observed in posterior cells. In stronger allelic combinations such as cg3 /cg3 and cg2/cg2, lower levels of both endogenous and ectopic Kn were observed. This may reflect the lower levels of both endogenous and ectopic Ci detected in stronger cg allelic combinations. The relatively high levels of Ci and Kn expression in the posterior compartments of wings from the weaker cg1/cg1 mutants may also explain the higher incidence of posterior compartment vein defects in this background (Svendsen, 2000).
The leg phenotypes of all allelic combinations were examined (except for cg2 homozygotes, which die prior to leg differentiation). All combinations exhibit an expansion of anterior dorsal and anterior ventral pattern elements, most notably in the tibia and in the tarsae. For example, two ventral anterior markers, the sex combs and the transverse rows, are abnormal in the male prothoracic legs in all cg mutants. The sex comb abnormalities range from a few extra bristles in cg1/cg1 through to highly disorganized structures consisting of as much as a threefold increase in the number of sex comb teeth in cg2/cg2 legs. The transverse rows are similarly affected. Duplications of dorsal structures such as the pre-apical bristle and occasional leg bifurcations are also observed. These overgrowths of ventral and dorsal anterior structures and pattern duplications are consistent with the changes in dorsal and ventral molecular marker gene expression observed in cg mutants. Owing to variability of the observed phenotypes, it was difficult to order the severity of the phenotype; however, cg1/cg1 clearly have the mildest defects, followed by cg1/cg2 and cg1/cg3, which have intermediate defects, followed by cg3cg3 and cg2/cg2, which are the most severe. This is the same order of allele strength as predicted by viability (Svendsen, 2000).
The effects of cg on wing development are very similar to the effects of gain-of-function ci alleles. However, even the mildest leg defects seen in cg mutants are more severe than the ciW gain-of-function mutants, which have normal legs. Using the N-terminal-directed antibody that recognizes Ci-155 and Ci-75, a decrease in overall Ci levels is observed in leg imaginal discs in the weakest cg mutant combinations and substantial decreases in the stronger cg allelic combinations. The overall reduction of anterior Ci staining in cg2/cg2 legs is less severe than that observed in cg2/cg2 wings and, in contrast to wing discs, no ectopic Ci staining has been observed in posterior legs (Svendsen, 2000).
Can the changes in Ci levels also explain the observed defects in A/P patterning? The expression of En/Inv was examined in leg imaginal discs, as well as the expression of ci targets hh, ptc, wg and dpp. No ectopic expression of posterior markers is observed using anti-En/Inc, hh-lacZ or anti-Hh sera. Leg imaginal discs from cg mutants are expanded in the A/P axis, and staining of hh-lacZ or En/Inv in cg2/cg2 leg discs shows that the expansion is largely due to a substantial increase in the relative size of the anterior compartment. This overgrowth is associated with the ectopic expression of ptc, dpp and wg. There is increased expression of Ptc and ectopic expression of dpp-lacZ throughout the anterior compartment of cg2/cg3 mutant leg discs. Using a monoclonal antibody to Wg, weak ectopic expression was observed in the anterior ventral quadrant of cg2/cg3 mutant leg discs. To confirm that there is an expansion of Wg signaling, the expression of H15-lacZ, a target of Wg regulation in the ventral leg that is normally expressed in a ventral wedge centered around the Wg domain, was examined. The expression of H15-lacZ expands throughout the anterior ventral quadrant of the cg3/cg3 leg imaginal discs. The regulation of Hh-responsive genes is very similar in leg and antennal development. Antennae in cg mutants have an overgrown phenotype reminiscent of the defects seen in cg mutant legs and similar ectopic expression of ptc, dpp, wg and H-15 (Svendsen, 2000).
A likely explanation for the observed effects on the spatial distribution of dpp and wg in combgap mutant legs and antenna is that there is insufficient Ci-75 to repress the two genes ectopically expressed in anterior cells. This has been reported for loss-of-function mutations in ci. This was directly tested by seeing if additional ci expression in cg mutants using a transgene could rescue cg leg phenotypes. Since Ptc expression throughout the anterior compartments of cg mutant legs is still present, and indeed somewhat elevated, ptc-gal4 was used as a tool to further increase ci expression in its normal domain using UAS-ci. The expression of dpp-lacZ in discs of the genotype ptc-gal4 cg1/dpp-lacZ cg2;UAS-ci/+ was compared with ptc-gal4 cg1/dpp-lacZ cg2 controls. Control flies have increased expression of dpp-lacZ in the anterior compartment and exhibit the previously observed overgrowth of the disc in the A/P axis, while flies carrying the UAS-ci transgene show no upregulation of the dpp-lacZ reporter and have normal disc morphology. Control flies lacking the UAS-ci transgene have the leg overgrowth typical of cg1/cg2, while flies from the same cross carrying the UAS-ci transgene are similar to the milder phenotype seen in cg1/cg1 flies, the weakest cg mutant combination. A similar rescue was observed of the cg antennal phenotype and Wg and dpp-lacZ expression in antenna (Svendsen, 2000).
Screening through a collection of P-element-induced mutations generated by the BDGP has identified a line, l(2)07659, in which homozygous larvae possess leg discs morphologically identical to those generated by ubiquitous hh expression. This phenotype is not associated with ectopic expression of hh, and loss of Hh activity using a temperature-sensitive mutant has no effect on the phenotype of l(2)07659 discs. However, both dpp and wg are misexpressed in the anterior of these discs, accounting for the overgrowth and ectopic al expression. In the posterior, wg and dpp are repressed as in wild-type discs. Curiously, although dpp is misexpressed, the level of expression, even at the compartment border, is lower than in wild-type discs. Ubiquitous expression of Hh also induces ectopic dpp expression and overgrowth in the wing, but there is no overgrowth in l(2)07659 mutant wing discs and there is only very weak ectopic dpp expression (Campbell, 2000).
l(2)07659 was mapped to region 50E1-2 by the BDGP. Mobilization of the P-element results in a high frequency of excisions (>50%) that are completely wild type, indicating that the P-element is responsible for the lethality of this line. Complementation tests with genes in the same region have identified it as an allele of cg and it has been renamed cg07659 . A single viable allele, cg1 , had been identified previously with two main phenotypes: (1) the number of sex comb teeth on the male first leg is increased (an anterior compartment phenotype associated with ectopic wg expression); and (2) there is a gap in vein IV in the wing (a posterior compartment phenotype). cg1 /07659 heterozygotes are also viable with a slightly more severe phenotype than cg1 homozygotes. It appears that cg07659 is a very strong allele identical in severity to a putative null, cgA22 (Campbell, 2000).
Although cg07659 homozygotes phenocopy mutants such as ptc, Pka-C1 and costa (cos), which show hedgehog gain-of-function phenotypes, the posterior compartment in the latter mutants is unaffected, unlike cg adult wings, indicating that Cg function may not be restricted to Hh signal transduction. cg1 interacts strongly with en and some ci mutants. The ci mutants used in these studies are gain-of-function mutants in which ci is misexpressed in the posterior, resulting in defects in posterior wing vein patterning, most notably loss of vein IV. These genetic interactions were confirmed with cg07659 , as follows. ciW is a weak dominant mutation with heterozygotes showing a variable loss of vein IV tissue. The cg07659 /+;ciW /+ transheterozygotes have a much stronger phenotype with vein IV being completely absent in all flies. Many viable en loss-of-function mutants have gaps in vein IV and some also show this phenotype very occasionally as heterozygotes, but en59+/+cg07659 transheterozygotes show this at a much higher frequency. This suggests ci and possibly en activities may be disrupted in cg mutants (Campbell, 2000).
Analysis of cg mutant discs reveals Ci expression is abnormal. Instead of high levels in the anterior and no expression in the posterior, Ci is expressed at low levels throughout. An antibody that recognizes all Ci proteins reveals fairly uniform levels of expression in both anterior and posterior compartments of the leg and wing discs, but at much lower levels than are found in the anterior of wild-type discs. A Ci antibody that recognizes only the full-length form (stabilized by Hh signaling) reveals higher levels in the posterior than in most of the anterior. Clonal analysis was used to demonstrate that the effect of cg on Ci expression is cell autonomous. cg clones in the anterior show autonomous reduction in ci levels while clones in the posterior show autonomous gain of ci expression (Campbell, 2000).
En is required to repress ci in wild-type discs, but posterior En expression in cg mutant discs appears normal. This was clearly demonstrated by clonal analysis since En expression in cg mutant clones in the posterior is indistinguishable from that in surrounding wild-type cells. Thus, it appears that En is unable to repress ci expression normally in cg discs. Loss of cg had a minor effect on En expression, but this is in the anterior compartment of the wing. Although En is usually restricted to the posterior, in late third instars, en expression is activated in the anterior of the wing at the compartment boundary and this is Hh-dependent. cg mutant cells immediately anterior to the compartment border (defined by clonal analysis) do not express En in late third instar wing discs (Campbell, 2000).
Loss of ci activity in the wing results in Hh-independent dpp expression in the anterior compartment, so it appears possible that the ectopic dpp and wg in the anterior of cg leg discs may result directly from the lowered Ci levels found in these discs. Initially an investigation was carried out to see whether loss of ci in leg discs also results in a hedgehog gain-of-function phenotype as it does in the wing. This was achieved by analysis of hypomorphic ci loss-of-function allelic combinations. These are pupal lethal with larvae having wing and leg discs that phenocopy ubiquitous Hh expression. Ci levels are dramatically reduced in these discs and the mutant phenotype is Hh independent. dpp is misexpressed in the anterior wing pouch and in the dorsal anterior of the leg discs and wg is misexpressed in the ventral anterior of the legs. The ectopic dpp expression is repressed by raising ci levels with a wild-type UAS-ci transgene driven by a Gal4 line. Thus, normal dpp and wg expression requires high levels of Ci in the anterior because this functions to repress their expression away from the compartment border (Campbell, 2000).
To test whether the ectopic dpp and wg in cg leg discs is a direct result of the lowered ci levels in these discs, ci levels were raised in cg mutants with a ubiquitous Gal4 driver. The higher ci levels rescue the overgrowth and dpp misexpression phenotypes, indicating that these anterior compartment combgap phenotypes are the direct result of lowered ci levels and consequently that one of the functions of cg is to maintain high levels of ci expression in the anterior (Campbell, 2000).
Point mutant cgY4 was identified because en59 +/+ cgY4 adults show a loss of vein IV. cgY4 is associated with a substitution of a conserved amino acid in the third zinc finger and appears to be a dominant negative mutant because, even though the vein IV phenotype over en59 is more penetrant than cg07659 (100% compared with 25%), it has a weaker phenotype as a homozygote, showing no overgrowth in leg discs. In addition, cgY4 homozygotes have a weak dominant phenotype, having an increase in the number of sex comb teeth compared with wild type, and it is not fully rescued by the Cg247 genomic fragment. Point mutant cgA22 was identified as a revertant of the dominant negative activity in cgY4. en59 +/+ cgA22 transheterozygotes are indistinguishable from en59/+ heterozygotes with regard to vein IV. cgA22 is fully rescued by Cg247 and has a stop codon between the third and fourth zinc fingers, suggesting that it may be a null, although it is impossible to rule out that the first three zinc fingers provide some function. cgA22 homozygotes have a phenotype almost identical to that of cg07659, indicating that the latter is close to being a complete loss-of-function allele (Campbell, 2000).
A combgap mutation was recovered in a screen for mutants in which retinal axons form an aberrant pattern of projections in the brain. This screen monitored the pattern of retinal axon projections in the late third instar stage when about half of the full complement of retinal axons have reached their target positions. In sum, ~3100 mutagenized second chromosome lines were screened, and 157 candidate lines with abnormal retinal axon projection patterns were recovered. Candidate lines were screened for defects in optic lobe development by examining the spatial distribution of proliferating cells and the expression of cell typespecific markers. An interesting phenotype was observed in animals homozygous for the lethal PlacW transposon insertion in the strain l(2)k11504. l(2)k11504 contains a single lethal transposon insert at cytogenetic position 50D that reverted with precise excision of the transposon. A lethal deletion mutation, cgDelta10, was derived from imprecise excision of the insertion. Complementation analysis with loci in the 50D region revealed that these mutations are lethal alleles of the gene combgap (cg). combgap was first identified by Bridges in 1925 as a mutation with pleiotropic effects on bristle number, wing venation, and oogenesis. A single hypomorphic allele of combgap, cg1, has been described. cg1 homozygotes and cg1/cgk11504 heterozygotes display these defects, as well as a visual system phenotype. In the wild-type brain, retinal axons project in a crescent-shaped array into the lamina target field. In cgk11504 animals, ingrowing retinal axons form an irregular pattern of projections, with axons often straying outside the normal target field (Song, 2000).
Campbell, G. L. and Tomlinson, A. (2000). Transcriptional regulation of the Hedgehog effector Ci by the zinc-finger gene combgap. Development 127: 4095-4103. PubMed citation: 10976042
House, V. L. (1953). Some observations on the interaction of comb-gap with Hairless, engrailed and alleles at the cubitus interruptus locus in Drosophila melanogaster. Genetics 38: 669-670
House, V. L. (1961). Mutant effects in multiple heterozygotes of recessive venation mutants in Drosophila melanogaster. Genetics 46: 871
Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-3765. PubMed citation: 16077094
McClure, K. D. and Schubiger, G. (2008). A screen for genes that function in leg disc regeneration in Drosophila melanogaster. Mech. Dev. 125(1-2): 67-80. PubMed citation
Song, Y., Chung, S. and Kunes, S. (2000). Combgap relays Wingless signal reception to the determination of cortical cell fate in the Drosophila visual system. Mol. Cell 6: 1143-1154. PubMed citation: 11106753
Svendsen, P. C., et al. (2000). The combgap locus encodes a zinc-finger protein that regulates cubitus interruptusduring limb development in Drosophila melanogaster. Development 127: 4083-4093. PubMed citation: 10976041
Waddington, C. H. (1953). The interactions of some morphogenetic genes in Drosophila melanogaster. J. Genet. 51: 243-258
date revised: 30 July 2008
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