distal antenna & distal antenna-related
The overlap between Hth and Dll has been proposed to define antennal identity, because co-expression of the two proteins in ectopic locations can induce formation of ectopic arista structures in other discs. To ask whether Hth and Dll have a role in defining the non-overlapping expression domains of Cut and Dan/Danr, clones of cells were examined lacking hth or Dll activity in the antenna. Dan expression is lost in cells mutant for hthc1 in the region where the two expression domains overlap. This suggests that Hth activity is required for Dan expression. Likewise, clones of cells lacking Dll activity have lost Dan expression in the distal region of the disc. Many Dll mutant clones were found adjacent to the edge of the Dan domain, suggesting that loss of Dan may cause these clones to sort out proximally. Thus both Dll and Hth are required for Dan expression (Emerald, 2003).
Ectopic expression of Hth in the leg disc under dppGal4 control, induces Dan expression in distal, Dll-expressing cells. It is known that Hth-expressing cells sort out from the distal region of the disc. This is also visible in GFP labeled cells in this study. Nonetheless, dppGal4 directed expression of Hth induces Dan expression in distal cells. This raises the possibility of a non-autonomous effect of Hth expression leading to sustained Dan expression. Ectopic expression of Dll in the leg disc under dppGal4 control, induces Dan expression in proximal, Hth-expressing cells. In this case, ectopic Dan was limited to cells expressing the Gal4 driver (Emerald, 2003).
These observations indicate that the regulatory relationship between Hth, Dll and Dan (Danr) is complex. Dll and Hth are each required for Dan expression. However, it is clear that Dan is not expressed in every cell in which Hth and Dll are co-expressed in the antenna. All Dan-expressing distal antenna cells express Dll but not all express Hth. These observations point to a non-autonomous effect of Hth on Dan expression, which may explain how Hth can be required for sustained expression of Dan in distal cells where Hth is not expressed (Emerald, 2003).
ss is expressed in the distal antenna in a domain similar to that of Dan/Danr. ss mutants cause transformation of distal antenna to tarsus, suggesting a role in antennal identity. To determine whether ss regulates Dan and Danr, antenna discs were examined from spinelessaristapedia (ssa) mutants. ssa alleles appears to reduce ss activity specifically in the antenna. Dan expression was lost from the distal part of ssa mutant antennal discs. Loss of Dan expression in ssa mutant antenna discs coincides with ectopic expression of Antennapedia. These expression domains appear to be non-overlapping. Ectopic expression of Antennapedia is sufficient to cause transformation of antenna to leg. The observation of ectopic Antp in the distal part of the third antennal segment (within the Dan domain) may explain the homeotic transformation of distal A3 and arista in the ss mutant. It was next asked whether ss is sufficient to induce Dan and Danr expression in the leg disc. Ectopic expression of ss in randomly positioned clones of cells causes expression of Dan and Danr in the wing and leg discs. These observations suggest that ss defines the domain of Dan and Danr expression (Emerald, 2003).
Next, the relationship between ss and Cut was examined. Ectopic expression of ss using ptcGal4 causes ectopic expression of Dan and repression of Cut expression. Repression of Cut is stronger on one side of the disc and is associated with antenna duplication. Ectopic expression of Dan or Danr has no effect on Cut expression, suggesting that ss may act directly to regulate Cut. The ability of ss to repress Cut, contrasts with the observation that cut expression is normal in ss mutants. It is possible that there are redundant mechanisms for Cut repression, one of which is mediated by ss (Emerald, 2003).
To ask whether Cut regulates Dan and Danr, an examination was made of expression clones of cells lacking cut activity, generated using the null allele cut145 and the FLP-FRT system. cut145 mutant clones do not cause ectopic expression of Dan in proximal regions of the antenna disc, but rather show limited expansion of the Dan domain in the region where Dll is expressed. Comparable effects on Danr expression were seen in cut mutant clones. Ectopic expression of Cut in the Dan domain using Act>CD2>Gal4 causes repression of Dan (Emerald, 2003).
Taken together these results suggest that distal expression of ss limits the expression domain of Cut to the proximal antenna. ss is required for Dan and Danr expression in distal antenna. At present it is not possible to determine whether expression of Dan and Danr in response to ss is mediated directly or indirectly by repression of Cut. However, the view that it is direct is favored because removal of Cut does not cause extensive ectopic expression of Dan (Emerald, 2003).
Antibodies were raised to the predicted Dan and Danr proteins. Both are nuclear proteins, expressed in the eye-antenna disc. Double labeling with anti-Dan and anti-Danr shows that the two proteins are co-expressed in the antenna. Both proteins are also expressed in the brain and the eye region of the eye-antenna disc. Antibody labeling of other imaginal discs shows limited Dan expression in small groups of cells in leg and wing. These appear in the location of prominent sense organ progenitors at relatively late stages of disc development. Danr was not detected in other discs (Emerald, 2003).
To define the domain of Dan and Danr expression in the antenna, a series of double labeling experiments were performed with antibodies to other antennal proteins. Homothorax (Hth) is expressed in the presumptive head capsule and in antennal segments A1 to A3. Hth overlaps with the proximal part of the Dan domain. Distal-less (Dll) is expressed in a distal domain comprising A2, A3 and the arista. Dll overlaps the Dan domain, but extends further proximally. Cut is expressed in the proximal part of the antenna, in a domain that does not overlap expression of Dan. In optical cross-section there appears to be one row of cells with low expression of Dan at the interface between these domains. The domain of Dan/Danr expression appears to coincide with the domain in which ss transcript is expressed (Emerald, 2003). The relationship between Dan, Hth and Dll expression suggests that the Dan domain corresponds to segment A3 and the arista and that Cut is expressed in A1 and A2 as well as the head capsule. Thus, in addition to the broadly overlapping domains of Hth and Dll, the antenna is subdivided into adjacent and perhaps mutually exclusive proximal and distal domains reflected by ss, Cut and Dan/Danr expression. Although the view is favored that the reciprocal expression of Dan/Danr and Cut is likely to define the border between antenna segments A2 and A3, it is noted that it is difficult to be precise about the location of the border before overt segmentation. The possibility exists that Dan and ss expression may extend into distal A2 (Emerald, 2003).
To delimit more precisely the expression of the hern and fer (danr and dan respectively) genes in the antennal primordium, double staining was carried out of the MD634 and CES115 GAL4 lines (revealing hern and fer transcription, respectively) with genes expressed in restricted areas of mature eye-antennal discs, like Dll, hth, sal, and dac. Both lines gave the same results. Within the third antennal segment, the hern and fer expression is included within the Dll and hth domains. The hern and fer proximal limit of expression seems to coincide with that of dac, and their distal limit of strong expression in the third antennal segment with that of sal. It cannot be exclude that the GAL4 lines drive expression in a few cells from the second antennal segment (Suzanne, 2003).
The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc. The gene names 'Hernandez' and 'Fernandez' are an homage to twin brothers, characters in the Tintin comic-book series. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).
From a collection of P-GAL4 lines that drive expression in restricted areas of the adult cuticle, four lines were selected that specifically direct a similar expression of the yellow (y) gene: the third antennal segment, the arista, and a few bristles in the second antennal segment express the y gene. These lines, when crossed to a line carrying an UAS-lacZ reporter construct, show ß-galactosidase expression in three regions of the eye–antennal disc: in the third antennal segment, in the arista, and within the eye, in the differentiated eye and in a region just anterior to the morphogenetic furrow (Suzanne, 2003).
The insertion site of these four lines was located by inverse PCR: two lines (CES75 and MD634) are in the 5' upstream region of the predicted CG13651 transcription unit and the other two (AC116 and CES115) are in the 5' upstream region of the CG11849 transcription unit, according to the annotated Drosophila genome. The two transcription units are located in the chromosomal position 96C2-4, in the same orientation and nearly adjacent, separated by about 45 kb. The CG13651 transcription unit is 1.259 kb long, bears no introns, and produces a 1259-bp mRNA, whereas the CG11849 transcription unit is 7.175 kb long, has 3 exons, and makes a mRNA of 2232 bp. There is an EST for the CG11849 unit, indicating that this predicted gene is transcribed (Suzanne, 2003).
The CG13651 and CG11849 transcription units encode predicted proteins of 419 and 743 amino acids, respectively, that have 27% identity and 37% similarity between them. Both proteins contain similar 69-amino-acid regions, located in their N-terminal part, with 78% identity and 88% similarity between them. This region includes a previously characterized 48-amino-acid Pipsqueak (Psq) motif, present in several proteins in Drosophila and other species, including human. The Psq motif is a helix-turn-helix DNA-binding domain similar to the DNA-binding domain of prokaryotic recombinases, which, in turn, also show similarities with the homeodomain. The CG13496 predicted Drosophila gene codes for a protein with a Psq domain very similar to those of CG13651 and CG11849 genes. The CG13496 transcription unit is not expressed in the eye-antennal imaginal disc like the CG13651 and CG11849 genes (Suzanne, 2003).
In situ hybridization experiments show that, among all the imaginal discs, the CG13651 and CG11849 transcription units are only and equally expressed in the eye-antennal disc. Their expression is very similar to that observed with the P-GAL4 lines, except that, in the eye primordium, the RNA signal in the differentiated eye is very weak. The same antennal and eye expression persists in early pupal stages. Therefore, these genes present a similar sequence, expression in the eye-antennal disc, regulation, and function. These genes have been called hernandez (hern, CG13651) and fernandez (fer, CG11849), and together are referred to as the Tintin genes (Suzanne, 2003).
Since hern and fer are not expressed in the leg discs, they may be part of the mechanism that distinguishes legs from antennae. The Antennapedia (Antp) Hox gene, which is expressed in the leg but not in the antennal discs, prevents hth expression and antenna formation in the leg primordia. As expected, in Antp73b/+ flies, which show a strong transformation of antennae into legs, the expression driven by the MD634 GAL4 line is clearly reduced (Suzanne, 2003).
To characterize the function of hern and fer in normal development, the phenotype of flies without hern or fer activity was studied. One of the P-GAL4 lines, AC116, is mutant for the antennal function of the fer gene. Homozygous AC116 third instar larvae show no fer transcription in the antennal primordium but present normal fer expression in the eye primordium. AC116/Df adults show one or more bristles in the third antennal segment, normally devoid of them. To obtain more mutations in the hern and fer genes, the MD634 and CES115 P-GAL4 lines (the two closer to the hern and fer transcription units) were mobilized to isolate imprecise excisions of the transposons. ferI49-1, one w- derivative of the CES115 insertion, was isolated that in hemizygosis, shows a phenotype very similar to that described for the AC116/Df adults. PCR analysis of the mutation revealed that it is a small deletion of about 2.5 kb in the 5' upstream region of the fer transcription unit. Larvae homozygous for the ferI49-1 mutation present reduced expression of the fer RNA in the antennal primordium of mature eye-antennal discs, whereas the expression in the eye primordium is normal. It is concluded that the AC116 insertion and the ferI49-1 deletion may have affected an antennal regulatory region. This regulatory region would control only the fer gene, since in AC116 and ferI49-1 homozygous larvae, no change was detected in hern expression. No mutation has been obtained for the hern gene (279 w- derivatives analyzed), although the MD634 line used for this purpose is closer to the origin of transcription of the hern gene than the CES115 line is to the transcription unit of the fer gene (Suzanne, 2003).
The weak phenotype of the AC116 and ferI49-1 mutations suggests that there may be a partial functional redundancy between hern and fer, and that the inactivation of both genes may result in a stronger phenotype. To check this, to ascertain the phenotype of hern inactivation, and to study the effect of the absence of Tintin products in the eye, double-stranded (ds)-mediated RNA interference (RNAi) was used to inactivate hern and fer functions. The inactivation in the antennal primordium of either the hern or the fer genes with a Dll-GAL4 (MD23) driver causes a phenotype similar to that described for the AC116/Df and ferI49-1/Df adults: there is one or more bristles in the third antennal segment and the base of the arista is slightly enlarged. However, when the ds-hern and ds-fer RNAs are induced together by the Dll-GAL4 driver, a clear transformation of part of the distal antenna into leg is observed: the third antennal segment and the proximal arista are substantially enlarged and covered with bristles; those in the base of the arista bear bracts, indicating a transformation into leg.. These results indicate that both hern and fer are required to develop part of the antenna as opposed to leg and that these two genes are partially redundant in this function (Suzanne, 2003).
To test whether hern and fer are sufficient to induce eye or antennal development, they were expressed ectopically using the GAL4/UAS system. When either the hern or the fer genes are misexpressed in the leg discs with dpp-GAL4 or Dll-GAL4 (EM212) drivers, distal legs are transformed to aristae. These transformations are accompanied by the ectopic expression of hth, sal, and ss, three genes expressed in the antennal primordium but not in the distal region of mature wild-type leg disc. Clones expressing either the hern or the fer genes in the leg or wing disc have smooth borders and frequently activate the sal and hth genes cell-autonomously. In dpp-GAL4/UAS-fer or ptc-GAL4/UAS-hern leg (or wing) discs, the expression of ss is also activated. Curiously, although ss is downstream of hth in the antenna and leg, ectopic ss in the leg disc can also activate hth in a few cells (Suzanne, 2003).
The hth or ss genes, together with Dll, are sufficient to develop ectopic distal antennae when expressed in different regions of the adult. The hern or fer genes are also able to elicit this transformation in the leg and they activate hth and ss. Conversely, when high levels of the Hth or Ss products are induced in the leg discs, ectopic expression of the hern and fer genes is found. To study the interactions between these genes in normal development, the relationship between Dll, hth, ss, and hern/fer in the antennal primordium was examined. A reduction of Hth activity using a dominant negative form of hth (UAS-EN-HTH1-430) results in a decreased activity of the MD634 and AC116 GAL4 lines, which reveal hern and fer expression, respectively. Similarly, in antennal discs of a Dll strong hypomorph or a ss null mutation, the expression of hern and fer disappears. These results suggests that hth, Dll, and ss are required to maintain hern and fer expression in the antenna. By contrast, high levels of hern or fer may reduce hth expression. In dpp-GAL4/UAS-fer or dpp-GAL4/UAS-hern larvae, the expression of hth (and sal) in the third antennal segment is eliminated or strongly reduced dorsally (where levels of hern and fer are high) and does not change or is ectopically activated ventrally (where levels of hern and fer are low). Similarly, fer-expressing clones are able to downregulate hth expression in the antennal primordium. These results suggest that levels of hern and fer expression may be important for a normal antennal development (Suzanne, 2003).
hern and fer genes are required for normal eye development and form eye tissue and activate ey when ectopically expressed. To study the role of the hern and fer genes in eye development, the eye phenotype was examined when either the hern or fer genes are inactivated by RNAi or are expressed ectopically. Expression of ds-hern or ds-fer RNA in the eye primordium with a GMR-GAL4 driver causes a slightly rough eye, with some bristles irregularly positioned. Curiously, the phenotype is not increased if the ds-hern and ds-fer RNAs are induced in the same fly. Misexpression experiments also suggest that both hern and fer are involved in eye development. Thus, the expression of either hern or fer with different GAL4 drivers causes the appearance of ectopic eye tissue in the third antennal segment or rostral membrane. These transformations are accompanied by the ectopic expression of ey, although this effect may also indicate the maintenance of a previous ey expression. Conversely, the misexpression of ey activates the hern and fer genes ectopically. Both hern and fer also activate embryonic lethal abnormal vision (elav), a marker of neuronal differentiation, when ectopically expressed. The analysis of clones expressing the fer gene in the leg, eye-antennal, or wing discs shows that elav activation is strictly nonautonomous, and only occurs in some cells adjacent to some of these clones (Suzanne, 2003).
The formation of the morphogenetic furrow in the eye is limited laterally by wg signaling. hern and fer expression within the eye primordium includes the more lateral wg-expressing regions. Interestingly, both hern and fer activate wg transcription when ectopically expressed. In ptc-GAL4/UAS-hern or dpp-GAL4/UAS-fer flies, the wings show several alterations, including the appearance of marginal bristles in the middle of the wing blade. This phenotype is characteristic of ectopic wg signaling, and in fact, wg is ectopically expressed in the wing discs of these larvae. Clones expressing the fer genes in the eye-antenna, leg, or wing discs also show induction of wg, mostly within but also outside the clone. The elav gene is also induced nonautonomously by these clones. Cells ectopically expressing elav do not coincide with those expressing wg and this reproduces the wild-type situation in the eye (Suzanne, 2003).
Signaling pathways can modify the activity of selector genes and are needed for proper organ formation. N signaling, for instance, is needed for eye formation and can activate ey when ectopically activated. Moreover, N has been implicated in the decision of making eye or antenna, directing eye development, and suppressing antenna formation. Therefore, whether N signaling could alter the ey and elav expression induced by the Tintin genes was examined. The coexpression of the hern gene and a dominant negative form of the Notch receptor substantially reduces ey and eliminates elav ectopic signals. Accordingly, no ectopic eyes are formed in this genetic combination. This indicates that the effect of hern on ey expression and eye formation requires N signaling (Suzanne, 2003).
The concept of selector genes in Drosophila has evolved from a precise and restricted definition to a more loose interpretation. Selector, or selector-like genes, are now considered as those required to make a particular structure and able to form it in different positions when the gene is expressed ectopically. hern and fer fit this definition as selector genes for the distal antenna. They also can make ectopic eyes, although their requirement for eye development is not so evident as that for antenna formation (Suzanne, 2003).
The differentiation of legs or antennae depends on the activity of the hth and Antp genes. The ss gene, however, is also able to transform distal leg (and also maxillary palp and rostral membrane) into distal antenna, and the absence of ss, like that of hth, transforms antenna into leg. Although ss seems to be downstream of Dll and hth in antenna specification, ectopic ss can activate hth in some cells of the leg disc. Similarly, misexpression of ss in the rostral membrane induces Dll expression. It seems, therefore, that ss can trigger an antennal genetic program when misexpressed in certain places (Suzanne, 2003).
The fer and hern genes are both required and sufficient to make part of the distal antenna. Four different genes, hth, ss, hern, and fer, are able to form distal antenna, together with Dll, when ectopically expressed. Their mutual regulation seems to differ when misexpressed in the leg disc or when normally expressed in the antennal primordium. In the leg disc, hern or fer activates hth and ss and, reciprocally, hth and ss induce hern and fer expression. Moreover, even ss can promote hth transcription, although just in a few cells. Taken together, these results suggest that the four genes can form distal antenna by activating each other's transcription when ectopically expressed (Suzanne, 2003).
In the third antennal segment, Dll, hth, and ss are required to activate hern/fer expression. Since ss is downstream of Dll and hth in the antenna, the activation of hern/fer by Dll and hth could be mediated by ss. It is noted, however, that the levels of hern and fer may modulate hth expression. Moderately increased levels of fer can activate hth in dpp-GAL4/UAS-fer discs but, when the levels of hern or fer in the antenna are highly increased, the transcription of hth is prevented. These results suggest that the total amount of hern and fer expression may be regulated in the antennal primordium. Accordingly, in clones mutant for danr (hern), the expression of dan (fer) is upregulated. Also supporting the conclusion that levels of hern and fer have to be regulated, it was found that, in ey-GAL4/UAS-hern or ey-GAL4/UAS-fer flies, where levels of either hern or fer are highly increased in the eye–antennal disc, both the eye and the antenna disappear (Suzanne, 2003).
Several eye-specifying genes have been identified, and they fulfill two conditions: they are required to make the eye and they can form ectopic eyes when expressed in different parts of the body. The hern and fer genes probably form part of this network of 'eye-specification' genes: (1) they are expressed in the eye primordium, with higher levels of expression anterior to the morphogenetic furrow; (2) they activate ey and elav and make ectopic eyes when expressed ectopically; (3) ey also activates the hern and fer genes when ectopically expressed. hern and fer genes have also been identified as downstream of ey in eye ectopic formation (Michaut, 2003). However, the inactivation of both hern and fer genes by RNAi with the GAL4 driver does not grossly affect eye development, as do mutants in the eye-specification genes. The nonautonomous induction of elav when hern or fer are ectopically expressed reproduces the wild-type situation, in which high levels of hern and fer are observed adjacent to the differentiating, elav-expressing, photoreceptor cells. Another similarity of hern and fer with some of the 'eye-specification'genes is that ectopic eye tissue is obtained in the antennae. The eye-specification genes eya and dac also form eyes predominantly, when ectopically expressed, in this same position. This is perhaps due to ey being expressed in the antennal primordium in late embryos, thus providing a favorable genetic context for eye formation. In accordance, when either the hern or the fer gene is ectopically expressed, ectopic ey expression is detected only in the antennal disc. Eyes are also obtained in the rostral membrane when ectopically expressing the fer gene. This may be due to the absence of hth, since high levels of either hern or fer repress hth and removal of this gene in the rostral membrane forms ectopic eyes (Suzanne, 2003).
The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).
Transformations are observed of third antennal segment, where hern and fer are normally transcribed, to eye tissue, in Dll-GAL4/UAS-hern or dpp-Gal4/UAS-fer flies. This suggests that the levels of Hern and Fer products may be important in inducing or maintaining ey expression and distinguishing eye from antenna. Accordingly, when Hern or Fer products are increased in the antennal primordium, the expression of hth, an inhibitor of eye development, is eliminated. It is also noted that, in the wild-type eye-antennal discs, hern and fer show higher levels of expression in the eye primordium than in the antennal one, where these genes are coexpressed with hth. However, the amount of Tintin product is not the only factor in this distinction, since, for instance, in Dll-GAL4/UAS-hern eye-antennal discs, the area of ectopic ey transcription in the antenna is smaller than the area of hern overexpression. The activity of other genes will probably contribute to the formation of either eye or antenna. Thus, the ectopic expression of either hern or fer induces wg, an inhibitor of morphogenetic furrow formation, and this probably limits the places where the eye can develop (Suzanne, 2003).
Two recent models have been proposed to explain the specification of eye and antenna within the eye-antennal disc. Both models suggest that the activation of the N signaling pathway is a key element in this process. It has been suggested that N signaling activates both ey and Dll in the eye and antennal primordia; subsequently, ey represses Dll in the eye and perhaps the hth and extradenticle genes repress ey in the antenna. In this way, the exclusive expression of ey (in the eye) and Dll and hth (in the antenna) determine eye and antenna identity, respectively. It has been proposed that the N and Egfr signaling pathways (together with the hedgehog and wg genes) are instrumental in the decisions to make eye or antenna. N signaling has been proposed to promote eye development and prevents formation of the antenna, whereas Egfr signaling does the opposite. Ectopic expression of either hern or fer in the antenna induces ectopic eyes and activates ey and elav, but the coexpression of hern and an N dominant-negative protein does not result in ectopic eyes and almost eliminates ey and elav activation. This suggests that N function impinges on hern activity to form ectopic eyes. As in other cases, the combined activity of signaling pathways and selector genes determine the specification of different structures (Suzanne, 2003).
To assess the roles of dan and danr in antenna development in detail, deletions that remove one or both genes were examined. Larvae homozygous for these deletions are viable. danrex35 is a small deletion that removes part of the Danr coding sequence. Antibody staining reveals loss of Danr protein in clones of cells mutant for danrex35. Interestingly, Dan protein is upregulated in these clones. Expression of both proteins is lost in homozygous dan danrex56 mutant clones; this loss of expression shows that ~45 kb between the two original P-element insertions have been removed. In both deletion homozygotes, the third antennal segment is reduced in size and develops ectopic bristles. In dan danrex56 animals the third antennal segment is generally smaller and more ectopic bristles are produced. In addition, the basal cylinder of the arista are enlarged and produce bracted bristles. Bracted bristles are typical of the distal leg and suggest partial transformation of antenna towards leg in the mutant tissue. A similar transformation of the basal cylinder was observed in mosaic antennae derived from ey-FLP danrex35/Minute heterozygous animals. The transformation associated with dan danrex56 homozygous clones was similar, but slightly stronger(Emerald, 2003).
Although many dan danr double mutant excisions were recovered, none was singly mutant for dan alone. To generate a dan mutant a screen was performed for EMS-induced revertants of the dan gain-of-function phenotype in the wing. One allele was recovered. Sequence analysis revealed a change of amino acid residue 45 from glutamate to lysine. This alteration lies in the conserved pipsqueak domain and affects a residue thought to be important for DNA binding of a related protein. When expressed in the leg disc under control of DllGal4, danems3 causes loss of the claws, but does not cause transformation to arista, suggesting a weaker gain of function phenotype than the wild-type protein. Thus, danems3 appears to be a hypomorphic allele that reduces but does not eliminate Dan activity. danems3 homozygotes were viable and show a mild antenna defect, including an occasional ectopic bristle in the third antennal segment. A stronger ectopic bristle phenotype was obtained when Dan activity was reduced using a Gal4 inducible construct that directs expression of a double-stranded dan RNA (DllGal4; sympUAST-dan). To verify that the inducible RNAi causes reduction of Dan protein levels, sympUAST-dan was expressed in the antenna disc using dppGal4. Dan protein levels were reduced in the RNAi-expressing cells, but Danr levels were unaffected (Emerald, 2003).
Emerald, B. S., Curtiss, J., Mlodzik, M. and Cohen, S. M. (2003). distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila. Development 130: 1171-1180. 12571108
Michaut, L., Flister, S., Neeb, M., White, K. P., Certa, U. and Gehring, W. J. (2003). Analysis of the eye developmental pathway in Drosophila using DNA microarrays. Proc. Natl. Acad. Sci. 100(7): 4024-9. 12655063
Siegmund, T. and Lehmann, M. (2002). The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins. Dev. Genes Evol. 212: 152-157. 11976954
Suzanne, M., Estella, C., Calleja, M. and S·nchez-Herrero, E. (2003). The hernandez and fernandez genes of Drosophila specify eye and antenna. Dev. Bio. 260: 465-483. 12921746
date revised: 10 November 2003
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