Expression of ac-sc in proneural clusters is regulated by independently-acting cis-regulatory enhancers. The enhancer responsible for activation of ac-sc in the cluster giving rise to the DC bristles has been characterized in detail. The activity of this enhancer in a reporter construct was examined using lacZ expression. The activity of this enhancer is modified in pad1. The domain of expression of lacZ appears wider. At the same time, the anterior limit of the cluster is retracted in a posterior direction. It is possible that this is in part due to the slight distortion of the overall shape of the notum seen in pad1 mutants. Interestingly, the ectopic bristles do not arise within the misshapen proneural cluster. They are therefore formed independently of the activity of the DC enhancer used for activation. In fact, the aDC, as well as the ectopic DC precursors, are both clearly situated outside the DC cluster. Another characterized enhancer of ac-sc, the L3-TSM enhancer involved in the formation of the sensilla on the anterior wing margin, anterior cross vein and third vein was examined and no significant modification was observed. These results suggest that poils au dos does not act through the cis-regulatory sequences controlling expression in the proneural clusters (Gibert, 2005).
To determine which regions of the AS-C are required for the formation of the ectopic bristles in pad, the pad1 mutant was placed in various ac-sc mutant backgrounds. These included several deletions generated by excision of the P-element in the line NP-6066. In(1)ac3, an inversion separating sequences located 1 kb upstream of ac, including the DC enhancer, was used, as well as Df(1)91B (which deletes 45 kb from a position 10.3 kb upstream of sc that includes ac and the DC enhancer); Df(1)115 (which deletes 7.8 kb between the positions 14.5 and 6.7 kb upstream of the scute ATG), and In(1)sc4 (an inversion with a breakpoint 7-8 kb downstream of sc). None of these rearrangements prevent formation of the ectopic bristles present in pad1. In(1)sc4 causes a loss of all scutellar bristles, because the relevant enhancer, located 40 kb downstream of sc, is translocated elsewhere and is thus not able to drive the expression of ac-sc in the scutellum. However, occasional scutellar bristles form in In(1)sc4; pad1 flies at the position normally occupied by the anterior scutellar bristle. In contrast to the rearrangements cited above, no, or very few, ectopic bristles are formed in scbald; pad1 flies. This hypomorphic sc allele carries the remains of a P element located 10 kb upstream of sc and displays a high frequency of missing SC, aDC and orbital bristles. Together, these results indicate that the target sequences are probably located in a fragment that extends 6.7 kb upstream and 7-8 kb downstream of sc (Gibert, 2005).
In order to visualize the precursors of the ectopic bristles in pad1, an antibody against Senseless, a marker of neural precursors, was used. A transgene was used driving the expression of LacZ under the control of the achaete/scute Sensory Organ Precursor enhancer (SOP-lacZ). The minimal SOP enhancer of 500 bp drives expression of lacZ exclusively in the bristle precursors and contains binding sites for Ac-Sc/Da (E boxes), as well as sites for the binding of repressors. It was observed that the precursors of ectopic bristles appear between 0 and 2 h after puparium formation. This is about the same time as the formation of the precursors for the anterior DC (aDC) bristles in wild-type flies. The posterior DC (pDC) precursors appear much earlier, around 24 to 12 h before puparium formation. In situ hybridization with a probe to sc, indicated that sc is expressed ectopically in third instar wing discs. Expression of ac was examined using an anti-Achaete antibody and is also significantly up-regulated in pad1. In both cases, the proneural clusters that give rise to the wild-type bristle precursors are clearly visible at wild-type locations, but they appear to be enlarged. In addition, many more cells express high levels of ac-sc outside the proneural clusters. These are mainly located in the future anterior and central regions of the notum, consistent with the fact that ectopic macrochaetes are found here. Weak sc expression can be detected in these areas in wild-type discs but does not give rise to sense organs. Ectopic expression in pad1 is particularly visible in the region of the presutural, DC and PSA bristles where many ectopic bristles form (Gibert, 2005).
To better visualize the regions of ectopic expression, the reporter construct EE4 containing an artificial SOP enhancer composed of four E-boxes and the binding sites for the Ac and Sc proteins was used. The EE4 construct lacks the sequences required for repression and so it is very sensitive to the levels of Ac-Sc and can be used to measure the increased amounts of Ac-Sc in the pad mutant. It was observed that expression driven by this enhancer in pad1 is significantly different from that seen in the wild type. In the wild type, it is expressed exclusively in the cells of the proneural clusters where it is present at high levels. In pad1, expression in the PSA region expands medially and expression in the DC region expands anteriorly. Some of the ectopic precursors appear within this expanded anterior region (Gibert, 2005).
Using in situ hybridization, the expression pattern of pad was examined in embryos and third instar larval wing discs. In embryos, transcripts accumulate in the central nervous system: staining can be clearly detected above background levels shortly before stage 16. This is consistent with the findings of Brody (2002). No staining could be detected in the larval peripheral nervous system. No staining was detected in the wing discs. This may reflect low levels of ubiquitously expressed transcripts. It is nevertheless believed that pad is expressed in the wing disc since pad1 mutant clones autonomously display ectopic bristles on the notum (Gibert, 2005).
Flies were collected in the garden of Marrakech University in 1999 (Chakir, 2002). A population founded by more than 30 females was selected in bulk for an increased number of thoracic macrochaetes at 17°C for the first few generations and at 25°C later. Chromosomes were extracted from one female Drosophila with a high number (16) of ectopic bristles using balancer chromosomes. It was observed that all the variation is due to the third chromosome. An isogenic line, A10, with the X and second chromosome from a wild-type stock (Oregon R) and the third chromosome from this female was used for the following analysis. This homozygous line is perfectly viable and fertile. The phenotype is recessive: homozygotes have a marked bristle phenotype. At 25°C, females show 13.0 (±2.28) and males 9.38 (±2.55) ectopic bristles. Ectopic bristles are mainly located in the dorso-central (DC) and presutural (PS) regions. The anterior scutellar (aSC), posterior post-alar (pPA) and posterior supra-alar bristles (pSA) are also frequently duplicated in females. Additional bristles are found laterally but less frequently. These are usually slightly shorter and thicker. The density of microchaetes is also increased. There are often four or five sensilla campaniformia on the third vein (L3) of the wing (average 3.45, n = 22) instead of three, and at the location of the twin sensilla of the anterior wing margin (TSM), there are often three sensilla (Gibert, 2005).
The multiply marked third chromosome ru cu ca was used for recombination mapping and a single segment was identified between curled (86D) and stripe (90E) that is responsible for the phenotype. Single nucleotide polymorphism (SNP) mapping, using 30 chromosomes with a break point between curled and striped was used and the location was refined to region 88C-89E. A study of deficiencies showed that Df(3R)sbd26 (89B9-10; 89C7-D1) and Df(3R)P115 (89B13; 89E7) do not complement A10 for the bristle phenotype. The phenotype is thus due to one (or several) loss of function mutations in gene(s) located in the region common to both deletions: 89B13-89D1. There are about 40 genes in this region which spans around 200 kb. In order to map the mutation(s) more precisely, new recombinants were used. To select the potentially informative ones, P insertions were used with a w+ marker located on the left [line MD237 (pnr-Gal4) and tara1] or on the right (insertion in CSN5 and line E7439) of the mutation(s). Females w/w; Pw+/A10 were crossed with males w; A10/A10. More than 5000 flies were screened for each P insertion and flies Pw+A10/A10 and +/A10 were selected. The recombination point was mapped by SNP analysis in these heterozygous flies. Identified and used were polymorphic sites located in the gene sulf1 (SF4, MspI), between sulf1 and CG6901 (ST1, NdeI), between CG17930 and SF2 (CSF, BalI), between CG10817 and ss (SS3, DraI) and between ss and CG31279 (SS5, SspI). The mutation(s) were localized between CSF and SS3. This segment is 36.3 kb long and contains eight genes, none of which had previously been shown to have a role in bristle development. One of them, CG10309, encoding a zinc-finger transcription factor, had been identified (Brody, 2002) in a differential screen for genes highly expressed in the embryonic nervous system (Gibert, 2005).
4289 bp encompassing the CG10309 gene in A10 were sequenced. The sequence is identical to the allele of CG10309 in the publicly available sequence of D. melanogaster genome except for a deletion of 29 bp from position 976 to 1004 (inclusive) downstream of the A of the predicted ATG . A cDNA recently sequenced by the Berkeley Drosophila Genome Project (clone IP01015p) corresponds exactly to the predicted mRNA. The deletion, in the third exon, induces a frameshift and introduces 20 new codons followed by a stop codon. The resulting truncated protein is thus predicted to be 308 aa long instead of 925 aa and would lack the four zinc fingers located in the C-terminal part. This gene was named poils-au-dos (pad) for 'hairy back' in French and A10 is now referred to as pad1 (Gibert, 2005).
In order to verify that the pad phenotype is indeed caused by the mutation in the gene CG10309, and that no other linked mutation contributes to the phenotype, complementation tests were performed. Since no other mutants of CG10309 were available, new alleles were generated by mobilizing a P-element inserted 400 bp from the predicted ATG in the line P(SUPor-P)KG08729 created by the Drosophila Genome Disruption project. Three independent mutant lines were recovered that failed to complement pad1 for the bristle phenotype. All three mutants deleted a large 5' portion of CG10309 transcription unit including the region encoding the ZAD domain. The mutants were named pad2, pad3 and pad4. All three are late pupal lethals, with a few escapers in pad2. In pad3 and pad4, the transcription unit of the neighboring gene SF2 is also disrupted, which correlates with the higher lethality of these mutants compared to pad2. To ascertain whether SF2 is affected in pad1, the whole coding frame of SF2 in the chromosome carrying pad1 was sequenced and no differences were found in the DNA sequence with the published genome sequence. The three new pad alleles have a more extreme bristle phenotype than that of pad1 and, unlike pad1 flies, they also have twisted legs (Gibert, 2005).
The generalized increase in ac-sc expression suggests that poils au dos is involved in the repression of ac-sc. Interactions between pad and other known repressors of ac-sc were tested. pad1 interacts moderately with emcpel and very strongly with hairy1. In h1 homozygotes grown at 18°C, ectopic bristles are occasionally found anterior to the aDC, whereas none were seen at 25°C. In h1 pad1 homozygotes, many ectopic bristles were observed at 25°C at positions where none were seen in either of the single mutants. These include DC bristles closer to the thoracic midline and additional bristles between the anterior and posterior scutellars. Interestingly, most of these ectopic bristles are located in the posterior half of the notum whereas the visible effect of pad alone is in the anterior part of the notum (Gibert, 2005).
Mutations in very few other genes have been shown to induce ectopic bristles in the anterior region of the notum. Some ectopic bristles can be induced in this region by reduction in Dpp signaling late in development. A genetic interaction between pad and Dpp signaling was tested using mutations in the receptors punt (put) and thickveins (tkv). A strong genetic interaction was observed between pad1 and putP1. Trans-heterozygous putP1/pad1 flies have ectopic DC bristles whereas each of the single heterozygotes displays a wild-type pattern. Flies homozygous for the hypomorphic mutation tkv1 occasionally have ectopic bristles anterior to the aDC at 18°C. The phenotype is strongly enhanced in the anterior region of the notum of double mutant tkv1; pad1 flies grown at 25°C. In particular, many more ectopic bristles are visible around the prescutal suture than in pad1 alone (Gibert, 2005).
Reference names in red indicate recommended papers.
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Gibert, J. M., Marcellini, S., David, J. R., Schlotterer, C. and Simpson, P. (2005). A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor Poils-au-dos, a repressor of achaete-scute. Dev. Biol. 288(1): 194-205. 16216235
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date revised: 5 March 2005
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