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The Drosophila daughterless gene autoregulates and is controlled by both positive and negative cis regulation

Department of Biology, Gilmer Hall, University of Virginia, P.O. Box 400328, Charlottesville, VA 22904-4328, USA

View larger version (41K): [in a new window]   Fig. 1. In the somatic ovary, dalyh is a null allele. (A) Diagram of the anterior Drosophila ovariole. The long bracket indicates the germarium, where stem cell divisions occur and follicle formation takes place. The somatic cells are shaded; the positions of the germline and somatic stem cells are indicated. (B-E) Ovarioles stained with the nuclear dye DAPI. The wild-type ovariole (B) illustrates normal ovarian morphology. By contrast, homozygous (dalyh/dalyh, C) and hemizygous (dalyh/da-: not shown) mutant ovaries from mature females exhibit gross disruptions in oogenesis, including the formation of multicyst follicles, failure of follicle individualization, and degeneration of late-stage cysts. The identification of dalyh as an ovarian null allele is evident from a comparison of dalyh/dalyh and dalyh/da2 ovaries (da2 is a lethal null allele.). Even in ovaries from newly eclosed mutant females, where the phenotype is least severe, dalyh/da2 (D) and dalyh/dalyh (E) mutant ovaries are indistinguishable. In each panel anterior is upwards or towards the left; the magnification of D,E is approximately twice that of B,C.

View larger version (42K): [in a new window]   Fig. 2. Insertion of a springer retrotransposon is responsible for the dalyh mutation. (A) A wild-type ovariole (upper) and a dalyh mutant ovariole (lower) stained for Da protein. Arrows indicate the anterior end of each ovariole. Da protein is apparent in nuclei of wild-type somatic cells of the ovariole; no nuclear-localized protein is seen in dalyh mutant ovarioles. (B) Graphical representation of the da genomic region. The dalyh chromosome has a 7.5 kb springer element inserted within the single intron. Below the map are the extents of the Frag 5 DNA probe (Cronmiller et al., 1988), used for the northern blot shown in D, and the genomic fragment used in construction of the da.G32 reporter (Wodarz et al., 1995), used in the real-time RT-PCR analysis shown in Fig. 5. (C) Comparison of the conceptual translation products of the springer and gypsy retrotransposons. A complete springer nucleotide sequence was assembled from the ends of the dalyh insertion and the Drosophila genome project sequences. Orientation of the springer is opposite of that in B. (D) Transcriptional analysis of dalyh. Poly(A)+ RNA from wild-type and mutant dalyh adults was probed with the da fragment 5 (B) to detect da RNA and the 3' adjacent fragment 6 (Cronmiller et al., 1988) to detect Mdh1 RNA as a loading control. Both da transcripts and the single Mdh1 transcript are indicated. RNA sizes are in kilobases.

View larger version (82K): [in a new window]   Fig. 3. Unlike da null alleles, dalyh completely complements stla16. DAPI stained ovarioles dissected from various da mutant genotypes. (A) Ovaries of flies doubly heterozygous for stla16 and the null allele da2 contain severe morphological defects in all ovarioles. (B) Similar defects are present in ovarioles that are doubly heterozygous for stla16 and the hypomorph das22. (C) By contrast, dalyh completely complements stla16. (D) In 15% of ovarioles from flies with an ectopic copy of da+, mild defects include mislocalization of the oocyte nucleus (arrow). The remaining 85% of the ovarioles are completely normal, indicating that the wild-type allele of da does not have to be on the homologous chromosome to provide full function. In each panel, the anterior end of the ovariole is at the top.

View larger version (59K): [in a new window]   Fig. 4. Increased doses of da+ produce a gain-of-function phenotype. (A-D) Ovarioles stained with the nuclear dye DAPI; (E-H) ovarioles double stained for Vasa (germline, red) and Hts (adducin, present in somatic cytoskeleton and germline spectrosome and fusome, green). (A) Wild-type ovariole. (B) Ovarioles from flies with three copies of da+ contain abnormally long interfollicular stalks (arrows). The genotype of the ovariole shown here included a tandem duplication of da+ (da+/Dp(2;2)da+); similar morphology was observed when the extra da+ copy was provided by a transposition, carried on the Y chromosome (not shown). (C-E) Ovaries of homozygous dalyh flies, carrying a duplication of da+ on the Y chromosome, exhibit a more extreme phenotype. In these ovarioles, smaller germaria are attached directly to maturing follicles. (E) The staining for Vasa and Hts highlights the gap in follicle stages that is observed between germline in the germarium and the closest individual follicle. (F) These defects are also seen in flies homozgyous for dalyh with two copies of a heat-inducible da+ transgene at 32°C. (G) Likewise, the defects are phenocopied by 37°C pulses of the heat-inducible da+ transgenes in a wild-type background. (H) These defects are not seen in wild-type flies exposed to the same heat shock regimen. In each panel anterior is at the top (C,E-H) or towards the left (A,B,D).

View larger version (63K): [in a new window]   Fig. 5. An in vivo transcriptional reporter indicates Da can transactivate the da promoter. Flies were generated that carried a heat shock inducible da+ transgene and a reporter transgene (da.G32), consisting of the da promoter fused to the gal4-coding region. Real-time RT-PCR was used to determine relative levels of gal4 transcript in adult females maintained at 25°C or after heat shock treatment. Both graphs plot the second derivative of SYBR green fluorescence for each replicate (blue, without heat shock; red, with heat shock). The most accurate measure of transcript level is the threshold cycle number (Ct), which is identifiable for each plot as the cycle corresponding to the second derivative peak. The mean threshold cycle numbers are indicated. The gal4 graph shows a significant decrease of 1.48 cycles with heat shock induction of Da protein, indicating at least a two- to threefold increase in RNA. The Mdh1 graph shows no significant change in the threshold cycle number with heat shock: control levels of RNA are unaffected by the treatment.

View larger version (71K): [in a new window]   Fig. 6. Su(lyh) dominantly suppresses dalyh phenotypes, both loss- and gain-of-function. (A-D) DAPI-stained ovaries. The dalyh loss-of-function phenotype (A) is completely suppressed by a single copy of Su(lyh) (B) (genotype, dalyh/dalyh; Su(lyh)26H6/Su(lyh)+). The dalyh-associated gain-of-function phenotype (C) (genotype, Tp(2;Y)da+; dalyh/dalyh) is also dominantly suppressed by Su(lyh)26H6 (D). Note that the reduced germaria (asterisks) that characterize the da+ overexpression phenotype are not present in the ovaries that carry the suppressor genotype.

View larger version (29K): [in a new window]   Fig. 7. A model for da transcriptional regulation. (A) Wild-type transcriptional regulation of da depends on both positive and negative control. An ovary-specific enhancer that is required for initiation of transcription in the somatic ovary is located within the 1.5 kb da intron. The location of this enhancer must be downstream of the dalyh insertion site (indicated in B) and also within the sequence included in the da.G32 promoter reporter transgene (extents indicated by the HindIII and BglII sites). Within this region (500 bp into the intron), a STAT-binding site is one enhancer candidate. At least in this tissue, the initial expression of da leads to the production of Da protein, which subsequently acts to maintain the transcription of da. The binding partner of Da for this autoregulation is not known; however, numerous target E-box-binding sites (E) are dispersed throughout the genomic sequence. To keep da transcript levels from becoming too high, there is also a negative regulatory element, which must be located downstream of the dalyh insertion site. (B) In the dalyh allele, a springer retrotransposon acts as a transcriptional insulator that is capable of blocking the effects of both the positive and negative regulatory elements without disrupting Da autoregulation. In most tissues, the function of this allele appears to be normal, indicating that there is generally no problem with its transcriptional activation. In the somatic ovary, however, transcriptional activation fails because the promoter is insulated from the downstream ovary enhancer. But, dalyh can be transactivated by Da protein from another allele. The springer element also insulates the negative regulatory element, so that the amount of da transcript produced from the dalyh allele is higher than that from a wild-type allele. The insulating effects of springer depend on the Su(lyh) gene product. By analogy to Su(Hw)-mediated gypsy insulation, Su(lyh)-mediated insulation may result from direct binding of the Su(lyh) protein to springer sequences.