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First published online May 1, 2006
Journal of Experimental Biology 209, 1803-1815 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02202

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Transcriptional regulation of neuropeptide and peptide hormone expression by the Drosophila dimmed and cryptocephal genes

Sebastien A. Gauthier^1,* and Randall S. Hewes^1,2

¹ Department of Zoology, Stephenson Research and Technology Center, University of Oklahoma, Norman, OK 73019, USA
² Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

View larger version (9K): [in a new window]   Fig. 1. Genomic map of the 39D1 region, showing the locations of three genes (crc, dimm and Tsp39D), the P element insertion in dimmKG02598 (arrowhead), a single non-conservative base substitution in the crc1 allele, and two local deletions (Rev8 and Rev4). The box on Rev8 indicates the proximal breakpoint uncertainty region.

View larger version (50K): [in a new window]   Fig. 2. Quantification of mRNA levels in hatchling larvae by qRTPCR. (A–C) Mean gene Ct values for (A) Rev8/+ vs Rev8/Rev8 (N=5), (B) dimmKG02598/Rev4 vs dimmKG02598/+ (N=6) and (C) crc1/crc1 vs crc1/+ larvae (N=5). The N values represent the number of independent mRNA extractions. (A'–C') Levels of transcripts in homozygous or transheterozygous mutants in A–C expressed as a percentage of the levels in heterozygous controls. During each cycle of the qRTPCR, the Ct value increases by 1 as the quantity of qRTPCR product is doubled. Therefore, the percentage change in each mRNA shown in A'–C' was calculated as 1/2(Ct experimental–Ct control). *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA, sequential Bonferroni post-hoc test.

View larger version (70K): [in a new window]   Fig. 3. Reduced Dms transcript levels in the CNS of dimm mutant, but not crc mutant, hatchling larvae. (A) In situ hybridization with a Dms antisense probe in dimmKG02598/+ CNS. (B–D) Intensity of Dms in situ hybridization for the MP2, SE and SP cells in (B) dimmKG02598/Rev4 (N=9) vs dimmKG02598/+ (N=11), (C) Rev8/Rev8 (N=12) vs Rev8/+ (N=13), and (D) crc1/crc1 (N=5) vs crc1/+ (N=11) larvae. Paired genotypes were processed for in situ hybridization in parallel within each experiment (e.g. Rev8/Rev8 vs Rev8/+) but not between experiments (e.g. B vs D), and the baseline in situ hybridization intensities between experiments cannot be directly compared. *P<0.05; ***P<0.001; one-way ANOVA. Scale bars: 25 µm (A); 2.5 µm (B–D).

View larger version (47K): [in a new window]   Fig. 4. Reduced Lk transcript levels in the CNS of dimm, crc double mutant hatchling larvae. (A) In situ hybridization with a Lk antisense probe in a wild-type CNS. (B) Intensity of Lk in situ hybridization for selected neurons in Rev8/Rev8 (N=17) vs Rev8/+ (N=12) larvae. **P<0.01; ***P<0.001; one-way ANOVA. Scale bars: 50 µm (A); 2.5 µm (B).

View larger version (50K): [in a new window]   Fig. 5. Reduced ETH transcript levels in the endocrine Inka cells of crc mutant third instar larvae. (A,B) Intensity of in situ hybridization with an ETH antisense probe in the Inka cells on tracheal metameres 5 (TM5) and 8 (TM8) of the tracheae in (A) dimmKG02598/Rev4 (N=9) vs dimmKG02598/+ (N=8) and (B) crc1/crc1 (N=9) vs crc1/+ (N=10) larvae. **P<0.01; ***P<0.001; one-way ANOVA. Scale bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 6. Reduced ETH reporter gene expression in crc mutant third instar larvae. Expression of EGFP was driven under the control of a 382 bp promoter sequence from the ETH gene. (A,B) Intensity of Inka cell (TM5 and TM8) EGFP fluorescence in (A) Rev4, ETH-EGFP/dimmKG02598 (N=9) vs Rev4, ETH-EGFP/+ (N=4) and (B) Rev4, ETH-EGFP/crc1 (N=9) vs Rev4, ETH-EGFP/+ (N=11) larvae. ***P<0.001; one-way ANOVA. Scale bar, 5 µm.

View larger version (35K): [in a new window]   Fig. 7. Comparative genomic analysis of the 382 bp ETH gene regulatory region. (A) VISTA plot of the D. melanogaster assembly in pairwise alignments with five other Drosophila species. The gray bar, with tick marks at 50 bp intervals, shows the extent of the 382 bp region. The percent identity from 50–100% (vertical axis) in a 20 bp window sliding in 1 bp increments is displayed for each alignment (horizontal axis). Windows (excluding gaps) that were at least 70% identical with D. melanogaster are highlighted (non-coding sequences in pink). The conservation track (bottom plot) shows phastCons scores for the three-way MULTIZ alignment of D. melanogaster, D. yakuba and D. pseudoobscura. Two highly conserved regions (CR1 and CR2) exceeded the 0.4 score threshold (broken line). Arrows, direction of transcription; asterisks, start ATG of the ETH gene [the 5' UTR of ETH is predicted to be 14 bp long (Park et al., 1999)]; purple box, ETH coding sequence; turquoise box, Orc4 3' UTR. (B) MULTIZ alignment of CR1 and CR2. Bases that were identical in at least seven Drosophila species are indicated with asterisks with the consensus sequence shown directly below. Positions marked x below the consensus denote ATF4, DR4 and E74A binding sites predicted by rVISTA. Four selected transcription factor binding sites (see Results) are also shown at the bottom of the alignment, and bases matching the CR1 or CR2 consensus are highlighted in blue.