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- Table 1.
Paired genotype comparisons used in the qRTPCR analysis
Allele Allele class Control genotype Test genotype Rev8 Null crc allele and strong dimm hypomorph yw; Rev8/CyO, y+ yw; Rev8/Rev8 dimm KG02598 Weak crc allele and strong dimm hypomorph yw; dimmKG02598/+ yw; dimmKG02598/Rev4 Rev4 Null allele for crc, dimm and Tsp39D crc 1 Strong crc hypomorph yw; crc1/CyO, y+ yw; crc1/crc1 - 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.
- Table 2.
Genes selected for qRTPCR analysis
Category Gene Known or predicted function(s) Protein levels in dimm-/- mutants Neuropeptide biosynthetic enzymes Furin 1 (Fur1) Neuropeptide endoprotease (Roebroek et al., 1991) Reduceda Peptidylglycine-α-hydroxylating monooxygenase (Phm) Neuropeptide amidation (Jiang et al., 2000) Reduceda amontillado (amon) Neuropeptide endoprotease (PC2) (Siekhaus and Fuller, 1999) Reducedb Secretory granule proteins ia2 Protein tyrosine phosphatase (Walchli et al., 2000) ND Calcium activated protein for secretion (Caps) Secretory granule protein (Renden et al., 2001) ND dimm region dimmed (dimm) bHLH transcription factora ND cryptocephal (crc) bZIP transcription factor (Hewes et al., 2000) ND Tetraspanin 39D (Tsp39D) Tetraspanin (Todres et al., 2000) ND Neuropeptides Pigment—dispersing factor (Pdf) Neuropeptidef Normala,c FMRF amide-related (Fmrf) Neuropeptidef Reduceda Dromyosuppressin (Dms) Neuropeptidef Reduceda,d Drososulfakinin (Dsk) Neuropeptidef Reduceda,d Leucokinin (Lk) Neuropeptidef Reduceda Cardioacceleratory peptide (Ccap) Neuropeptidef ND Eclosion hormone (Eh) Neuropeptidef Normala Ecdysis triggering hormone (ETH) Peptide hormones (ETH1 and ETH2)f Reduceda,e Biogenic amine synthetic enzymes Dopa decarboxylase (Ddc) Dopa decarboxylase (Livingstone and Tempel, 1983) Normala pale (ple) Tyrosine hydroxylase (Neckameyer and White, 1993) ND Ribosomal Ribosomal protein L32 (RpL32) Ribosomal protein (O'Connell and Rosbash, 1984) ND -
ND, not done.
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↵a (Hewes et al., 2003)
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↵b (Park et al., 2004)
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↵c Native expression pattern only.
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↵d Inferred from immunostaining of the MP1 and MP2 neurons with the PT-2 antiserum, which detects —RFamide-containing peptides (Taghert, 1999), likely including DMS and DSK. The MP1 cells express Dsk, and the MP2 cells are immunopositive for DMS (Nichols et al., 1997).
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↵e Inferred from immunostaining of the endocrine Inka cells with the anti-myomodulin (MM) antiserum (Hewes et al., 2003). This antiserum likely cross-reacts with ETH, since both peptides share a PRL-amide C-terminal motif (Zitnan et al., 2003).
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↵f For reviews see (Hewes and Taghert, 2001; Vanden Broeck, 2001).
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- Fig. 2.
Quantification of mRNA levels in hatchling larvae by qRTPCR. (A–C) Mean gene ΔCt 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(ΔCt experimental–ΔCt control). *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA, sequential Bonferroni post-hoc test.
- 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: 25μ m (A); 2.5 μm (B–D).
- 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: 50μ m (A); 2.5 μm (B).
- 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.
- 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.
- 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.
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