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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

^* Author for correspondence (e-mail: seb-usa{at}ou.edu)

Accepted 4 March 2006

	Summary

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
The regulation of neuropeptide and peptide hormone gene expression is essential for the development and function of neuroendocrine cells in integrated physiological networks. In insects, a decline in circulating ecdysteroids triggers the activation of a neuroendocrine system to stimulate ecdysis, the behaviors used to shed the old cuticle at the culmination of each molt. Here we show that two evolutionarily conserved transcription factor genes, the basic helix-loop-helix (bHLH) gene dimmed (dimm) and the basic-leucine zipper (bZIP) gene cryptocephal (crc), control expression of diverse neuropeptides and peptide hormones in Drosophila. Central nervous system expression of three neuropeptide genes, Dromyosuppressin, FMRFamide-related and Leucokinin, is activated by dimm. Expression of Ecdysis triggering hormone (ETH) in the endocrine Inka cells requires crc; homozygous crc mutant larvae display markedly reduced ETH levels and corresponding defects in ecdysis. crc activates ETH expression though a 382 bp enhancer, which completely recapitulates the ETH expression pattern. The enhancer contains two evolutionarily conserved regions, and both are imperfect matches to recognition elements for activating transcription factor-4 (ATF-4), the vertebrate ortholog of the CRC protein and an important intermediate in cellular responses to endoplasmic reticulum stress. These regions also contain a putative ecdysteroid response element and a predicted binding site for the products of the E74 ecdysone response gene. These results suggest that convergence between ATF-related signaling and an important intracellular steroid response pathway may contribute to the neuroendocrine regulation of insect molting.

Key words: bHLH, bZIP, Drosophila, ecdysis, stress, ETH

	Introduction

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Neuropeptides and peptide hormones are small chemical transmitters that carry physiological messages to the attention of specific target cells. They are present in vertebrates and invertebrates and control diverse processes, including growth, stress responses, reproduction, homeostasis and memory (Nässel, 2002; Strand, 1999). A central feature of neuroendocrine signaling is the regulation of the synthesis and secretion of neuropeptides by inputs to neurosecretory cell afferents (Burbach, 2002). In many systems, the mechanisms regulating neuropeptide expression and secretion, and the genes and cell signaling pathways underlying these processes, are largely unknown.

The Drosophila melanogaster dimm gene encodes a bHLH protein (DIMM) in the Atonal family of transcription factors (Hewes et al., 2003). This family includes NeuroD, Neurogenin, Mist1 and Olig, which play essential roles in the determination and execution of cell fate decisions in many tissues (Hassan and Bellen, 2000). Likewise, DIMM determines secretory protein levels in diverse neuropeptidergic cells. The dimm gene is highly expressed in neuroendocrine cells, and dimm mutant animals display strikingly reduced cellular levels of various neuropeptides, neuropeptide biosynthetic enzymes (Hewes et al., 2003) and a dopamine receptor (Park et al., 2004). In contrast, dimm mutations do not disrupt cell survival or the differentiation of neuropeptidergic cell types, and the functions of dimm are largely restricted to development of the neuropeptide secretory pathway (Hewes et al., 2003). Does dimm regulate the expression of other transcription factors or structural proteins required for secretory granule biosynthesis, or does dimm directly regulate the expression of many secretory proteins?

In the present study we examined whether dimm is required for normal expression of neuroendocrine genes. We monitored 16 genes encoding neuropeptides, peptide hormones, neuropeptide biosynthetic enzymes, secretory granule proteins, and enzymes involved in synthesis of biogenic amines. Levels of these transcripts in dimm mutants and in control genotypes were measured by quantitative real-time polymerase chain reaction (qRTPCR) and in situ hybridization. To disrupt dimm expression, we used several genetic aberrations that differentially disrupt dimm and/or a neighboring gene, crc. crc encodes a basic-leucine zipper (bZIP) transcription factor that is orthologous to activating transcription factor-4, ATF-4 (Hewes et al., 2000), an important mediator of the unfolded protein response to endoplasmic reticulum stress (Blais et al., 2004). We have previously tested several crc alleles (crc¹, crc^E98, crc⁹²⁹ and Df(2L)TW1) by immunostaining with anti-neuropeptide antisera (myomodulin and –RFamide) and anti-neuropeptide biosynthetic enzyme antisera (Furin 1 and Amontillado), and in each case, the levels of these markers were unaffected by disruption of crc (Hewes et al., 2003; R.S.H., unpublished). Therefore, we predicted that secretory protein mRNAs would be found at normal levels in crc¹/crc¹ larvae, and we included this genotype as a control for the qRTPCR experiments.

Levels of three neuropeptide mRNAs, Dromyosuppressin (Dms), FMRFamide-related (Fmrf) and Leucokinin (Lk), were all reduced by disruption of dimm and not crc. However, crc was required for expression of the ETH gene in the endocrine Inka cells. Comparative genome sequence analysis revealed putative recognition elements in the ETH promoter for factors in the ecdysteroid response pathway and CRC. Our results suggest that DIMM controls the transcription of multiple neuroendocrine genes. Additionally, the molting defects in animals bearing the crc¹ mutation, a classical allele first discovered in 1942 (Hadorn and Gloor, 1943), result from loss of a key endocrine regulator of ecdysis behavior.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Fly strains and genetic manipulations
Drosophila melanogaster Meigen stocks were cultured on standard cornmeal–yeast–agar media at 22–25°C. The following alleles were used to disrupt genes in the 39D1 region of chromosome 2L (Fig. 1, Table 1, and Table S2 in the supplementary material): Df(2L)Rev8 (Rev8), Df(2L)Rev4 (Rev4) and crc¹ (Hewes et al., 2000); P{SUPor-P}dimm^KG02598 (dimm^KG02598) (Hewes et al., 2003); and P{EPgy2}dimm^EY14636 (Bellen et al., 2004). The ETH-EGFP reporter line (Park et al., 2002) was kindly provided by Michael Adams (University of California, Riverside). Mutations were balanced over CyO-y⁺ or CyO, P{Ubi-GFP.S65T}PAD1 (CyO, Ubi-GFP). Oregon-R was used as the wild-type strain.

View larger version (9K): [in this window] [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.

RNA extraction
RNA extractions were performed on 24.5°C collections of 50 hatchling larvae on apple juice–agar plates supplemented with yeast paste. At this stage, the central nervous system (CNS) fills approximately 20–30% of the total body volume. The CNS volume:body volume ratio decreases with larval growth, and early collections maximized the relative yield of CNS mRNAs that could be obtained from whole animals. In addition, several of the neuropeptide transcripts measured in this study are expressed exclusively or primarily in the CNS (e.g. Fmrf and Dms) (Nichols, 2003; Schneider et al., 1991). Larval genotypes were distinguished by scoring for yellow (y) or UBI-GFP.

Tissues were disrupted by Polytron homogenization (Brinkmann, Westbury, NY, USA) on speed 5 for 5 min on ice. Total RNA was extracted in Trizol (Invitrogen, Carlsbad, CA, USA), in two extractions separated by a DNase treatment (RQ1 DNase kit; Promega, Madison, WI, USA). We synthesized cDNA from total RNA using random hexamer primers with the ISCRIPT kit (Bio-Rad, Hercules, CA, USA). One complete reaction and one `No Enzyme' (NoE) reaction was performed for each RNA sample, with 50 ng (by spectrophotometry) of total RNA per reaction (reverse transcriptase was omitted from the NoE reactions).

qRTPCR
Three sets of PCR primers were designed using Primer3 (Rozen and Skaletsky, 2000) for each gene in our analysis (Table 2). Based on product quality and purity using genomic DNA templates (judged by the presence of a single band of the correct size in 2% agarose electrophoresis gels and by the homogeneity of amplicon T_m values in qRTPCR dissociation curves), the best pair of primers was then selected (see Table S1 in the supplementary material). Primer concentrations were picked according to the nearest neighbor thermodynamic parameters method with salt corrections (SantaLucia, Jr, 1998) to match the conditions of the ABI qRTPCR cycle protocol (50 cycles: 15 s at 95°C followed by 1 min at 60°C on an ABI 7000; Applied Biosystems, Foster City, CA, USA).

Gene-specific qRTPCR reactions were performed with 1 µl of the reverse transcriptase mix, a pair of gene-specific primers, and SYBR green dye (ABI SYBR green PCR master mix). Each qRTPCR run was performed on a 96-well plate, providing transcript level information for 11 genes and the Ribosomal protein L32 (RpL32) control (see below) for two experimentally paired genotypes. For each gene on the plate, we performed three technical qRTPCR replicates per genotype and one `No Template' (NoT) reaction. NoT reactions lacked cDNA and were used to detect potential template-independent PCR amplification. For each genotype, we included two technical replicates with the RpL32 primer set and the NoE control to check for potential genomic DNA contamination. In all cases, PCR products in NoE and NoT reactions were at least 50-fold less concentrated than the gene-specific qRTPCR products. Thus, contamination with genomic DNA and primer-related templates was negligible. In addition, melting temperatures of the gene-specific amplicons were always consistent across the technical and biological replicates and across all genotypes (data not shown).

We performed relative quantitation analysis on qRTPCR data using the housekeeping gene, RpL32 (rp49), as a control. Levels of RpL32 mRNA were not significantly different between paired genotypes (data not shown) and were therefore not affected by mutations in the dimm region. For each PCR reaction, we obtained a Ct value representing the number of PCR cycles necessary to reach a threshold amplicon concentration. Ct values were normalized to RpL32 to obtain Ct values (Ct=Ct_{test gene}–Ct_RpL32), which were then averaged across the three technical replicates. By comparing levels of each transcript to RpL32, we confirmed consistency of the mRNA extraction, cDNA synthesis, and loading for the two paired genotypes within each experiment. In addition, normalization of test gene Ct values to those of RpL32 allowed us to compare transcript levels across experiments.

Tissue preparation and image analysis
Anti-Manduca pre-ecdysis triggering hormone (anti-PETH) immunostaining (Park et al., 2002; Zitnan et al., 1999) and ETH-EGFP imaging, preparation of digoxigenin-labeled DNA probes (from genomic templates), and whole-mount larval or CNS in situ hybridization were performed as described (Hewes et al., 2003). Control and experimental genotypes were always processed in parallel within a given experiment, using the same reagents, to minimize variability. In addition, for the in situ hybridization analysis, all reactions were stopped at the same time (when the most intense signals first became dark to prevent overstaining). We then measured the intensity of each cellular signal (intensity index) as described (Hewes at al., 2003). Briefly, confocal (fluorescence) and CCD (brightfield) images were obtained after adjusting exposure settings to optimize detection without saturating the signal. For a given neuron, identical settings were used for all preparations and genotypes, and the mean pixel intensity for the area covering each soma (S), and the neighboring background (B), was measured using Adobe Photoshop (San Jose, CA, USA). The intensity index=(S–B)/B. Images shown in the figures were chosen to best represent the mean intensity index values.

Statistical analysis
Statistical analyses were performed using NCSS 2001 (Kaysville, UT, USA). Sequential Bonferroni corrections were performed to minimize type I errors in multiple pair-wise comparisons (Rice, 1989). We used parametric statistics, because the data generally followed a normal distribution. All values are means ± s.e.m.

Comparative genomic analysis of the 382 bp ETH regulatory region
Drosophila genome sequences were visualized with VISTA (VGB2.0) (Frazer et al., 2004), using AVID and SLAGAN alignments, on the UCSC Genome Browser at http://genome.ucsc.edu/ (Karolchik et al., 2003) and with the MAVID multiple alignment server at http://baboon.math.berkeley.edu/mavid/ (Bray and Pachter, 2004). The alignments included sequences from eight Drosophila genomes: D. melanogaster (January 2003 assembly) (Celniker et al., 2002); D. pseudoobscura (July 2003) (Richards et al., 2005); D. yakuba (April 2004) and D. simulans (December 2004; Genome Sequencing Center, Washington University School of Medicine); D. ananassae (July 2004; The Institute for Genomic Research); D. mojavensis (August 2004), D. erecta (October 2004) and D. virilis (July 2004; Agencourt Bioscience Corporation). Consensus sequences (IUPAC code) were obtained using the TRANSFAC (see below) adaptations of the Cavener rules (Cavener, 1987). The code was capitalized when the nucleotide was present in at least seven sequences in the eight-species alignment.

The conservation track (phastCons) in the UCSC Genome Browser was based on a MULTIZ alignment of the D. melanogaster, D. yakuba and D. pseudoobscura genomes. These scores present an estimate of evolutionary conservation based on phylogeny, nucleotide substitution rates and autocorrelation of conservation levels along the genome (Siepel and Haussler, 2005). Putative transcription factor binding sites were identified using rVISTA (Loots et al., 2002), using the TRANSFAC Professional 7.4 library of binding site sequences (BIOBASE Biological Databases, Wolfenbüttel, Germany).

	Results

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Differential effects of aberrations in 39D1 on dimm, crc and Tsp39D expression
We used qRTPCR to analyze neuropeptide gene expression because of the sensitivity of this method. This was true even with whole-animal RNA samples, because a large majority of the cells that express dimm-dependent neuroendocrine peptides in our analysis (e.g. the populations of cells that express LK and ETH; Table 2) are affected in dimm mutant animals (Hewes et al., 2003). Based on our earlier immunocytochemical studies, we chose the neuropeptide and peptide biosynthetic enzyme genes for this analysis based on whether they were expressed in patterns largely or completely overlapping with dimm, and whether they showed reductions in protein levels in dimm mutant larvae (Hewes et al., 2003). However, we also included neuropeptide genes (e.g. Pdf, Eh) encoding proteins that are known not to be affected in dimm mutants as internal controls.

Because all of the loss-of-function alleles of dimm were also loss-of-function alleles of the crc gene (Fig. 1), we used three different paired genotype comparisons, in order to reveal the effects of dimm specifically on levels of secretory protein mRNAs (Table 1). First, we performed qRTPCR to monitor transcript levels in Rev8/Rev8 larvae and Rev8/+ controls. The Rev8 deletion is a null allele of crc and a strong loss-of-function allele of dimm (Hewes et al., 2003; Hewes et al., 2000). Second, we compared dimm^KG02598/Rev4 mutants to dimm^KG02598/+ controls. The Rev4 deletion is a null mutation of both crc and dimm. In contrast, the dimm^KG02598 mutation is a strong dimm loss-of-function allele, but a weak loss-of-function allele of crc (Hewes et al., 2003). Therefore, in both of the first two experiments we tested the effects of double-mutant combinations of dimm and crc, but in the second experiment, the crc defects were much less severe. In the third experiment, we compared crc¹/crc¹ larvae to crc¹/+ controls. crc¹ is a strong crc loss-of-function allele, but it does not disrupt dimm (Hewes et al., 2003; Hewes et al., 2000).

We first examined the effects of the above genotypes on genes in the 39D1 region: dimm is flanked by crc and a second gene, Tetraspanin 39D (Tsp39D). As expected, dimm and crc transcript levels were reduced in Rev8/Rev8 larvae (Fig. 2A). Rev8 deletes the crc gene (Fig. 1), resulting in a dramatic decrease in crc mRNA levels (although some crc mRNA is maternally loaded) (Hewes et al., 2000). Rev8/Rev8 mutants also display markedly reduced dimm mRNA levels (Hewes et al., 2003), presumably due to the deletion of dimm gene regulatory regions. In dimm^KG02598/Rev4 mutants, levels of crc, dimm and Tsp39D transcripts were all lower than in the heterozygous controls (Fig. 2B). This result is consistent with our earlier observation that KG02598 is not only a strong hypomorphic allele of dimm but also a weak hypomorphic allele of crc (Hewes et al., 2003). We suspect that the broad effects of this insertion on genes in 39D1 are due to chromosomal insulator elements contained within the KG02598 element (Roseman et al., 1995). Finally, dimm, crc and Tsp39D transcript levels were not significantly different between crc¹/crc¹ and crc¹/+ (Fig. 2C). This result was expected because crc¹ is a missense mutation specific to crc, and because our earlier crc in situ hybridization analysis of crc¹/crc¹ animals showed no change in crc mRNA levels (Hewes et al., 2000). Thus, the levels of dimm, crc and Tsp39D transcripts behaved as predicted in the three qRTPCR experiments.

View larger version (50K): [in this window] [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.

Three neuropeptide transcripts are downregulated in dimm and crc mutants
Only three neuropeptide mRNAs, Dms, Fmrf and ETH, varied significantly between paired genotypes in at least one of the three qRTPCR experiments. Dms transcript levels were reduced by 46% in dimm^KG02598/Rev4 animals (Fig. 2B). Levels of Dms were also down by 44% in Rev8/Rev8 mutants (Fig. 2A), although this difference was not statistically significant after the Bonferroni correction (P=0.047). In contrast, Dms transcript levels were normal in crc¹/crc¹ animals (Fig. 2C). We obtained similar results for Fmrf. Fmrf mRNA levels dropped 83% in Rev8/Rev8 (Fig. 2A), and they were down 51% in dimm^KG02598/Rev4 (Fig. 2B), although the latter difference was not statistically significant (P=0.13). Fmrf transcript levels were normal in crc¹/crc¹ animals (Fig. 2C). Based on our in situ hybridization data (see below), the relatively low P values, and the conservative nature of the Bonferroni correction, it appears likely that the reductions of Dms in Rev8/Rev8 and of Fmrf in dimm^KG02598/Rev4 were incorrectly judged as not significantly different due to type II error (false negatives). Notably, we previously observed reduced in situ hybridization with an Fmrf probe in dimm^KG02598/Rev4 larval CNS (Hewes et al., 2003). Therefore, the combined qRTPCR results suggested an effect of dimm, but not crc, on levels of Dms and Fmrf mRNA. These findings are consistent with the cellular reductions in immunocytochemical staining for the neuropeptide products of these two genes (Table 2).

The last of the three affected neuropeptide/peptide hormone mRNAs was ETH, which was reduced by 90% in the Rev8/Rev8 mutants (Fig. 2A) and by 60% in the crc¹/crc¹ mutants (Fig. 2C). While the reduction in ETH levels caused by the Rev8 chromosome was consistent with our previous studies (Table 2), the reduction in crc¹/crc¹ animals was novel, and we explored this relationship further (see below).

In the qRTPCR experiment comparing Rev8/+ and Rev8/Rev8, we did not observe significant differences in transcript levels for three neuropeptide genes, Pdf, Ccap and EH, two genes that encode known or putative components of secretory granules in neuropeptidergic cells (ia2 and Caps), and two genes, Ddc and ple, encoding enzymes involved in synthesis of biogenic amines (Fig. 2A). For Pdf and Ddc, these results are consistent with previous immunostaining data (Table 2). Thus, these seven transcripts were not affected by disruption of either dimm or crc, and we excluded them from the subsequent qRTPCR analysis of dimm^KG02598/Rev4 and crc¹/crc¹ (Fig. 2B,C).

Finally, there were five genes, amon, Dsk, Fur1, Lk and Phm, for which we observed no change in mRNA levels (Fig. 2) despite marked reductions in levels of their protein products (Table 2). In some cases, these differences may be due to indirect regulation of protein levels by dimm, such as through transcriptional regulation of other elements of the regulated secretory pathway (see Discussion).

dimm is required for normal Dms expression
The pattern of in situ hybridization with a Dms probe was similar to the reported immunostaining pattern (Nichols, 2003). Dms was expressed in 14–16 cells, with one pair in the subesophageal region (SE) and at least three pairs in each brain lobe (LB, MP2 and SP) (Fig. 3A). Additional, faintly labeled cells were sometimes visible. In dimm^KG02598/Rev4 larval CNS, we observed significantly less signal in two cell types, SP and SE, than in the dimm^KG02598/+ controls (Fig. 3B). There was also a reduction in Dms levels in the MP2 cells, although this trend was not statistically significant. In contrast, we found no significant variation in the intensity of Dms hybridization between Rev8/Rev8 and Rev8/+ (Fig. 3C) or between crc¹/crc¹ and crc¹/+ (Fig. 3D). The reason for the effect of dimm^KG02598/Rev4 but not Rev8/Rev8 on Dms transcript levels is unclear, although dimm^KG02598 may simply be a stronger dimm allele than Rev8. However, these results are in general agreement with the qRTPCR data, and we conclude that dimm, and not crc, likely upregulates Dms gene expression and/or increases the stability of the Dms mRNA.

View larger version (70K): [in this window] [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).

Lk neurons are differentially regulated by dimm
Previously, we found a marked reduction in levels of anti-LK immunostaining in Rev8/Rev8 mutants (Hewes et al., 2003). The qRTPCR results here, however, showed no change in Lk transcript levels in Rev8/Rev8 mutants, indicating that the regulation of LK protein levels in this genotype may be post-transcriptional. Therefore, we performed Lk in situ hybridization on Rev8/Rev8 larvae to further test this hypothesis. In first instar larval CNS, we detected hybridization with an Lk antisense DNA probe in a pair of cells (Br1) in the brain lobes, in two pairs of cells in the subesophageal region (SE), and seven pairs of more weakly Lk-expressing cells (A1–A7) in the ventral nerve cord (VNC) (Fig. 4A). This pattern of expression appears to be identical to the immunostaining pattern (Hewes et al., 2003). In the A1–A7 cells of Rev8/Rev8 mutant larvae, the strength of Lk hybridization was strongly reduced relative to Rev8/+ controls (Fig. 4B). In contrast, levels of Lk in the SE and Br1 cells appeared to be increased in Rev8/Rev8 animals, although the increase observed in the Br1 cells was not statistically significant. These results are consistent with our qRTPCR data, since increased Lk mRNA levels in the six Br1 and SE cells likely masked a decrease in Lk levels in the 14 more weakly Lk-expressing A1–A7 cells.

View larger version (47K): [in this window] [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).

crc regulates ETH expression
To further test the dependence of ETH levels on crc, we performed in situ hybridization with an ETH probe in crc mutant larvae. To facilitate preparation of larval fillets, we used third instar larvae, and we observed strong ETH hybridization in seven pairs of Inka cells (O'Brien and Taghert, 1998; Park et al., 2002). ETH-positive cells were located on the dorsal-longitudinal tracheal trunks in tracheal metameres 1 and 4–9 (TM1, TM4–TM9) (Manning and Krasnow, 1993). Compared to heterozygous controls, we found reduced ETH hybridization in dimm^KG02598/Rev4 (Fig. 5A). The cause of the difference in the results for dimm^KG02598/Rev4 in the qRTPCR versus the in situ hybridization analysis was not determined, but these experiments were performed on different larval stages, and the cumulative effects of dimm^KG02598/Rev4 on crc-dependent processes may be more pronounced in older animals. Notably, ETH in situ hybridization was markedly reduced in crc¹/crc¹ larvae (Fig. 5B), consistent with the qRTPCR results (Fig. 2C). In addition, we observed a severe reduction in anti-PETH immunostaining (Park et al., 2002) in crc¹/crc¹ Inka cells (data not shown). This antiserum interacts with ETH-like peptides from diverse insect species (Zitnan et al., 2003), and it labels peptides in the Drosophila Inka cells that are presumably ETH1 and/or ETH2 (Park et al., 2002). These results provide strong additional evidence for an important role of crc in regulating ETH expression.

View larger version (50K): [in this window] [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.

In dimm^EY14636/dimm^EY14636 larvae, we observed a marked reduction in CNS levels of anti-LK immunostaining relative to dimm^EY14636/+ controls (data not shown). We also observed a small decrease in the intensity of anti-PETH immunostaining in the Inka cells in dimm^EY14636/dimm^EY14636 larvae, although the strength of ETH in situ hybridization was unaffected (see Fig. S1 in the supplementary material). Thus, crc and dimm regulate ETH through distinct mechanisms. crc controls ETH transcription, whereas dimm can regulate ETH levels without altering ETH mRNA expression.

crc interacts with a 382 bp ETH regulatory region
Park et al. defined a 382 bp ETH enhancer region that is sufficient to direct expression of an ETH-Enhanced green fluorescent protein (ETH-EGFP) transgene specifically to the 14 Inka cells (Park et al., 2002). To determine whether this regulatory region is sensitive to regulation by crc, we monitored EGFP fluorescence in crc¹/Rev4, ETH-EGFP and dimm^KG02598/Rev4, ETH-EGFP third instar larvae. In dimm^KG02598/Rev4, ETH-EGFP CNS, we observed slightly reduced levels of EGFP relative to +/Rev4, ETH-EGFP controls (Fig. 6A), but this difference was not statistically significant (P=0.056, Inka^TM5; P=0.35, Inka^TM8). We observed a much stronger reduction in EGFP fluorescence in crc¹/Rev4, ETH-EGFP larvae (Fig. 6B). These findings, together with the qRTPCR and in situ hybridization results, demonstrate crc-dependent control of ETH gene expression.

View larger version (39K): [in this window] [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 this window] [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.

We compared the Drosophila genus consensus sequence for the predicted ATF-4 sites in CR1 and CR2 to the ATF-4 binding site in the rat Grp78 promoter (ATF4-Grp78) (Luo et al., 2003), the CAATT-enhancer binding protein (C/EBP)-activating transcription factor (ATF) composite site in the hamster chop promoter (ATF4-chop) (Fawcett et al., 1999; Ma et al., 2002), and the cAMP response element (CRE) in the rat phoshpenolpyruvate carboxykinase (PEPCK) gene (PEPCK CRE) (Vallejo et al., 1993) (Fig. 7B). All of these confirmed ATF-4-binding sites were imperfect matches to the ATF4 rVISTA hits in the Drosophila sequences. The best match (7 of 8 nucleotides) was between the CR1 hit and the PEPCK CRE. The latter has been shown to bind ATF-4-C/EBPß heterodimers (Vallejo et al., 1993). Thus, there is strong conservation of two sequences in the ETH promoter that are close, but imperfect matches to known binding sites for ATF-4, the mammalian ortholog of CRC. We predict that one or both of these putative CRC binding sites is required for CRC-dependent expression of ETH.

	Discussion

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
dimm controls levels of Lk, Fmrf and Dms neuropeptide mRNAs
DIMM has been proposed as a direct regulator of neuroendocrine gene expression in most neuropeptidergic cells (Allan et al., 2005; Hewes et al., 2003). Here we present qRTPCR results, supplemented by in situ hybridization, showing that DIMM upregulates the levels of mRNAs derived from at least three neuropeptide genes, Fmrf, Lk and Dms. These findings provide strong support for DIMM as a key regulator of multiple neuroendocrine genes.

The LIM-homeodomain gene apterous (ap) also controls Fmrf and Lk gene expression (Allan et al., 2005; Allan et al., 2003; Benveniste et al., 1998; Herrero et al., 2003; Park et al., 2004). ap acts cell-autonomously to stimulate dimm expression, but the AP and DIMM proteins can also physically interact, and they may function together in regulating Fmrf (Allan et al., 2005). Several other factors, including the transcriptional co-factors encoded by dachshund and eyes absent (Miguel-Aliaga et al., 2004), the zinc-finger gene squeeze, and the retrograde bone morphogenetic protein (BMP) pathway, act in combinatorial fashion with dimm and ap to control Fmrf expression (Allan et al., 2005; Allan et al., 2003). However, other neuropeptidergic cells appear to use only portions of this code. For example, ap and dimm appear to contribute to the expression of Lk in Fmrf-negative cells (A1–A7 and possibly Br1). Even within the population of Lk cells, loss of dimm results in very different effects in different neurons, with a reduction in Lk transcript levels in cells A1–A7, and an increase (or no change) in Lk levels in the Br1 and SE neurons (Fig. 4). How do these relatively widely expressed factors interact with other regulatory proteins to produce cell type-specific patterns of neuropeptide gene expression? It will be of interest to determine which other elements of the combinatorial pro-Fmrf code are used to control Lk and Dms expression, and to identify additional factors that interact with dimm to control expression of these neuropeptide genes.

Does dimm control neuropeptide levels through an additional indirect mechanism?
We did not detect changes in levels of three neuropeptide biosynthetic enzyme mRNAs, Phm, Fur1 and amon, in the qRTPCR analysis. This is in contrast to our earlier immunocytochemical studies, in which we observed a marked reduction in the protein products of these genes in dimm mutant CNS (Hewes et al., 2003). In some cases, these differences may reflect the spatial insensitivity of the qRTPCR methods, as was confirmed by our in situ hybridization analysis of Lk expression (Fig. 4). Phm, in particular, may belong in this category. Although levels of PHM and DIMM expression are strongly correlated (Allan et al., 2005; Hewes et al., 2003), PHM is also highly expressed in many other tissues (Jiang et al., 2000) that do not express dimm. Any dimm-dependent change in Phm expression may have been obscured by the much larger pool of dimm-independent Phm mRNA in our whole-animal qRTPCR analysis.

DIMM may regulate levels of other neuroendocrine proteins through a route that does not involve interactions between DIMM and cis-regulatory elements in the respective genes. We obtained the first evidence in support of this hypothesis in our earlier analysis of an ectopically expressed neuropeptide in dimm mutant cells; levels of ectopic PDF protein were strongly reduced while dimm had no effect on levels of the cognate Pdf mRNA (Hewes et al., 2003). Here, we show that larvae homozygous for a specific loss-of-function mutation in dimm displayed reduced levels of endogenous ETH-like protein(s), but not ETH mRNA, in the endocrine Inka cells (see Fig. S1 in the supplementary material), a site of dimm gene expression (Hewes et al., 2003). This may occur simply through a dimm-dependent change in levels of one secreted protein, such as PHM, that may disrupt the formation of multi-protein aggregates required for neuropeptide sorting into secretory granules (Arvan and Castle, 1998; Brakch et al., 2002). Alternatively, recent studies on the mouse ortholog of dimm, Mist1, suggest that dimm may play a more direct role in the management of secretory granule budding from the trans-Golgi network. In Mist1 knockout mice (Mist1^KO), pancreatic exocrine cells display reduced intracellular organization (Pin et al., 2001). Moreover, the Mist1^KO phenotype is partially phenocopied in animals mutant for the Rab3D gene, a small GTPase involved in secretory granule trafficking (Johnson et al., 2004). Further studies on the regulation of ETH, PHM and Rab3-like proteins, and on the biochemical interactions among them, may shed light on the cellular mechanisms underlying the indirect actions of DIMM.

crc controls expression of ETH through a 382 bp 5' region
Mutations in the crc gene result in pleiotropic defects in ecdysone-regulated events during molting and metamorphosis (Hewes et al., 2000). Many of the morphological defects are associated with a failure of the insect to properly complete ecdysis, a stereotyped set of behaviors required for shedding of the old cuticle at the culmination of each molt. Several neuropeptides and peptide hormones, including ETH, play critical roles in organizing and triggering ecdysis behavior (Ewer and Reynolds, 2002).

Here we provide four independent lines of evidence that demonstrate a crucial role for crc in the upregulation of ETH mRNA levels. First, we observed a marked reduction by qRTPCR in levels of ETH transcripts [but not in mRNAs encoding CCAP or EH, two other neuropeptides involved in the neuropeptide hierarchy controlling ecdysis (Ewer and Reynolds, 2002)] in crc mutant larvae (Fig. 2). Second, in situ hybridization revealed a strong reduction in ETH mRNA levels in the endocrine Inka cells in crc mutant larvae (Fig. 5). Third, the intensity of anti-PETH immunoreactivity was markedly reduced in crc¹/crc¹ homozygotes. Fourth, EGFP fluorescence driven by an ETH-EGFP reporter gene was reduced in crc mutant larvae (Fig. 6). Therefore, CRC is a strong activator of ETH gene expression, and loss of CRC results in a corresponding reduction in levels of the ETH protein.

Despite the molecular identification of the crc locus (Hewes et al., 2000), almost six decades after the original description of the first crc allele (Hadorn and Gloor, 1943), the causes of the molting and metamorphosis defects in crc mutants remained unclear. Our current results suggest a simple model to explain the crc mutant phenotype. Strong hypomorphic or null mutations in crc and ETH both severely disrupt ecdysis. These defects include weak, irregular and slower ecdysis contractions and a failure to shed old cuticular structures, leading to retention of two and sometimes three sets of mouthparts into the next larval stage (Chadfield and Sparrow, 1985; Park et al., 2002). These similarities in molting defects, taken together with our observation that crc is required for normal expression of ETH mRNA and ETH protein, point to the loss of ETH signaling as the likely proximate cause of the ecdysis defects observed in crc mutants.

Despite the specific effects of crc on ETH transcription in the Inka cells, crc is widely expressed (Hewes et al., 2000), suggesting a cellular housekeeping function. The vertebrate ATF-4 protein is also ubiquitously expressed (Hai and Hartman, 2001). In addition, the upregulation of ATF-4 constitutes a milestone of many cellular stress response pathways including oxidative stress, amino acid deprivation (Rutkowski and Kaufman, 2003), and hypoxia (Blais et al., 2004). In the tobacco hornworm, Manduca sexta, levels of ETH fluctuate during the molts and are regulated by circulating ecdysteroids (Zitnan et al., 1999). We hypothesize that CRC contributes to the regulation of ETH gene expression during this period, perhaps in response to signals from the tracheae.

Predicted CRC binding sites in the ETH promoter region
Peaks in circulating levels of the ecdysteroid hormone, 20-hydroxyecdysone (20HE), initiate and coordinate each molt. A subsequent decline in 20HE levels is required for ecdysis, and the activation of these behaviors involves a hierarchical cascade of peptide hormone and neuropeptide signals that is triggered by ETH (Ewer and Reynolds, 2002). Is CRC required in order to maintain ETH expression, or is CRC involved in regulating transcription of the ETH gene during the molts? While it is not known whether ETH mRNA levels fluctuate during Drosophila post-embryonic development, the regulation of ETH levels by ecdysteroids in molting Manduca sexta, and our analysis of the CR1 and CR2 sequences, provides tantalizing clues to possible coordinate regulation of ETH gene expression by CRC and ecdysone response genes. There is substantial overlap between the predicted CRC binding site in CR1 and a putative ecdysteroid response element (EcRE) (cf. Park et al., 1999). In addition, we found a potential binding site in CR2 for products of the E74 early ecdysone-inducible gene. E74 expression is induced directly by 20HE, and it encodes transcription factors that regulate other ecdysone response genes (Fletcher and Thummel, 1995). Mutations that specifically disrupt E74B, which likely binds the same consensus as E74A (Urness and Thummel, 1990), display defects associated with pupal ecdysis that closely phenocopy crc. In future studies we hope to determine if ETH expression is regulated by elements in both CR1 and CR2 in an ecdysteroid-dependent manner, and whether CRC, E74B and other factors in the ecdysone-response pathway interact competitively or cooperatively at these sites.

	List of abbreviations

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 

20HE

20-hydroxyecdysone

amon

amontillado

ap

apterous

ATF-4

activating transcription factor-4

B

background

bHLH

basic helix-loop-helix

BMP

bone morphogenetic protein

bZIP

basic-leucine zipper

C/EBP

CAATT-enhancer binding protein

Caps

Calcium activated protein for secretion

Ccap

Cardioacceleratory peptide

CNS

central nervous system

CR

ETH promoter conserved region

crc

cryptocephal

CRE

cAMP response element

Ct

cycles to threshold amplicon concentration

Ddc

Dopa decarboxylase

dimm

dimmed

Dms

Dromyosuppressin

Dsk

Drososulfakinin

EcRE

ecdysteroid response element

EGFP

enhanced green-fluorescent protein

Eh

Eclosion hormone

Eip74EF (E74)

Ecdysone-induced protein 74EF

ETH

Ecdysis triggering hormone

Fmrf

FMRFamide-related

Fur1

Furin 1

Lk

Leucokinin

NoE

No Enzyme

NoT

No Template

Orc4

Origin recognition complex subunit 4

Pdf

Pigment–dispersing factor

PEPCK

phospenolpyruvate carboxykinase

PETH

pre-ecdysis triggering hormone

Phm

Peptidylglycine--hydroxylating monooxygenase

ple

pale

qRTPCR

quantitative real time polymerase chain reaction

RpL32 (rp49)

Ribosomal protein L32

S

soma

TM

tracheal metamere

T_m

melting temperature

Tsp39D

Tetraspanin 39D

VNC

ventral nerve cord

y

yellow

	Acknowledgments

 
We thank Chad Hargrave for statistical guidance, Mike Adams for fly stocks and antisera, David Durica for comments on the manuscript, and Audrey Kennedy, Kendal Milam, Matthew Mote, Jeremiah Smith and Elizabeth Pearsall for technical assistance. This work was supported by grants from the National Science Foundation (NSF IBN0344018), NSF EPSCoR and the Oklahoma State Regents for Higher Education (NSF-0132534), and the Oklahoma Center for the Advancement of Science and Technology (HR03-048S) to R.S.H.

	Footnotes

 
Supplementary material available online at http://jeb.biologists.org/cgi/content/full/209/10/1803/DC1

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