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. 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 (Gauthier, 2006).
DIMM has been proposed as a direct regulator of neuroendocrine gene expression in most neuropeptidergic cells. Quantitative RTPCR results, supplemented by in situ hybridization, show 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. 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. Several other factors, including the transcriptional co-factors encoded by dachshund and eyes absent, 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. 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 (the segmental cells A1-A7 and possibly the brain lobe cells 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 the subesophageal SE neurons. 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 (Gauthier, 2006).
Does dimm control neuropeptide levels through an additional indirect mechanism? No changes were detected in levels of three neuropeptide biosynthetic enzyme mRNAs, Phm, Fur1 and amon, in the qRTPCR analysis. This is in contrast to earlier immunocytochemical studies, in which a marked reduction was observed in the protein products of these genes in dimm mutant CNS. In some cases, these differences may reflect the spatial insensitivity of the qRTPCR methods, as was confirmed by in situ hybridization analysis of Lk expression. Phm, in particular, may belong in this category. Although levels of PHM and DIMM expression are strongly correlated, PHM is also highly expressed in many other tissues 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 whole-animal qRTPCR analysis (Gauthier, 2006).
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. Evidence was obtained in support of this hypothesis in an 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. This study showed 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, a site of dimm gene expression. 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. 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 (Mist1KO), pancreatic exocrine cells display reduced intracellular organization. Moreover, the Mist1KO phenotype is partially phenocopied in animals mutant for the Rab3D gene, a small GTPase involved in secretory granule trafficking. 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 (Gauthier, 2006).
Mutations in the crc gene result in pleiotropic defects in ecdysone-regulated events during molting and metamorphosis. 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 (Gauthier, 2006).
This study provides four independent lines of evidence that demonstrate a crucial role for crc in the upregulation of ETH mRNA levels: (1) a marked reduction by qRTPCR is observed in levels of ETH transcripts [but not in mRNAs encoding CCAP or EH, two other neuropeptides involved in the neuropeptide hierarchy controlling ecdysis in crc mutant larvae; (2) in situ hybridization revealed a strong reduction in ETH mRNA levels in the endocrine Inka cells in crc mutant larvae; (3) the intensity of anti-PETH immunoreactivity was markedly reduced in crc1/crc1 homozygotes; (4) EGFP fluorescence driven by an ETH-EGFP reporter gene was reduced in crc mutant larvae. 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 (Gauthier, 2006).
Despite the molecular identification of the crc locus, almost six decades after the original description of the first crc allele, the causes of the molting and metamorphosis defects in crc mutants remained unclear. The 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. These similarities in molting defects, taken together with the 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 (Gauthier, 2006).
Despite the specific effects of crc on ETH transcription in the Inka cells, crc is widely expressed, suggesting a cellular housekeeping function. The vertebrate ATF-4 protein is also ubiquitously expressed. In addition, the upregulation of ATF-4 constitutes a milestone of many cellular stress response pathways including oxidative stress, amino acid deprivation, and hypoxia. In the tobacco hornworm, Manduca sexta, levels of ETH fluctuate during the molts and are regulated by circulating ecdysteroids. It is hypothesized that CRC contributes to the regulation of ETH gene expression during this period, perhaps in response to signals from the tracheae (Gauthier, 2006).
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. 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 the analysis of the conserved region sequences CR1 and CR2 (located 91-171 bp upstream of the ETH translational start site), 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). In addition, 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. Mutations that specifically disrupt E74B, which likely binds the same consensus as E74A, display defects associated with pupal ecdysis that closely phenocopy crc. In future, studies will focus on whether 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 (Gauthier, 2006).
in situ hybridization was performed to determine the expression pattern of crc mRNAs. In wild-type, wandering stage third instar larvae, specific hybridization was seen in several tissues. The imaginal discs and CNS displayed the strongest signals. There was strong, relatively uniform staining in the T1-T3 leg discs and detectable, though weaker, staining in the wing discs, labial discs, and in large cells associated with the anterior spiracles. Within the CNS, the strongest expression was observed in or near the optic lobe proliferation zones. The rest of the brain and ventral nerve cord showed strong, uniform hybridization, although less hybridization was observed in the posterior abdominal neuromeres. Specific hybridization was seen in patches of small cells located throughout the midgut (Hewes, 2000).
The effects of several crc alleles on the pattern of crc in situ hybridization was examined in the CNS of feeding third instar larvae. There was strong hybridization in the CNS of +/Rev8 larvae. By contrast, no signal was detected in larvae bearing a complete deletion of the crc locus (Rev8/Rev4), nor was there signal in R6/Rev8 and R1/Rev8 larvae. Both R1 and R6 delete portions of crc-a, but R1 and R6 may have differential effects on crc-b/c. Thus, under the hybridization and detection conditions used for this experiment, it appears that the crc-a isoform accounts for most if not all of the visible signal, while crc-b and crc-c were below detection. Finally, the pattern of hybridization in crc1/Rev8 larvae was the same as the pattern observed in the control, +/Rev8 larvae. Thus, crc expression in the CNS appeared to be normal in crc1 mutants, consistent with the interpretation that a defect at the protein level (Q171R) likely accounts for the crc1 mutant phenotype (Hewes, 2000).
crc alleles were generated using imprecise P-element excision and male recombination. Six partial or complete deletions of crc were generated. crcRev8 (Rev8) is a complete null; it removed all of the crc exons and several exons from gene Y. The remaining five deletions (R1, R2, R6, E85, and E98) are all partial disruptions of crc. At least two of these alleles, R2 and E98, also disrupt gene Y. E85 appears to be a specific CRC-D mutant, since it affects only exon 4b. By contrast, R1, R2, and E98 disrupt both CRC-A and CRC-D. R6, which deleted the exons encoding the bZIP domain, disrupts CRC-A and CRC-B. Because exons 1-4 remain intact in R6, this allele may not disrupt CRC-D and two small 5' RNAs (crc-a and crc-f). R1, R2, R6, and E98 each retained some or all of c929, the P element used for the mutant screens. In E85, c929 appears to have been excised completely. An additional recombinant line, R20, contained a precise excision of c929 (Hewes, 2000).
Complementation analysis revealed at least three lethal groups in 39C2-4, two of which (the 5' group and 3' group) were associated with deletions of crc exons. The 5' group includes R1, R2, E85, and E98, all deletions of 5' crc exons, as well as crc929 (929). The 3' group includes crc1 and R6, a deletion of the 3' crc exons. A third lethal complementation group was associated with disruptions of gene Y (Hewes, 2000).
With the exception of crc1, all of the crc mutant alleles share the same parental chromosome, 929. Precise excision of the c929 P element (R20) fully restored the viability of animals bearing this chromosome in trans over Rev8, a lethal deletion of the entire crc locus, and over Rev4, a larger deletion of 39C. Thus, the parental 929 chromosome displayed no lethality in 39C2-4 independent of the P-element insertion (Hewes, 2000).
The crc 5' complementation group is associated with isoform-specific disruptions of the crc gene. For example, 929 is semilethal in trans over TW161, Rev4, and Rev8 (all of which completely delete crc) but not over TW1, which leaves intact the entire crc gene as well as ~15 kb of DNA upstream of the putative crc-a transcriptional start site. Since the 929 P element is inserted in an intron of crc upstream of the putative crc-b/c transcriptional start site, the lethality caused by 929 may reflect a specific disruption of the crc-a mRNA isoform. Similar results were obtained with R1, which deletes all of the 5' exons of crc (leaving the exons encoding crc-b and crc-c intact). Both R1 and 929 display similar lethality (with variable penetrance) in crosses to the deficiencies TW161, Rev4, and Rev8. R1 is semilethal in homozygotes, whereas 929 homozygotes are fully viable. Thus, R1 appears to be a slightly more severe allele. This difference may stem from the fact that R1 disrupts the crc-d-f mRNAs in addition to crc-a. Consistent with this hypothesis, E85, a smaller deletion that disrupts an exon specific to crc-d, displayed significant lethality in trans with TW161 and Rev4. The E85 chromosome also appears to bear a lethal mutation at a second, distant site: E85 homozygotes displayed greater lethality than E85 hemizygotes, and in contrast with the larger R1 deletion, E85 displayed some lethality in trans with TW1. Finally, there are two stronger lethal alleles, E98 and R2, and the degree of lethality associated with these alleles is correlated with the distal extent of these deletions (Hewes, 2000).
The crc 3' complementation group is associated with disruptions of both of the major crc mRNA isoforms, crc-a and crc-b. R6 deletes all of the 3' crc exons shared by these two mRNAs. crc1 and R6 both were lethal in trans with deletions of the crc locus, and crc1 and R6 fail to complement each other. By contrast, crc1 was fully viable over deletions that extend distally from the c929 P-element insertion site. Thus, the wild-type function(s) of the crc gene must include contributions by transcription units located proximal to the c929 insertion, such as crc-b and crc-c. Up to 2% adult escapers were observed among hemizygous crc1 progeny. Hemizygous R6 adult escapers were never observed. Thus, although both crc1 and R6 are very strong hypomorphs, it is concluded that R6 is a more severe allele. R6 is not a complete crc amorph, since it complements E85 (Hewes, 2000).
Crosses revealed two largely distinct phenotypes, each generally associated with only one of the crc complementation groups. This result further indicates that the 5' and 3' groups represent distinct genetic functions. For the 3' group (crc1 and R6), there were several lethal phases during larval, pupal, and adult development. Both crc1 and R6 hemizygotes displayed 15-50% of their lethality after pupariation. At each stage, the R6 allele displayed a more severe phenotype than crc1 (Hewes, 2000).
The molts between successive larval stages are disrupted in crc1 mutants, and this phenotype is accompanied by significant lethality (Chadfield, 1985). A comparable phenotype was observed in the R6 mutants, and the presence of supernumerary mouthparts was strongly correlated with larval lethality. In addition to the larval molting defects, R6 hemizygotes showed delayed and defective pupariation. By contrast, crc1 hemizygotes pupariated normally (cf., Hadorn, 1943), consistent with the weaker hypomorphic phenotype of crc1. Although ~5% of the hemizygous R6 puparia were indistinguishable from wild type, the rest were aberrant to varying degrees. These defects included a failure to evert the anterior spiracles and a retention of a larval shape, which was thinned, elongated, and sometimes curved to one side. In the most severe cases, the abdominal gas bubble, which normally forms ~6 hr after pupariation, did not appear, although the larval mouthparts were later expelled (Hewes, 2000).
crc1 and R6 mutant pupae display a range of defects associated with pupation and subsequent development, as previously described for the crc1 allele (Hadorn, 1943; Fristrom, 1965; Chadfield, 1985). The pupal phenotypes of these two alleles were similar. The mutants often failed to expel or translocate the abdominal gas bubble. Head eversion failed or was incomplete, and the leg and wing discs did not completely elongate. In addition, segmentation and differentiation of the abdomen usually failed, although in some cases the anterior abdominal segments differentiated. Other aspects of adult development proceeded normally, resulting in the appearance of mature eye pigments and darkened macrochaetes and differentiation of the wings and legs (Hewes, 2000).
Adult, hemizygous crc1 females displayed markedly decreased fecundity. In addition, 5-50% (depending upon the genetic background) of the hemizygous crc1 adults of both sexes failed to expand their wings and fully tan the adult cuticle. Other hemizygous crc1 adults displayed more subtle defects involving the wings, legs, scutellum, scutellar bristles, halteres, and dorsal thorax. Many of these defects could be explained by incomplete tanning of the adult cuticle after eclosion (Hewes, 2000).
The lethal phase for the 5' group of crc alleles (R1, R2, E85, E98, and 929) was primarily after pupariation, since the number of dead pupae observed on the sides of the vials was approximately equal to the total amount of lethality. Larvae were never observedwith multiple mouthparts, and the puparia were normal in size and shape. In addition, gas bubble translocation, expulsion of the larval tracheae and mouthparts, and head eversion were all completed successfully. The 5' group of alleles displayed defects in leg and wing disc elongation that were similar to those observed for the 3' group, but they also caused novel defects in adult development ('head/abdomen-collapsed' phenotype. In contrast to crc1, the distal portions of the everted leg discs often darkened abnormally and did not differentiate further. After pupation, the abdomen shrank markedly and withdrew to a dorsal position. Subsequently, the head collapsed partially or completely into the thoracic cavity. Despite these events, many pupae developed eye pigmentation and other signs of adult differentiation (Hewes, 2000).
Although most of the mutations within the 5' group fully complemented the 3' group, R6 was an exception. R6 uncovered crc1 (3' group) as well as R1, R2, E98, and 929 (5' group). In addition, R6 mutants displayed a cryptocephal phenotype when crossed to crc1 and the head/abdomen-collapsed phenotype when crossed to alleles from the 5' group. When placed in trans with R6, the 929, R1, R2, and E98 alleles each displayed a similar degree of lethality (independent of deletion size), indicating that each of these crc 5' alleles displayed comparable defects in the function of the crc gene (Hewes, 2000).
Interestingly, the 5' group alleles and R6 (but not crc1) also displayed the head/abdomen-collapsed phenotype when heterozygous over either CyO, y+ or a second balancer, In(2LR)SLM. This dominant effect was associated with variable, but significant pupal lethality. Because the CyO, y+ and In(2LR)SLM chromosomes were created independently, it appears unlikely that these chromosomes share dominant enhancers of the head/abdomen-collapsed phenotype. Rather, this result suggests that the 5' group alleles and R6 are crc haploinsufficient in some genetic backgrounds (Hewes, 2000).
Because deletions distal to c929 complemented crc1, the genomic regions containing exons 5 and 6 and exons 7 and 8 were sequenced from crc1/crc1 larvae. Nine differences were identified between the crc1 and wild-type sequences. Of these, six corresponded to wild-type polymorphisms, and two were conservative substitutions. The remaining substitution (GATGCACAGCCAAAA; the underlined residue is G in crc1) results in a nonconservative change from glutamine to arginine (Q171R). Because Q171R was the only nonconservative substitution in the crc1 coding sequence, it is speculated that it is the cause of the associated phenotypic defects (Hewes, 2000).
To confirm the molecular identification of the crc gene, the lethality of mutant crc alleles was rescued using germ-line transformants expressing a crc cDNA. On the basis of the complementation analysis, it is predicted that ectopic expression of the CRC-B protein isoform (encoded by the crc-c mRNA) would rescue crc1 lethality. To test this hypothesis, multiple independent germ-line transformants were made with crc-c under the control of a GAL4 upstream activating sequence (UAS-crc). In six of seven lines, UAS-crc rescued 8%-21% of the lethality in crc1/Rev8 heterozygotes. The rescue was constitutive (without heat shock), presumably reflecting basal expression of UAS-crc. Heat-shock-induced expression of UAS-crc under the control of an hs-GAL4 driver caused substantial lethality in an otherwise wild-type background; thus the hs-GAL4 driver only lowered the degree of rescue seen. The degree of rescue also was influenced by the parental genotype; for insertion 9-2, ~40% rescue was obtained when both parental stocks were balanced with CyO, y+ (Hewes, 2000).
Attempts were made rescue of 5' group functions. 929 was chosed as a representative 5' group allele for two reasons. First, the complementation and molecular analyses indicated that 929 displayed lethality due to disruption of crc-a without any confounding disruption of gene Y. Second, c929 is a GAL4 enhancer trap P element, which allowed heterogeneous expression of the UAS-crc transgene in 929 mutants. The c929 GAL4 reporter gene is expressed in larvae in peptidergic central nervous system (CNS) neurons, intrinsic cells of the ring gland, salivary gland, fat body, patches of the epidermis, the PM peritracheal cells, and a few other scattered locations. By contrast, there is very restricted c929 reporter gene expression in the imaginal discs and no detectable expression in the skeletal muscles and abdominal histoblasts. Several independent UAS-crc insertions partially or completely rescued the lethality observed in 929 hemizygotes. Thus, there were two separable crc functions, and both were rescued by transgenic expression of CRC-B. Moreover, given the inclusion of numerous neurosecretory neurons in the pattern of c929 reporter gene expression, it is speculated that crc may function in close association with ecdysone biosynthesis/secretion (Hewes, 2000).
Search PubMed for articles about Drosophila Cryptocephal
Bartsch, D., et al. (1995). Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change. Cell 83(6): 979-92. Medline abstract: 8521521
Chadfield, C. G. and Sparrow, J. G. (1985). Pupation in Drosophila melanogaster and the effect of the lethalcryptocephal mutation. Dev. Genet. 5: 103-114. FlyBase link.
Estes, S. D., Stoler, D. L. and Anderson, G. R. (1995). Normal fibroblasts induce the C/EBP beta and ATF-4 bZIP transcription factors in response to anoxia. Exp. Cell Res. 220(1): 47-54. Medline abstract: 7664842
Fawcett, T. W., et al. (1999). Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response. Biochem. J. 339: 135-141. Medline abstract: 10085237
Fletcher, J. C., et al. (1995). The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone. Development 121: 1455-1465. Medline abstract: 7789275
Fristom, J. W. (1965). Development of the morphological mutant cryptocephal of Drosophila melanogaster. Genetics 52: 297-318 Flybase link
Gauthier, S. A. and Hewes, R. S. (2006). Transcriptional regulation of neuropeptide and peptide hormone expression by the Drosophila dimmed and cryptocephal genes. J. Exp. Biol. 209: 1803-1815. Medline abstract: 16651547
Hadorn, E., and Gloor, H. (1943). Cryptocephal ein spat wirkender Letalfaktor bei Drosophila melanogaster. Rev. Suisse Zool. 50: 256-261. FlyBase link
Hewes, R. S,. Schaefer, A. M. and Taghert, P. H. (2000). The cryptocephal gene (ATF4) encodes multiple basic-leucine zipper proteins controlling molting and metamorphosis in Drosophila. Genetics 155(4): 1711-23. Medline abstract: 10924469
Kawai, T., et al. (1998). ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol. Cell. Biol. 18: 1642-1651. Medline abstract: 9488481
Mielnicki, L. M., et al. (1996). Mutated Atf4 suppresses c-Ha-ras oncogene transcript levels and cellular transformation in NIH3T3 fibroblasts. Biochem. Biophys. Res. Commun. 228: 586-595. Medline abstract: 8920955
Sparrow, J. C. (1981). The recovery and preliminary examination of a temperature sensitive suppressor of the cryptocephal mutant of Drosophila melanogaster. Genet. Res. 38(3): 297-314. Medline abstract: 6800886
Sparrow, J. C. and Chadfield, C. G. (1982). Chitin biosynthesis during pupal development of Drosophila melanogaster and the effect of the lethalcryptocephal mutation. Dev. Genet. 3: 235-245. FlyBase link
date revised: 2 September 2007
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