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. First, 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. Second, in situ hybridization revealed a strong reduction in ETH mRNA levels in the endocrine Inka cells in crc mutant larvae. 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. 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).
G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous 'orphan' GPCRs have become available for functional analysis. This study analyzes two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins (FXPRXamide), Cap2b-like peptides (FPRXamide), or ecdysis triggering hormones (PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (CCAP), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors (Park, 2002b).
Genes encoding Drosophila signaling peptides having PRXa C-terminal motifs were located by using BLASTP and TBLASTN searches with parameters for finding short matching sequences. Various insect PRXa peptides previously described were used for query sequences. Mature peptides were predicted by the C-terminal sequence motif PRXG(K/R): G for amidation followed by a mono- or di-basic cleavage site. N termini were predicted after the dibasic cleavage sites (K/R)(K/R) in upstream positions proximal to the PRXG(K/R) motif. A total of three genes encoding seven mature peptides were predicted. It was not possible to identify sequences similar to AVP or to the locust AVP-like insect diuretic hormone in database searches with similar search parameters as above (Park, 2002b).
The PRXa C-terminal motif is found in a number of invertebrate and vertebrate peptides. In the invertebrates, these include the PBAN-like FXPRXa motif characteristic of the pyrokinin group, FPRXa exemplified by small cardioactive peptide and CAP2b, and PRXa of ETH. Vertebrate PRXa peptides consist of pancreatic polypeptide (36 aa with C-terminal NMLTRPRYa), AVP (NXPRXa), and NMU-25 or -8 (25 or 8 aa with C-terminal FXPRXa) (Park, 2002b).
The Drosophila genome database (www.fruitfly.org/blast/) was searched for all genes encoding peptides with C-terminal amino acid PRXa motifs and for G protein-coupled receptors likely to be activated by these ligands. The search for peptides yielded three genes: hugin (CG6371, GenBank accession no. AJ133105), cap2b-like (CG15520, capability, GenBank accession no. AF203878), and eth (CG18105; GenBank accession no. AF170922). The gene hugin encodes two peptides, referred to here as Hug and Drm-PK-2, whose C-terminal motifs are related to the insect pyrokinins. The cap2b-like gene encodes three putative peptides related to cardioacceleratory peptides (CAPs), referred to here as CAP2b-1, -2, and -3. CAP2b-1 and CAP2b-2 contain a common C-terminal motif (FPRXa), whereas the C terminus of CAP2b-3 (GLWFGPRLa) is identical to that of the diapause hormone of Lepidoptera. The peptides ETH1 and ETH2 encoded by the gene eth possess a C-terminal PRXa motif (Park, 2002b).
Analysis of the three vertebrate PRXa peptides, NMU, AVP, and pancreatic polypeptide (PP) shows that the PRXa motifs are strictly conserved in NMU and AVP, whereas that of PP is likely a consequence of converging evolution from NPY/PYY/PP family, which includes Drosophila neuropeptide F [C-terminal motif (PH)R(YF)amide]. In this fashion, the search for PRXa-activated GPCRs in Drosophila was narrowed to those related to the AVP and NMURs (Park, 2002b).
Phylogenetic analysis reveals that NMURs occur in a monophyletic clade with four Drosophila GPCRs: CG8784, CG8795, CG9918, and CG14575. Three Drosophila GPCRs homologous to AVP receptors are CG6111, CG11325 (also known as gonadotropin releasing hormone receptor), and CG10698. CG6111 is orthologous to the vasopressin/oxytocin receptor gene family (Park, 2002b).
Putative Drosophila GPCRs in the database were amplified by RT-PCR using primers based on gene predictions in the FGENESH gene finder. Conceptual translations of these genes aligned with other GPCRs present complete seven transmembrane domains. Sequences confirmed by at least two independent RT-PCR experiments revealed several polymorphic sites compared with the Celera Drosophila genomic sequences (Park, 2002b).
Oocytes injected with cRNAs for the GPCRs generated inward currents up to 2.5 µA upon activation with appropriate ligands. It is presumed that ligand-activated inward currents in these experiments result from Gq activation of phospholipase C, liberation of inositol trisphosphate, and activation of chloride current by mobilization of intracellular calcium stores (Park, 2002b).
Drosophila GPCRs in the NMUR clade were activated by PRXa peptides with various levels of sensitivity and specificity. CG14575 was the most selective within this group, responding only to CAP2b-1 (EC50 150 nM) and CAP2b-2 (EC50 230 nM), which have an identical C-terminal VFPRVamide motif. All other peptides were inactive on application at 10 µM. In contrast, CG8795 responds to a relatively wide range of ligands, including Drm-PK-2, hug, CAP2b-3, and ETH1, listed in order of decreasing potency. Drm-PK-2 and Hug appear to have highest potency, but also induce the most severe receptor desensitization. The high level of desensitization complicated efforts to produce quantitative determinations of potency for these ligands. In contrast, ETH1 and CAP2b-3 treatment produces little or no desensitization.
CG9918 and CG8784 were insensitive to most ligands applied. CG9918 responded only to the highest concentration of CAP2b-3 applied (10 µM), and was otherwise insensitive to all other ligands applied at this concentration. Similarly, CG8784 was activated only by Drm-PK-2 or Hug applied at 10 microM (Park, 2002b).
Thus Drosophila GPCRs in the NMUR group respond to the PRXa peptides, Hug, Drm-PK-2, CAP2b-1 to -3, and ETH. Non-PRXa peptides such as proctolin, FMRFamide, and diuretic hormone produced no response at 10 µM, the highest concentration tested. The range of ligand concentrations sufficient to activate each GPCR ranged from low nanomolar to micromolar. CG14575 was the most ligand-selective receptor in this group, responding only to low nanomolar concentrations of CAP2b-like peptides CAP2b1 and CAP2b-2 having FPRXa motifs, whereas CAP2b-3, a mature peptide from the same gene having FXPRXa motif had no effect on CG14575 at 10 microM (Park, 2002b).
It seems likely that CG14575 is involved in ion transport functions associated with diuresis in Drosophila. It has been shown that Drosophila CAP2b-1 and -2 act on principal cells of Malphighian tubules, stimulating fluid secretion through the calcium-nitric oxide-cGMP pathway. It will be interesting to determine whether CG14575, the putative CAP2b-1/CAP2b-2 receptor from this study, is expressed in Malpighian tubules (Park, 2002b).
CG8795 responds to a different set of nonoverlapping PRXa peptides, being most sensitive to Hug and Drm-PK-2. These peptides produce activation at low nanomolar concentrations accompanied by marked receptor desensitization, making it difficult to ascertain a reliable EC50 value for these peptides. CG8795 also shows moderate sensitivity to ETH1 and CAP2b-3, responding to mid- to high nanomolar concentrations. Interestingly, ETH2 had no effect at 10 µM. The responses of CG8795 to a wide range of peptides were unexpected. Although Drm-PK-2 was most active, Hug, ETH1, and CAP2b-3 also produced robust responses. The ligands active on this receptor also include Manduca sexta MasETH and Heliothis virescens HezPBAN at 10 microM concentration. However, some obvious selectivity was apparent, with no responses registered to CAP2b-1 and -2, ETH2, and Manduca PETH applied at 10 microM (Park, 2002b).
Activation of CG8795 by both Hug and ETH1 raises the possibility of its involvement in ecdysis. Such a possibility is indicated not only by its sensitivity to ETH1 (which is known to be obligatory for ecdysis signaling). Ecdysis deficiency is induced by ectopic expression of the hugin gene. Furthermore, the hugin gene product Hug mimics ETH1 by inducing ecdysis behavior in wild-type flies and by rescuing ecdysis deficiency in buttoned-up eth null mutants. Given that Hug and ETH1 activate both CG8795 and ecdysis behavior, several interpretations are possible. CG8795 may be involved in ecdysis signal transduction, and both Hug and ETH1 are ecdysis signaling molecules. Alternatively, CG8795 is not involved in ecdysis, but can be activated by relatively high concentrations of ETH1 acting as a Hug agonist. According to this alternative scenario, CG8795 could be involved in other physiological functions such as pheromone biosynthesis as a Hug and/or Drm-PK-2 receptor. Further work is needed to clarify an authentic role for CG8795 and function of Hug in the ecdysis signaling pathway (Park, 2002b).
The remaining GPCRs in the NMU group, CG8784 and CG9918, respond only to high levels (10 microM) of Hug and Drm-PK-2, and CAP2b-3, respectively. It is possible that the endogenous signal transduction machinery in the Xenopus oocyte is inappropriate for mediation of functional receptor activation for CG8784 and CG9918. This assay system generates a presumed calcium-activated chloride current known to be activated exclusively by Gq coupled pathways. GPCRs can be coupled to a variety of G proteins, including Gi/o, Gs, and Gq, with various degrees of efficiency and specificity. Poor coupling of heterologously expressed GPCRs to Gq in the Xenopus oocyte clearly could result in artifactually low affinity estimates. In particular, CG9918 and CG8784 were found to be largely insensitive to all ligands tested (Park, 2002b).
The functions of PRXa peptides known thus far in the vertebrates include activation of ion transport and contractile activity in intestine and arterial musculature via the NMUR. In invertebrates, functions for many of the PRXamide peptides remain uncertain, biological activity having been inferred from standard assays for visceral muscle contraction. For example, early demonstrations of activity for the pyrokinins (FXPRXa) were based on stimulation of gut, oviduct, and heart, whereas more recent data implicating them in pheromone biosynthesis and cuticle melanization are more suggestive of authentic physiological functions. The FPRXa peptides, including small cardioactive peptides and cardioacceleratory peptide (CAP2b), were isolated based on their activity in heartbeat modulation but may be involved in water and ion transport. Finally, although all other PRXa peptides are produced in the central nervous system, ETH (PRXa) is produced peripherally in epitracheal Inka cells and acts on CNS to trigger central pattern generators leading to ecdysis behavior. Knowledge of the expression patterns of the receptor GPCRs will likely provide new insights into the true physiological functions for the PRXa peptides (Park, 2002b).
Immunohistochemical staining using antisera raised against Drosophila ETH1 has revealed segmentally repeated cells in both larval and adult stages. These cells appear to be homologous to 'Inka cells' previously identified in M. sexta (Zitnan, 1996), and henceforth are referred to by the same name. An identical staining pattern was observed using an antiserum raised against the C-terminus of the M. sexta peptide, MasPETH (Zitnan, 1999). In larvae, cells exhibiting ETH-like immunoreactivity (ETH-IR) occur along each of the two dorsal tracheal trunks at the main branch points of transverse connectives. A total of seven Inka cell pairs occur consistently in each tracheal metamere Tr1 and Tr4 through Tr9 in the larval stage. In adults, cells showing ETH-IR also occur at homologous positions, but vary in shape and location. Depletion of ETH-IR is observed at each larval ecdysis (Park, 2002a).
Antisera to Drosophila ETH1 and MasETH also stain ~20 neurons and axons in the CNS. Staining in the CNS presumably results from cross-reactivity with neuropeptides containing the conserved C-terminal sequence motif -PRXamide, which is shared by ETHs (Drosophila ETH1, DDSSPGFFLKITKNVPRLa; Drosophila ETH2, GENFAIKNLKTIPRIa; MasPETH, SFIKPNNVPRVa; MasETH, SNEAISPFDQGMMGYVIKTNKNIPRMa), the cardioactive peptide CAP2b, pheromonotropic and diapause hormones in moths, and the Drosophila neuropeptides CG15520 and CG6371 (Park, 2002a).
To examine the cellular expression pattern of ETH, a fly line carrying the chimeric transgene 2eth3-egfp was constructed. This transgene occurs on the 2nd chromosome and contains the sequence of ETH up to the 3rd amidation site with chimeric egfp encoding the enhanced green fluorescent protein. EGFP fluorescence in 2eth3-egfp flies is observed in both larval and adult stages, but is confined to the constellation of Inka cells showing ETH-IR. No EGFP fluorescence occurs in any other cell or tissue in larvae or adults. These data are consistent with cell-specific expression of eth. Observations under laser confocal microscopy revealed an identical distribution of EGFP fluorescence and ETH-IR in Inka cells of wandering 3rd instar, suggesting that EGFP and processed ETHs are located in the same subcellular compartments (Park, 2002a).
In 1st instar larvae, peak EGFP fluorescence occurs at dVP (the time of appearence of douple vertical plates), and declines sharply to 16±3% of peak emission just before tracheal inflation. A further drop of EGFP emission to 11±3% occurs by the squeezing wave stage. Loss of EGFP fluorescence suggests that ETH is released naturally in vivo during the time interval between dVP and tracheal collapse (Park, 2002a).
In insects, ecdysis is thought to be controlled by the interaction between peptide hormones; in particular between ecdysis-triggering hormone (ETH) from the periphery and eclosion hormone (EH) and crustacean cardioactive peptide (CCAP) from the central nervous system. The behavioral and physiological functions of the first two of these peptides was studied in Drosophila melanogaster using wild-type flies and knockout flies that lacked EH neurons. ETH from Manduca sexta (MasETH) was used to induce premature ecdysis and the responses of the two types of flies were compared. The final release of EH normally occurs approximately 40 min before ecdysis. It is correlated with cyclic guanosine monophosphate (cGMP) production in selected neurons and tracheae, by an elevation in the heart rate and by the filling of the new tracheae with air. Injection of developing flies with MasETH causes all these events to occur prematurely. In EH cell knockouts, none of these changes occurs in response to MasETH, and these flies show a permanent failure in tracheal filling. This failure can be overcome in the knockouts by injecting them with membrane-permeant analogs of cGMP, the second messenger for EH. The basis for the 40 min delay between EH release and the onset of ecdysis was examined by decapitating flies at various times relative to EH release. In flies that had already released EH, decapitation is always followed within 1 min by the start of ecdysis. Immediate ecdysis is never observed when the EH cell knockout flies were decapitated. It is proposed that EH activates both ventral central nervous system elements necessary for ecdysis (possibly the CCAP cells) and descending inhibitory neurons from the head. This descending inhibition establishes a delay in the onset of ecdysis that allows the completion of EH-activated physiological processes such as tracheal filling. A waning in the inhibition eventually allows ecdysis to begin 30-40 min later (Baker, 1999).
At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. To elucidate neural substrates underlying the ecdysis sequence, neurons expressing ETH receptors (ETHRs) have been identified in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. These findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles (Kim, 2006a).
Analysis of the pupal ecdysis behavioral sequence: In Drosophila, pupal ecdysis is preceded by pupariation, whereby the prepupa contracts its body into a barrel shape to form the puparium composed of the old larval cuticle. The underlying new pupal cuticle then separates from the puparium during pupal ecdysis ~12 hr later. The stereotypic nature of pupal ecdysis and reliable developmental markers make it a favorable model for the behavioral analysis and neural imaging (Kim, 2006a).
Pupal ecdysis consists of three centrally patterned behavioral subunits performed sequentially: pre-ecdysis (~10 min), ecdysis (~5 min), and postecdysis (~60-70 min). The behavioral sequence was examined through the semitransparent puparium ('puparium-intact'), but it was found that the puparium obscures and places constraints on some movements. This made it particularly difficult to discriminate differences in abdominal swinging movements during ecdysis and postecdysis. Therefore, a 'puparium-free' preparation was used by surgically removing the puparium immediately after pre-ecdysis onset. The improved visibility and room for movement in this preparation allowed for a more complete analysis of natural and ETH-induced behavior. The following description of the natural pupal ecdysis sequence resulted from comparison of behaviors observed in both puparium-intact and puparium-free prepupae (Kim, 2006a).
Pre-ecdysis: About 5 min after in vivo ETH release, preecdysis commences with the abrupt appearance of an air bubble at the posterior end of the prepupa (time zero). Pre-ecdysis involves anteriorly directed rolling contractions along the lateral edges of the abdomen, alternating on the left and right sides of the animal. These contractions move the air bubble anteriorly to separate pupal cuticle from the puparium. This behavior is completed within ~10 min and is followed by ecdysis behavior. Preecdysis behavior is the same in puparium-intact and puparium-free animals (Kim, 2006a).
Ecdysis: In higher Diptera including Drosophila, the incipient adult head develops within the prepupal thorax. During pupal ecdysis, head eversion results from lateral swinging movements of the abdomen occurring along with anteriorly directed peristaltic contractions. In puparium-intact preparations, head eversion occurs ~1 min after the onset of ecdysis swinging and is completed within ~5s. After completion of head eversion, ecdysis contractions continue for ~15 min, facilitating expansion of wing pads and legs to their final size. The frequency of ecdysis swinging (~5 swings/min) decreases markedly after head eversion (~2 swings/min). In puparium-free animals, head eversion occurs sooner, and the duration of ecdysis behavior lasts only ~5 min. Later in ecdysis, anteriorly directed swinging contractions are often interrupted by posteriorly directed ones, indicating a transition to postecdysis (Kim, 2006a).
Postecdysis: Postecdysis behavior consists of two behavioral subroutines: postecdysis swinging and stretch-compression movements of the abdomen. Postecdysis swinging occurs along with posteriorly directed peristaltic contractions and alternates with longitudinal movements of the abdomen, referred to as 'stretch-compression.' The frequency and intensity of postecdysis contractions wane gradually until they are detected mainly in the anterior part of the abdomen; they cease w100 min after pre-ecdysis onset. Postecdysis behavior concludes with compression of the pupa at the posterior end of the puparium (Kim, 2006a).
ETH release coincides with initiation of the ecdysis sequence: To confirm the role of ETH in initiation of the pupal ecdysis sequence, its release from endocrine Inka cells was monitored in vivo by using time-lapse EGFP fluorescence imaging in pharate pupae (prepupae) carrying the chimeric transgene 2eth3-egfp. In this transgenic fly, EGFP is expressed as part of a fusion protein with the ETH propeptide precursor, and loss of EGFP fluorescence indicates ETH release. Because pharate pupae generally are immobile and Inka cells are located immediately below the semitransparent puparium, in situ imaging of Inka cell in intact pharate pupae is feasible. Two to three Inka cells were monitored simultaneously in each experiment (Kim, 2006a).
Depletion of ETH-EGFP occurs in about 50% of monitored Inka cells shortly before pre-ecdysis onset (time zero). The time course of secretory activity for each Inka cell was variable. The mean value for the timing of ETH release was 4.5 min prior to pre-ecdysis onset, and the duration of ETH secretion was 4.4. In contrast, 40% of Inka cells showed no sign of secretory activity by pre-ecdysis onset. After initiation of pre-ecdysis contractions, it was usually impossible to continue monitoring loss of EGFP fluorescence as a result of movement artifacts. All Inka cells are depleted of ETH-EGFP by the end of ecdysis sequence (Kim, 2006a).
Injection of ETH induces the ecdysis sequence: Because ETH release coincides with onset of pupal preecdysis, it was of interest to determine whether ETH injection would trigger the pupal ecdysis sequence. The Drosophila gene eth encodes a precursor producing one copy each of two peptides, ETH1 and ETH2, which share similar structure and biological activity. In vivo experiments were carried out primarily in puparium-free preparations (Kim, 2006a).
Injection of ETH1 alone into pharate pupae (~1-2 hr prior to natural ecdysis) induced within 1-3 min strong pre-ecdysis contractions followed by ecdysis and postecdysis contractions sequentially. ETH-induced pre-ecdysis showed a strong dose dependence, with higher doses inducing shorter pre-ecdysis duration and higher frequency of contractions. Similar but somewhat less pronounced dose-dependent effects were observed during ecdysis behavior, whereas the frequency of postecdysis contractions showed little or no dose dependence during the first 10 min of behavior (Kim, 2006a).
Injection of ETH2 was less efficacious for induction of the behavioral sequence compared to ETH1. ETH2 generated prolonged pre-ecdysis behavior lasting to 50 min or more, but no ecdysis behavior. In contrast, injection of the same dose of ETH1 (0.4 pmol) produced the complete behavioral sequence consisting of pre-ecdysis, ecdysis, and postecdysis. Higher doses of ETH2 (20 pmol) generated a behavioral sequence comparable to that induced by 4 pmol ETH1 in terms of pre-ecdysis duration and frequency of contractions. Behaviors after injecting a cocktail of ETH1 and ETH2 (0.4 pmol of each peptide) were also examined. Because the two peptides are processed from the same precursor, it is likely that these peptides are coreleased under natural conditions. The duration of the behavioral sequence induced by injection of the cocktail was similar to the naturally occurring sequence or one induced by 0.4 pmol ETH1 alone. It is estimated that a 0.4 pmol ETH injection into a prepupa w10 hr after puparium formation results in a concentration of ~300 nM in vivo (Kim, 2006a).
ETH receptors are expressed in diverse ensembles of peptidergic neurons: ETH acts directly on the CNS to initiate the ecdysis behavioral sequence in moths and flies via unknown signaling pathways within the CNS. A starting point for elucidation of these downstream signaling pathways is identification of primary neuronal targets of ETH. The ETH receptor gene (CG5911), first identified in Drosophila, encodes two G protein-coupled receptors, ETHR-A and ETHR-B, via alternative splicing. In situ hybridization was used for identification of central neurons expressing ETHR-A and ETHR-B by using DNA probes specific for each receptor subtype. ETHR-A and ETHR-B transcripts were located in mutually exclusive populations of neurons distributed throughout the CNS, suggesting that two subtypes of ETH receptors likely mediate different functions. Further analysis revealed that most ETHR-A neurons are peptidergic. Neurons expressing ETHR-B have not been identified thus far (Kim, 2006a).
Multiple ensembles of ETHR-A neurons are classified according to specific neuropeptides they express. Peptides expressed in these ensembles were identified by using GAL4 transgenes under control of neuropeptide promoters to drive UAS-GFP or UAS-GCaMP expression (GAL4::GFP or GAL4::GCaMP). Expression of neuropeptides in these cells was confirmed by combining immunohistochemical staining and in situ hybridization. The first ETHR-A ensemble comprises six pairs of lateral abdominal neurons producing kinin, also known as drosokinin. These cells project axons posteriorly along the lateral edge of the neuropile and then turn anteriorly along the midline of ventral nerve cord, where they arborize and form possible central release sites. Axons of these cells also exit the CNS through nerve roots, suggesting peripheral kinin release. The second ETHR-A ensemble contains three pairs of ventrolateral FMRFamide neurosecretory cells (Tv1-3 or T1-3) in the thoracic neuromeres TN1-3. These cells project axons into the dorsomedial neurohemal organs (NHOs) specialized for peptide release into the hemolymph (Kim, 2006a).
The third class of ETHR-A neurons comprises the eclosion hormone (EH)-producing VM neurons in the brain, which project one axonal branch anteriorly into the neurohemal ring gland and a second posteriorly along the dorsal midline of the entire ventral nerve cord. The fourth ETHR-A ensemble is composed of paired dorsolateral neurons producing CCAP, MIPs, and bursicon in subesophageal, thoracic, and abdominal neuromeres (SN1-3, TN1-3, AN1-7, respectively). These cells are likely homologs of moth neurons 27/704, on the basis of their anatomy, peptide coexpression profile, and functional roles during pupal ecdysis. These neurons are referred to as Drosophila neurons 27/704 and them were subdivided on the basis of peptide coexpression. In AN1-4, CCAP is colocalized with MIPs and the heterodimeric peptide hormone bursicon (composed of burs and pburs subunits). In TN2-3 and AN5- 9, CCAP is colocalized with burs, but pburs is not expressed in these neurons. Finally, CCAP is colocalized with MIPs in large paired neurons of AN8,9, but ETHR-A expression has not been confirmed in these cells. The presence of MIP mRNA in abdominal neurons 27/704 was further confirmed by in situ hybridization (Kim, 2006a).
Ca2+ imaging of primary ETH targets in transgenic flies: Having shown that ETH receptors occur in diverse ensembles of peptidergic neurons, it was asked whether these cells are activated by ETH and whether this activity coincides with specific behavioral steps of the ecdysis sequence. Calcium dynamics in each group of ETHR-A neurons was monitored by driving expression of the GFP-based Ca2+ sensor, GCaMP [23, 24], in genetically defined sets of neurons with the binary GAL4/UAS system. Ca2+ elevation induces a conformational change of GCaMP, increasing its GFP fluorescence. Using optical imaging of GFP fluorescence, [Ca2+]i dynamics of ETHR neurons were monitored, and these events were associated with each behavioral phase induced by ETH (Kim, 2006a).
An abundance of evidence indicates that the ecdysis behavioral sequence in insects is centrally patterned. In particular, the onset and duration of each behavior in the sequence (pre-ecdysis I, pre-ecdysis II, ecdysis) is the same whether observed in vivo or as fictive behavior recorded from the isolated CNS in vitro. On the basis of this evidence, [Ca2+]i dynamics of ETHR-A neuron ensembles of the isolated CNS were associated with behaviors observed in puparium-free preparations (Kim, 2006a).
FMRFamide neurons and their neurohemal endings become active early in pre-ecdysis: [Ca2+]i levels were monitored in ETHR-A/FMRFamide Tv neurons by preparing transgenic flies doubly homozygous for FMRFa-GAL4 and UAS-GCaMP. Prior to ETH1 exposure (4-6 hr prior to ecdysis), Tv cell bodies and neurohemal endings in the dorsomedial NHO exhibit low levels of basal GCaMP fluorescence (Kim, 2006a).
Exposure of the CNS to ETH1 (600 nM) elicits robust increases in calcium-associated fluorescence in cell bodies and axon terminals of all Tv neurons. At this concentration of ETH1, calcium dynamics typically are characterized by transient, spike-shaped fluctuations superimposed upon a slow upward shift of the baseline, beginning ~8 min after exposure to the peptide. This response lasts ~10-15 min, after which weaker spike-like fluctuations continue without baseline changes until the end of recordings (~40 min). It is estimated that a concentration of 600 nM ETH1 results from a dose of w0.4 pmol of the peptide in vivo. Thus the major calcium response of Tv neurons coincides with the early phase of pre-ecdysis, and weaker activity persists through ecdysis and postecdysis. In contrast, ETH2 alone (600 nM) generates Ca2+ responses after a longer delay comparable to one following exposure to 60 nM ETH1. The longer delay of Ca2+ responses after ETH2 fits with the observations of in vivo behavior, where ETH2 is a less potent agonist than ETH1. The cocktail of ETH1 and ETH2 (600 nM each) evokes Ca2+ dynamics after a delay similar to that induced by ETH1 alone. Overall, [Ca2+]i dynamics observed in Tv neurons are synchronized. In many preparations, Tv neurons from the same neuromere appear to be strongly coupled, given that they produce precisely synchronized Ca2+ dynamics. Transient Ca2+ signals are obvious in the terminal processes of Tv neurons in NHO, the release sites of FMRFamides (Kim, 2006a).
Lower concentrations of ETH1 elicit calcium dynamics after a somewhat longer delay. Interestingly, calcium dynamics are obvious first in neurohemal endings of the NHO, followed by fluctuations in cell bodies. This was particularly evident at 60 nM ETH1, where a robust calcium response in the NHO was accompanied by only a weak response in the Tv2 cell body. No calcium responses are observed in Tvs exposed to 6 nM ETH1 (Kim, 2006a).
EH neurons reach peak activity at ecdysis: VM neurons producing eclosion hormone (EH) have been implicated as primary ETH targets during fly and moth ecdysis. Expression of ETHR-A was demonstrated in VM neurons, confirming that they are primary ETH targets. To determine whether ETH elicits activity in EH neurons, transgenic flies were prepared doubly homozygous for EHup-GAL4 and UASGCaMP, that show GCaMP fluorescence only in these cells (Kim, 2006a).
EH neurons are highly sensitive to ETH1, exhibiting robust [Ca2+]i dynamics upon exposure to concentrations as low as 6 nM. No detectable fluorescence responses are observed after exposure to 0.6 nM ETH1 over a period of 50-60 min. The latency to Ca2+ responses is inversely proportional to the concentration of ETH1; higher ETH1 concentrations evoke faster responses. The cocktail of ETH1 and ETH2 (600 nM each) elicited Ca2+ responses after a ~10-15 min delay (Kim, 2006a).
Close examination of these ETH-evoked fluorescence responses reveals two components distinguished by slow and fast kinetics. The slow component is characterized by a gradual increase in baseline levels of Ca2+ followed by a decrease over 20-30 min, whereas the fast component is composed of transient, spike-like activity. Fast components have durations ranging from 5-20 s. Peak DF/F responses are quite variable, even among a group of neurons exposed to the same ETH1 concentration. No significant concentration dependence could be detected in peak response (Kim, 2006a).
Distinct subsets of neurons 27/704 are active during different phases of the ecdysis sequence: ETH-evoked Ca2+ signals of neurons 27/704 were examined in transgenic flies carrying CCAP-GAL4 and UAS-GCaMP. Use of the CCAP promoter to drive GCaMP expression resulted in a reporter pattern identical to that described previously. Upon exposure to 600 nM ETH1, distinct subsets of neurons 27/704 exhibited reproducible, stereotypic Ca2+ responses in terms of peak intensity, latency, and termination of Ca2+ dynamics. According to the magnitude of peak fluorescence intensity (peak DF/F), neurons 27/704 fall into three major groups: strong responders, weak responders, and nonresponders. The strong-responder group includes neurons 27/704 in AN1-4 (CCAP/MIPs/bursicon), AN8,9 (CCAP/ MIPs), and TN3 (CCAP). Weak responders are neurons 27/704 in SN2-3, TN1-2, and AN7 producing CCAP only. Neurons in the brain, SN1, and AN5,6 showed no reproducible Ca2+ dynamics in response to 600 nM ETH1 (Kim, 2006a).
In response to ETH1, neurons 27/704 in TN3 and AN8,9 become active within 10-15 min, whereas neurons 27/704 in AN1-4 are activated after a 15-25 min delay. Neurons in TN3 and AN8,9 are therefore activated just prior to ecdysis onset, indicating their possible roles in initiation and maintenance of ecdysis behavior. In addition, Ca2+ dynamics observed in AN8,9 neurons terminated early in postecdysis, supporting this interpretation. In contrast, Ca2+ dynamics of neurons in AN1-4 begin during ecdysis and increase in intensity during the entire postecdysis period, suggesting their roles in these events. The cocktail of ETH1 and ETH2 (600 nM each) evoked Ca2+ dynamics similar to those induced by ETH1 alone. Two groups of neurons 27/704 in abdominal neuromeres (AN1-4 versus AN8,9) exhibit differences in sensitivity to ETH and in their patterns of [Ca2+]i dynamics. It was found that 6-60 nM ETH1 activates neurons in AN1-4 (n = 4), whereas higher concentrations of ETH1 (R600 nM) are required to activate neurons in AN8,9. In addition, neurons in AN8,9 generate transient (1-2 min) Ca2+ spikes over a 15-20 min period after ETH1 activation, whereas neurons in AN1-4 generally produce slower, more persistent Ca2+ dynamics. These differences among subgroups of neurons 27/704 suggest their different functional roles during the ecdysis sequence (Kim, 2006a).
Targeted ablations of apecific ETHR neurons have behavioral consequences: To evaluate behavioral roles of specific ETHR neurons, phenotypes of the pupal ecdysis sequence were investigated in transgenic flies bearing targeted ablations of ETHR neurons, including Tv FMRFamide neurons, EH neurons, and CCAP neurons (27/704 homologs). In control flies carrying UAS-reaper and UAS-GCaMP, but lacking the GAL4 driver, pupal ecdysis was executed as in wild-type flies: pre-ecdysis (0-10 min), ecdysis (10-23 min), and postecdysis (23-100 min) (Figure 7). Given that puparium-intact animals were used, the duration of ecdysis behavior may have been overestimated. Transgenic flies bearing targeted ablations of Tv FMRFamide neurons (FMRF-KO) were generated by crossing females doubly homozygous for FMRFa-GAL4, UAS-GCaMP with homozygous UAS-reaper males. Pupal ecdysis of FMRFa-KO flies is very similar to that of control flies. Because FMRFa-GAL4 drives expression of GAL4 only in three pairs of thoracic Tv neurons and one pair of unidentified neurons in SN, FMRFa-KO flies lost only Tv neurons and not other FMRFamide neurons in the CNS. FMRFa-KO flies complete pupal ecdysis without any detectable abnormality, except that pre-ecdysis contractions appear weaker than in control flies. Pupal ecdysis of VM neuron knockout flies (EH-KO) was then examined. Behavioral analysis showed that, although they complete pupal ecdysis without any severe defects or lethality, ecdysis onset is delayed w4 min. As a result of this delay, EH-KO flies show longer pre-ecdysis than control flies. Additional parameters governing pre-ecdysis, ecdysis, and postecdysis are indistinguishable between EH-KO and control flies (Kim, 2006a).
Finally, CCAP-KO flies were generated in order to examine the functional roles of neurons 27/704 (CCAP neurons) in pupal ecdysis. As expected, CCAP-KO flies failed to initiate ecdysis contractions and could not complete head eversion. Instead, they show prolonged pre-ecdysis contractions for ~25 min, followed by weak random contractions of the abdomen (different from ecdysis and postecdysis contractions of control flies) for the next 50 min (Kim, 2006a).
Conclusions: This study has described orchestration of an innate behavior, the Drosophila pupal ecdysis sequence, by the endocrine peptide ETH. ETH release coincides with onset of behavior, and injection of ETH triggers the complete behavioral sequence, consistent with its role in ecdysis activation previously established in the moths Manduca sexta and Bombyx mori and in Drosophila larvae and adults. Absence of ETH causes lethal ecdysis deficiency, a phenotype that is rescued by ETH injection. ETH therefore functions as a 'command chemical' to orchestrate an innate behavior. Primary CNS targets of ETH was identified by using ETHR-specific in situ hybridization. ETHR-A occurs in multiple classes of peptidergic neurons producing EH, CCAP/MIPs/bursicon, FMRFamides, or kinin (Kim, 2006a).
Expression of ETHR-A was shown in VM neurons, which release EH. In response to ETH, VM neurons become active prior to ecdysis behavior and reach peak levels of activity during ecdysis. These results provide further support for a previously described positive-feedback signaling pathway between VM neurons and Inka cells. This feedback is thought to ensure depletion of ETH from Inka cells. These findings are striking because independent evidence indicates that homologous VM neurons in the moth Manduca are direct targets of ETH and that their secretory products regulate ecdysis behaviors downstream of ETH. For example, isolated EH neurons of Manduca respond to direct action of ETH with increased excitability and spike broadening. In response to ETH action, these neurons release EH, causing cGMP elevation and increased excitability in CCAP-containing neurons 27/704 of the thoracic and abdominal ganglia. CCAP and MIPs, cotransmitters produced by neurons 704, are implicated in eliciting ecdysis behavior (Kim, 2006a).
A homologous role for EH in activation of Drosophila 27/704 neurons has not been clearly demonstrated. For example, no cGMP elevation is observed in these neurons during the natural ecdysis sequence. This lack of cGMP elevation suggests that CCAP neurons are not directly targeted by EH in Drosophila. Nevertheless, EH-knockout flies exhibit a delay in ecdysis initiation, suggesting that EH may modulate excitability in 27/704 cells indirectly through release of additional factors within the CNS. It is therefore proposed that activation of EH neurons by ETH serves two purposes: (1) release of EH into the hemocoel functions as part of a positive-feedback pathway to ensure ETH depletion from Inka cells; (2) release of EH within the CNS synergizes direct ETH actions on different subsets of neurons 27/704 producing CCAP, MIPs, and bursicon, perhaps indirectly through release of downstream signals within the CNS (Kim, 2006a).
Neurons 27/704 expressing ETHR-A respond to ETH with unique patterns of Ca2+ dynamics. These neurons are subdivided by pattern of transmitter expression: CCAP/MIPs/bursicon in AN1-4; CCAP/MIPs in AN8,9; and CCAP in SN1-3, TN1-3, and AN5-7. Temporal patterns of Ca2+ dynamics were determined in each neuronal subset relevant to the behaviors observed. On the basis of these temporal patterns, it is proposed that direct action of ETH on neurons 27/704 in TN3 and in AN8,9 induces initiation and execution of ecdysis contractions and head eversion. In support of this, it is shown that ablation of CCAP neurons abolishes ecdysis contractions and head eversion. Parallel study in Manduca showed that neurons 704 expressing ETHR-A and their peptide cotransmitters, CCAP and MIPs, are implicated in control of the ecdysis motor pattern, supporting the homologous function of 27/704 neurons in Drosophila. Neurons 27/704 in AN1-4 produce CCAP, MIPs, and bursicon, and therefore a cocktail of these peptides is likely released within the CNS and into the hemolymph during postecdysis. It is suggested that centrally released peptides control postecdysis movements, whereas blood-borne CCAP/MIPs regulate heart beat and blood pressure for cuticle expansion and bursicon controls sclerotization of expanded new cuticle. Bursicon was recently identified as a heterodimeric peptide hormone regulating cuticle plasticization, sclerotization, and melanization (Kim, 2006a).
The Drosophila FMRFamide gene (FMRFa) encodes multiple FMRFamide-related neuropeptides, which are expressed in many different cell types, including neuroendocrine cells, interneurons, and perhaps motoneurons. Among these diverse FMRFamide-producing neurons, ETHR-A expression is confined to three pairs of thoracic neurosecretory neurons, Tv1-3. Results of the present study show that the Tv neurons are activated early in pre-ecdysis and that they remain active during ecdysis and postecdysis. However, FMRFa-KO flies show no differences in timing of the ecdysis behavioral sequence. Because FMRFamides enhance twitch tension of larval body-wall muscles through synaptic modulation at the neuromuscular junction, blood-borne FMRFamides released from Tv neurons likely facilitates pre-ecdysis, ecdysis, and postecdysis contractions. Thus the role of Tv neurons as primary ETH targets may be enhancement of muscle contraction during the behaviors. Further work to substantiate this is in progress (Kim, 2006a).
Expression of ETHR-A occurs in in kinin neurons of abdominal neuromeres of Drosophila. Drosophila kinin is known to be involved in water balance, but its central functions have not been described or considered. Expression of ETHR-A in kinin neurons appears to be a conserved mechanism in fly and moth; the Manduca ETHR-A is expressed in abdominal neurosecretory cells (L3,4), which produce kinins and diuretic hormones (DHs). It was further found that the isolated Manduca CNS generates the fictive pre-ecdysis motor pattern upon exposure to a cocktail of kinin and DHs. These findings suggest that ETH activates L3,4 neurons in Manduca to release kinins and DHs centrally, which initiate and execute pre-ecdysis. On the basis of the conservation between Drosophila and Manduca in spatial expression pattern of ETHR, it is proposed that ETH initiates pre-ecdysis behavior indirectly via central release of kinin in Drosophila (Kim, 2006a).
In Drosophila, pupal ecdysis is accomplished by sequential recruitment of three major behavioral units: pre-ecdysis (0-10 min), ecdysis (10-15 min), and postecdysis (15-100 min). Each behavioral unit is programmed in the CNS and sequentially activated by direct actions of ETH, which is synthesized and released from peripheral endocrine Inka cells. Around 4-5 min before pre-ecdysis onset, a sizeable portion (~50%) of Inka cells initiates secretion of ETH into the hemolymph, whereas the remaining portion completes secretion after onset of pre-ecdysis. Appearance of ETH in the hemolymph activates ETHR-A in neurons expressing neuropeptides including kinin, FMRFamides (Tv1-3), EH, or CCAP, MIPs, and bursicon, but they are not released until descending inhibition is removed at key times during the ecdysis sequence. Upon activation of ETHR, the central release of kinin initiates pre-ecdysis contractions, whereas Tv neurons secrete FMRFamides to enhance neuromuscular transmission. ETH activates neurons producing EH, CCAP, CCAP/MIPs, and CCAP/MIPs/bursicon at different times. EH cells in the brain and neurons producing CCAP in TN3 and CCAP/MIPs in AN8,9 become active ~10-13 min after pre-ecdysis initiation. EH participates in timing the activation of ecdysis neurons, whereas CCAP and MIPs from TN3 and AN8,9 control initiation and execution of the ecdysis motor program. At the end of ecdysis (25 min after pre-ecdysis onset), neurons in AN1-4 secrete a cocktail of CCAP, MIPs, and bursicon, which likely regulate postecdysis contractions and processes associated with cuticle expansion, hardening, and tanning (Kim, 2006a).
This study has mapped central ETH receptor neurons, and discovered that they comprise multiple peptidergic ensembles, which are recruited sequentially to generate each phase of the ecdysis sequence. Ensemble-specific knockout analysis supports this interpretation. Each step of the ecdysis sequence (pre-ecdysis, ecdysis, postecdysis) is driven by a central pattern generator (CPG) within the CNS in the absence of sensory input. It is known that amines and peptides can modulate and reconfigure neuronal circuits comprising CPGs so as to elicit a variety of motor patterns. It seems likely that the multiple peptidergic ensembles described in this study as targets for ETH may be involved in configuring and activating CPGs underlying each step of the ecdysis sequence (Kim, 2006a).
Processes in the brain that govern behaviors over longer time frames such as sleep, mood, sexual activities, and even learning and memory could be associated with coordinated release of neuromodulators such as peptides. Further work on activation of central peptidergic ensembles in the CNS may shed light on mechanisms underlying release of a variety of behaviors (Kim, 2006a).
To test whether ETH is required for ecdysis, gene deletions were generated by imprecise excisions of EP(2)1065, a P-element located 1427 bp downstream of the ETH polyA site. These efforts led to creation of three deletion lines, eth27, eth25b and eth196, all of which possess small excisions near or including ETH. The eth27 line has a deletion from the P-element site in the 5' untranslated region of reg-5 (rhythmically expressed gene 5) up to the 3' untranslated region of eth, 22 bp downstream of the ETH stop codon. This deletion does not disturb the coding sequence of eth, thus serving as a useful negative control for other deletions. The eth25b deletion removes virtually the entire ETH sequence, extending from the original P-element site up to the eth-coding region, leaving only part of the signal sequence (MRIITVLSV) (Park, 1999). The eth196 deletion occurs from the P-element site through ETH to the middle of the adjacent gene orc4 (Park, 2002a).
Loss of ETH in both eth25b and eth196 lines causes recessive lethality, while eth27 has no obvious phenotype. The fact that genotype eth25b/eth196 also shows the same ecdysis deficiency phenotype suggests that the ETH deletions cause this phenotype, rather than other unknown aberrations. Lethality is associated with ecdysis deficiencies, whereby double mouthhooks and dVP indicate failure to shed the old mouthparts. These animals show a shrunken body appearance, thick trachea and partial ecdysis of old cuticle both exteriorly and within the tracheal system. The phenotype resulting from eth-deletion is referred to as 'buttoned-up', which describes an inability to extricate old mouthhooks and vertical plates from the new sclerotized structures (Park, 2002a).
Further analysis has revealed disrupted respiratory dynamics and behavioral deficits in ETH null mutants. Tracheal collapse and inflation of new trachea are delayed for ~1.5 hours, and pre-ecdysis behaviors are completely absent. In the absence of these events, 'ecdysis-like' behavior occurs early, around the dVP stage with a large variation among individuals (4±23 minutes). Ecdysis-like behavior differs from wild-type ecdysis behavior in several respects. (1) Normal forward thrust movements to plant the old mouthparts in the substrate are absent. Instead, animals engage in swinging head movements, and repeated extensions and retractions of the mouth. (2) Strong backward thrust movements, which normally result in separation of the spiracles and ecdysis of trachei, are also absent. Although some backward movements are observed, animals are unsuccessful in detaching the old spiracles and tracheal linings. Some turning behavior resembling forward escape is observed, but animals are unsuccessful in this maneuver, owing to the fact that neither mouthparts nor spiracles have been detached. These ecdysis-like behaviors are repeated on an irregular basis for 1 to 3 hours. Some time after the occurrence of delayed tracheal collapse and inflation, ecdysis-like behaviors become more like normal ecdysis. Indeed, the majority of eth- mutants are able to move through an anterior dorsal opening in the old cuticle that appears after repeated ecdysis movements. This occurs on the average at 2 hours 17 minutes±40 minutes after the dVP stage. This type of exit from the old cuticle contrasts with that of wild-type flies, which ecdyse by moving through the anterior opening created by removal of the old mouthparts. Even though many mutant larvae are able to escape the old cuticle, their mouthparts remain 'buttoned-up'. The buttoned-up phenotype remains quiescent, does not feed and dies within 1 to 2 days. A small fraction of eth- larvae undergo successful ecdysis and development through the second instar (~2%), but all succumb following ecdysis failure at the 2nd to 3rd instar transition (Park, 2002a).
Properly timed injection of ETHs rescue ecdysis deficiencies in mutant flies and promotes successful ecdysis. Injection of Drosophila ETH1 (~ 1 fmol) into either eth25b or eth196 larvae at the dVP stage restores all missing steps in the ecdysis sequence. Specifically, Drosophila ETH1 injections induce tracheal collapse and inflation of trachea (3 and 4 minutes after the injection, respectively). Thereafter, pre-ecdysis behaviors appear, including weak anterior-posterior movements (7±2 minutes) followed by strong squeezing waves (9±2 minutes). A set of typical ecdysis behaviors, including forward and backward thrusts and forward escape, occurs at 18±2 minutes after injection. Rescued flies that succeeded in passing to the 2nd instar succumbed at the transition to the 3rd instar, owing to unsuccessful ecdysis (Park, 2002a).
Though mutants injected with Drosophila ETH1 showed a normal ecdysis behavioral sequence, some individuals were unsuccessful in completing ecdysis. Approximately 25% of eth25b and 41% of eth196 flies fail to successfully shed the old cuticle. Rescued eth25b mutants show no further mortality during the 2nd instar, but rescued eth196 mutants show significant mortality during the early 2nd instar; accumulated mortality rises from 41% to 73% (lethal phase i in the 2nd instar. The elevated mortality observed for eth196 mutants during the 2nd instar may result from partial deletion of the upstream gene orc4. Maternally deposited orc4 mRNA can promote survival enough the early 1st instar, but is insufficient for development through the early 2nd instar. Further examination of this question requires genetic rescue of the eth196 line (Park, 2002a).
Lethality can be reversed also by injection of DrmETH2 at relatively high doses (> 10 fmol). These treatments partially rescued behavioral deficits in eth25b flies, including induction of tracheal collapse, inflation of trachea and ecdysis. However, DrmETH2 injections failed to induce anteroposterior contractions and squeezing waves. Lower doses of DrmETH2 (~1 fmol) induced tracheal collapse and inflation of new trachea, but were not effective in eliciting either pre-ecdysis or ecdysis behaviors (Park, 2002a).
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date revised: 20 December 2006
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