The neurons that express the bursicon gene were identified using in situ hybridization. The bursicon antisense probe revealed one to two pairs of neurons in each of the thoracic and abdominal neuromeres of the larval CNS. Since Bursicon is co-expressed in some of the neurons that express the neuropeptide CCAP (see Drosophila CCAP) in a number of other insects (Honegger, 2002; Kostron, 1996), whether this relationship also held for Drosophila was examined. By double labeling with an antiserum directed against CCAP, it was found that bursicon transcripts are indeed expressed in a set of the neurons that contain CCAP. However, as in other insects (Honegger, 2002), not all CCAP neurons express bursicon. Bursicon transcripts were not detected in two pairs of CCAP-immunoreactive (ir) neurons in the brain or in two pairs in the first thoracic neuromere. Antibodies directed against amino acids 91-108 of the Bursicon sequence also labeled the CCAP-ir cells in the ventral nervous system (VNS). Using this antibody, a wild-type pattern of Bursicon-ir neurons was detected in the burs^Z5569 and burs^Z1091 mutants, indicating that these mutants produce Bursicon protein, although it has greatly reduced biological activity (Dewey, 2004).

A subset of Drosophila neurons that expresses crustacean cardioactive peptide (CCAP) makes the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Evidence is presented that CCAP-expressing neurons (N_CCAP) consist of two functionally distinct groups, one of which releases bursicon into the hemolymph and the other of which regulates its release. The first group, which it called N_CCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion that lie within the expression pattern of the enhancer-trap line c929-Gal4. Suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which has been called N_CCAP-R, consists of N_CCAP neurons outside the c929-Gal4 pattern. Because suppression of synaptic transmission and protein kinase A (PKA) activity throughout N_CCAP, but not in N_CCAP-c929, also blocks tanning and wing expansion, it is concluded that neurotransmission and PKA are required in N_CCAP-R to regulate bursicon secretion from N_CCAP-c929. Enhancement of electrical activity in N_CCAP-R by expression of the bacterial sodium channel NaChBac also blocks tanning and wing expansion and leads to depletion of bursicon from central processes. NaChBac expression in N_CCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. These results suggest that N_CCAP form an interacting neuronal network responsible for the regulation and release of bursicon and suggest a model in which PKA-mediated stimulation of inputs to normally quiescent bursicon-expressing neurons activates release of the hormone (Luan, 2006).

Tanning bioassays performed in blowflies and the hawkmoth, Manduca sexta, have determined that bursicon bioactivity is concentrated in the abdominal ganglia from which it is likely to be released into the hemolymph. Recent molecular characterization of bursicon, and the availability of antibodies to its two subunits, has allowed identification of neurons that make bursicon in several insects and confirmed previous findings that some of these coexpress CCAP, a peptide with cardioacceleratory activity. In Drosophila larvae, bursicon expression is restricted to a small number of CCAP-expressing neurons in the ventral nerve cord. Its distribution has now been mapped in late-stage pharate adults, a stage more relevant to its release into the hemolymph. Bursicon expression in the adult is broader but remains restricted to N_CCAP, with most bursicon-expressing neurons located in the abdominal ganglion. The 14 abdominal neurons (B_AG) are included in the expression pattern of the c929-Gal4 enhancer-trap line, whereas a pair of neurons that consistently express bursicon in the subesophageal ganglion (B_SEG) are not. Although bursicon immunoreactivity is occasionally observed in neurons other than these 16, the variability of its expression rendered these neurons unlikely substrates for the highly invariant developmental processes of cuticle tanning and wing expansion that bursicon mediates (Luan, 2006).

Functional evidence that the B_AG are responsible for release of bursicon into the hemolymph is provided by demonstrating that suppression of excitability in the c929-Gal4 pattern blocks bursicon release into the hemolymph. The inhibition of wing expansion by this manipulation suggests that wing expansion, like tanning, also requires bursicon in the hemolymph. This is consistent with a proposed role for bursicon in cuticle plasticization, a process required to render the wing extensible before expansion. The partially expanded wing phenotypes seen at lower levels of suppression may result from incomplete cuticle plasticization attributable to insufficient bursicon in the hemolymph (Luan, 2006).

The expression of c929-Gal4 in B_AG, but not B_SEG, may indicate functional distinctions between these two groups of neurons. The c929-Gal4 expression pattern has been extensively characterized and conforms primarily to that of dimmed, a gene that neighbors the Gal4 insertion site and is involved in upregulating peptide processing, and the coincidence of bursicon and c929-Gal4 expression in B_AG may reflect upregulation of peptidergic processing in N_CCAP neurons preparing to corelease both CCAP and bursicon into the hemolymph. B_SEG, which lie outside the c929-Gal4 expression pattern and are unlikely to contribute significantly to circulating levels of bursicon in the hemolymph after eclosion based on these Western blot data, may instead release the hormone within the CNS, where it may regulate behaviors required for wing expansion (Luan, 2006).

This study introduces a new tool for the targeted enhancement of cellular excitability; it was used to demonstrate that secretion of bursicon from B_AG must be regulated by a population of N_CCAP neurons outside of N_CCAP-c929 (i.e., by N_CCAP-R). Evidence is provided that B_AG are electrically quiescent before eclosion. The tool is a GFP-tagged version of the bacterial sodium channel NaChBac. Transgenic flies were made that express UAS-NaChBac-EGFP under the control of Gal4 drivers; it was shown that NaChBac-EGFP enhances photoreceptor excitability. (Nitabach, 2005), and the utility of the NaChBac channel in enhancing excitability was further demonstrated in other cell types (Luan, 2006).

This study shows that enhancement of excitability in N_CCAP using NaChBac-EGFP eliminates bursicon secretion into the hemolymph and blocks tanning and wing expansion. This observation is consistent with results (Hodge, 2005) that show that dominant-negative inhibition of Shaw K⁺ channel function in N_CCAP results in many animals exhibiting the juvenile phenotype, particularly females. Interestingly, a similar sexual dimorphism was observed in the effects of NaChBac in N_CCAP when using a 'weaker' insert that lacks the EGFP tag. The Shaw channel is expressed endogenously in N_CCAP, and its properties suggest that it acts to limit membrane excitability. Inhibiting Shaw should therefore enhance excitability like NaChBac-EGFP. Conversely, overexpressing Shaw should suppress excitability and, like EKO, cause tanning and wing expansion deficits (Luan, 2006).

The observation that bursicon secretion into the hemolymph is relatively unimpaired by expression of NaChBac-EGFP in N_CCAP-c929 implies that NaChBac-EGFP does not act by directly enhancing the excitability of B_AG. Instead, NaChBac-EGFP must alter B_AG activity when it is expressed in neurons in N_CCAP-R. The failure of NaChBac-EGFP to affect bursicon release when expressed by c929-Gal4 is unlikely to result from lower transgene expression levels in B_AG than are obtained with CCAP-Gal4, because both drivers cause similar levels of wing expansion failure when driving expression of varying copy numbers of the EKO transgene (Luan, 2006).

How enhancement of excitability in N_CCAP impairs bursicon release remains an open question. The observation that NaChBac-EGFP depletes bursicon immunoreactivity in central processes when expressed in N_CCAP-R suggests a constitutive enhancement of bursicon secretion. If the central processes derive from B_SEG, this effect may be a direct consequence of their enhanced excitability. Enhanced excitability in B_AG, by expression of NaChBac-EGFP in N_CCAP-c929, does not deplete central bursicon or alter secretion of the hormone into the hemolymph after eclosion. This observation implies that B_AG are quiescent until stimulated after eclosion. This conclusion supports electrophysiological data from Manduca, which indicates that bursicon-secreting neurons receive little synaptic input until after eclosion when synaptic activity becomes continuous (P. Taghert, personal communication to Luan, 2006). More work will be required to determine how NaChBac-EGFP expression in N_CCAP-R alters bursicon secretion into the hemolymph (Luan, 2006).

The current results strongly support a role for synaptic transmission in the regulation of bursicon secretion. Inhibition of both dynamin and synaptobrevin function, by expression of UAS-Shi^ts1 and UAS-TNT, respectively, had no effect on wing expansion when expressed in N_CCAP-c929 alone, indicating that bursicon secretion is not dependent on these molecules. The observation that both UAS-Shi^ts1 and TNT inhibit wing expansion when expressed throughout N_CCAP therefore demonstrates that synaptic blockade in N_CCAP-R, but not N_CCAP-c929, is necessary for bursicon release. Block of synaptic inputs onto bursicon-secreting neurons may mediate this effect, although the efficacy of blockade in inhibiting wing expansion when applied before, as well as after, eclosion suggests the involvement of multiple synapses (Luan, 2006).

The less penetrant effects of TNT on wing expansion compared with those of UAS-Shi^ts1 cannot currently be explained. Tetanus toxin may not completely eliminate synaptic transmission. Alternatively, compensatory developmental responses to constitutive, rather than transient, block of synaptic transmission may be attenuating the effects of TNT. Changes in cellular physiology in response to TNT expression in neurons have been described previously (Luan, 2006).

PKA activity is required within N_CCAP for bursicon release. Inhibition of PKA within N_CCAP-c929 blocks neither tanning nor wing expansion. PKA is thus required within N_CCAP-R. Although the targets of PKA in N_CCAP remain to be determined, one intriguing possibility is that PKA downregulates the Shaw channel, which has been proposed to be a PKA target in mushroom bodies, to increase excitability and signaling in N_CCAP-R (Luan, 2006).

Additional work will be required to validate the model of B_AG regulation and to determine the functional roles of specific neurons within N_CCAP-R. Related to this is the question of whether bursicon secretion from B_SEG and B_AG is coordinately or independently regulated. Interestingly, the B_SEG neurons belong to N_CCAP-R and may themselves participate in regulating B_AG function. The role of the four non-bursicon-expressing neurons within N_CCAP-c929 also requires additional investigation. The data rule out a role for these neurons in regulating B_AG by mechanisms sensitive to enhancement of excitability or suppression of PKA or neurotransmission, but it remains possible that they regulate B_AG by other means (Luan, 2006).

The functionally oriented approach established here has facilitated the demonstration that the molecularly related N_CCAP neurons comprise all or part of a neuronal network that regulates bursicon secretion. Using this approach with enhancer-trap lines other than c929-Gal4 should allow the functional identities of specific neurons within this network to be defined (Luan, 2006).

A neuropeptide hormone-signalling pathway controls events surrounding eclosion in Drosophila. Ecdysis-triggering hormone, eclosion hormone and crustacean cardioactive peptide (CCAP) together control pre-eclosion and eclosion events, whereas bursicon, through its receptor rickets (RK), controls post-eclosion development. Cuticular tanning is a convenient visible marker of the temporally precise post-eclosion developmental progression, and this study investigated how it is controlled by the ecdysis neuropeptide cascade. Together, two enzymes, tyrosine hydroxylase (TH, encoded by ple) and dopa decarboxylase (DDC, encoded by Ddc), produce the dopamine that is required for tanning. Levels of both the ple and Ddc transcripts begin to accumulate before eclosion, coincident with the onset of pigmentation of the pharate adult bristles and epidermis. Since DDC activity is high before the post-eclosion onset of tanning, a different factor must be regulated to switch on tanning. Transcriptional control of ple does not regulate the onset of tanning because ple transcript levels remain unchanged from 24 hours before to 12 hours after eclosion. TH protein present before eclosion is degraded, and no TH activity can be detected at eclosion. However, TH protein rapidly accumulates within an hour of eclosion, and evidence is provided that CCAP controls this process. Furthermore, TH is shown to be transiently activated during tanning by phosphorylation at Ser32, as a result of bursicon signalling. It is concluded that the ecdysis hormone cascade acts as a regulatory switch to control the precise onset of tanning by both translational and activational control of TH (Davis, 2007).

In Drosophila, the onset of tanning of the puparium occurs within 1 hour after the wandering larva becomes sessile. This requires metabolites of DA, the production of which is dependent on the actions of TH and DDC. Transcripts levels of both genes, and TH protein and activity levels, are all high in white pre-pupae (WPP). Unlike at eclosion, TH does not appear to be activated by PKA phosphorylation for the rapid tanning of the pupal case at pupariation. This is not unexpected because the ecdysis neuropeptides are not released until a full 12 hours APF. During the late third instar, the relatively insoluble tyrosine, which is indispensable for tanning, is stored as a more soluble derivative, tyrosine-O-phosphate (tyr-P). Tanning at pupariation is probably controlled by the release of tyrosine from tyr-P. No appreciable accumulation of tyr-P occurs before eclosion, suggesting that post-eclosion tanning is switched on by a different mechanism (Davis, 2007).

This study establishes a role for the ecdysis neuropeptide cascade in post-eclosion tanning by examining the regulation of two genes, ple and Ddc, which encode two enzymes with critical roles in tanning. Semi-quantitative RT-PCR was used to examine the profile of transcription after puparium formation. Levels of both transcripts are high in WPP, but they drop and then rise again before eclosion. Ddc levels begin to increase 60 hours APF, reach their peak 84 hours APF, and decline thereafter. DDC enzyme activity is required before eclosion for pigmentation of the pharate adult bristles and epidermis and after eclosion for tanning of the adult cuticle, and reaches a peak at eclosion. This indicates that Ddc is transcribed and translated before eclosion to ensure enzyme activity is present when substrate becomes available. This study investigated whether the control of substrate availability, and therefore the control of tanning, is effected by the transcriptional, translational, or post-translational regulation of TH (Davis, 2007).

Levels of ple transcripts are high during the 24 hour period spanning eclosion. The early appearance of ple transcripts is not surprising, because pigmentation of the pharate adult bristles and epidermis occurs between 84 and 96 hours APF. Both ple (and Ddc) transcription are normal in EH-KO, CCAP-KO, burs^Z1091 and rk⁴ flies. The accumulation of ple transcripts before eclosion, the maintenance of high levels of TH transcription until 12 hours after eclosion and the fact that neuropeptide mutant and ablation knockout flies exhibit normal ple transcription, led to the conclusion that the precise onset of tanning following eclosion is not due to regulation of ple transcription (Davis, 2007).

TH protein and activity levels are high before eclosion when pigmentation of the pharate adult bristles and epidermis occurs. Levels fall rapidly just before eclosion and rise thereafter. During this entire time, ple transcripts are present, suggesting that protein levels are being regulated. The drop in TH protein levels may occur through repression of translation from ple transcripts and/or increased turnover of the protein. The complete failure of CCAP-KO flies to accumulate TH protein following eclosion, although they transcribe ple normally, indicates a role for CCAP in this process. This could occur at the level of translation; alternatively, CCAP signalling may alter TH protein stabilisation. Since PKA signalling has been shown to regulate proteins involved in translational control, it is more likely that CCAP signalling activates PKA to cause translation, not stabilisation, of TH following eclosion (Davis, 2007).

EH-KO, burs^Z1091 and rk⁴ flies all appear to have relatively normal TH protein and activity profiles. Although all three exhibit a considerable range of activity in WPP, the pupal cases of these organisms tan normally. Despite the initial delay in TH accumulation in EH-KO flies following eclosion, these flies, and burs^Z1091 and rk⁴ mutants, maintain high levels of TH until 144 hours APF, a time when TH is undetectable in control flies. This persistence of TH indicates a delay in the execution of the neuropeptide hormone cascade. Interestingly, rk⁴ flies also show a delay in degradation of TH following pupariation. Perhaps there is a requirement for RK signalling to trigger TH degradation following tanning of the puparium (Davis, 2007).

Neck-ligation of flies at eclosion prevents tanning, whereas flies ligated 30 minutes after eclosion tan normally. Furthermore, tanning of flies neck-ligated at eclosion is rescued by injection of 8-Br-cAMP. TH protein begins to accumulate 1 hour after eclosion in control flies. Phosphorylation of the protein by PKA at Ser32 leads to enzyme activity rising between 1.5 and 3 hours after eclosion. It is concluded that the translational and activational state of TH is responsible for controlling tanning following eclosion. TH protein accumulates, but is not phosphorylated in flies neck-ligated at eclosion resulting in reduced TH activity. Interrupting neuropeptide signalling after eclosion reveals that the element that controls TH translation is released before eclosion. The loss of TH accumulation in CCAP-KO flies and the restoration of TH accumulation upon injection of CCAP suggests that CCAP is responsible for inducing TH translation (Davis, 2007).

Flies neck-ligated 30 minutes after eclosion, translate and phosphorylate TH normally. By allowing neuropeptide signalling after eclosion, it has been demonstrated that a factor is released within 30 minutes of eclosion that causes phosphorylation and therefore activation of TH. The reduced phosphorylation of Ser32 in burs^Z1091 flies, and complete loss of phosphorylation in rk⁴, burs^Z1091/burs^Z5569 and rk¹/rk⁴ flies suggests that bursicon signalling through RK controls this process. Activity levels of TH are significantly reduced in flies neck-ligated at eclosion compared with control flies. Flies ligated 30 minutes after eclosion show twofold higher levels than flies neck-ligated at eclosion and this difference probably accounts for the presence or absence of tanning. This suggests that a critical threshold of TH activity exists that is surpassed in the flies ligated at 30 minutes. Thus, although the activity present in these flies is significantly less than that in control flies, the organisms have sufficient TH activity to tan, whereas flies ligated at eclosion do not attain the threshold of activity required for tanning. Injection of 8-Br-cAMP into flies neck-ligated at eclosion rescues tanning by restoring phosphorylation and therefore activation of TH. Although injection of 8-Br-cAMP does not restore TH activity to control levels, it increases activity nearly sixfold, achieving the threshold of activity required for tanning following eclosion (Davis, 2007).

These results, taken together, suggest that at least two factors control the precise timing of tanning after eclosion. One, released before eclosion, causes translation of TH; the other, released after eclosion, causes phosphorylation and activation of TH. Both EH and CCAP are released before eclosion to control pre-ecdysis and ecdysis, respectively. EH-KO and CCAP-KO flies both exhibit extreme post-eclosion tanning defects. EH-KO flies take more than 9 hours to tan and CCAP-KO flies fail to tan. TH protein is undetectable in EH-KO flies immediately following eclosion, but these flies do eventually accumulate TH and tan. The complete failure of CCAP-KO flies to tan, combined with the fact that CCAP-KO flies fail to accumulate TH from the ple transcripts that are present at eclosion, suggest that CCAP is responsible for inducing TH translation. The initial failure of EH-KO flies to accumulate TH is probably caused by a failure to trigger the rapid release of CCAP. Presumably, enough CCAP is eventually released in these flies to effect the translation of TH and eventually tanning, because the EH genetic ablation is leaky. Consistent with this prediction, EH-KO flies that expand their wings accumulate TH normally, suggesting that CCAP is released normally in these flies. TH translation is restored in CCAP-KO flies injected with CCAP and rescue of TH accumulation and phosphorylation occurs when EH-KO and CCAP-KO flies are injected with 8-Br-cAMP. Rescue of both defects probably occurs because injection of 8-Br-cAMP activates PKA in CCAP target cells, thus circumventing the need for CCAP release, and also activates PKA in TH-expressing cells, leading to phosphorylation and activation of TH (Davis, 2007).

These data suggest that the post-eclosion factor causing the phosphorylation of Ser32 is the heterodimeric hormone bursicon. It is responsible for tanning and wing expansion and acts through its receptor RK. Consistent with the role of bursicon in the phosphorylation of TH, rk⁴ flies fail to phosphorylate TH and have reduced activity. These flies show a delay in tanning, taking up to 9 hours to tan. Injection of 8-Br-cAMP rescues tanning by restoring phosphorylation and therefore activation of TH (Davis, 2007).

Two mutants in the α subunit of bursicon have been identified, of which one (burs^Z5569) shows a delay in tanning in 40% of the progeny, whereas a delay is present in 82% of burs^Z1091/burs^Z5569 flies. The burs^Z1091 mutant does not show a delay in tanning, although phosphorylation of TH is reduced in these flies. Phosphorylation of Ser32 is undetectable in burs^Z1091/burs^Z5569 flies, probably causing the more severe tanning defect seen in these flies. The reduced phosphorylation of TH in burs^Z1091 flies corresponds to a minor loss of TH activity. Thus, it seems that the threshold TH activity required for proper tanning is achieved in burs^Z1091 flies, although they do not have wild-type levels of TH phosphorylation or activity. Normal tanning in these flies cannot be attributed to residual activity of the β subunit of bursicon, CG15284, proposed to be encoded by pu (S. McNabb and J. Truman, personal communication to Davis, 2007), because neither subunit independently confers bursicon activity. The burs^Z1091 allele is probably a hypomorph, and creation of a null allele would be useful. Additional studies on the activational state of TH in pu or burs null mutants will help to elucidate why tanning is not delayed in burs^Z1091 flies (Davis, 2007).

The data indicate that CCAP is responsible for initiating TH translation following eclosion. In Drosophila, translational regulation often occurs through microRNA (miRNA)-dependent RNAi-mediated repression through binding sites in the 3'UTR of transcripts. Three miRNAs - let-7, mir-iab-4-3p and mir-iab-4-5p - have been predicted to regulate TH translation in Drosophila. It is conceivable that one or more of these miRNAs, in association with the RISC complex, could bind to ple transcripts to cause the repression of translation through a miRNA-dependent RNAi-mediated mechanism. It is also plausible that PKA, activated by CCAP signalling, might relieve repression of TH translation by phosphorylation of one of the subunits of the RISC complex or associated proteins. Future work will establish whether there is a role for these miRNAs in the repression of TH translation before eclosion (Davis, 2007).

Neurons acquire their molecular, neurochemical, and connectional features during development as a result of complex regulatory mechanisms. This study shows that a ubiquitous, multifunctional protein cofactor, Chip, plays a critical role in a set of neurons in Drosophila that control the well described posteclosion behavior. Newly eclosed flies normally expand their wings and display tanning and hardening of their cuticle. Using multiple approaches to interfere with Chip function, it was found that these processes do not occur without normal activity of this protein. Furthermore, the nature of the deficit was identified to be an absence of Bursicon in the hemolymph of newly eclosed flies, whereas the responsivity to Bursicon in these flies remains normal. Chip interacts with transcription factors of the LIM-HD (LIM-homeodomain) family, and one member, dIslet, was identified as a potential partner of Chip in this process. These findings provide the first evidence of transcriptional mechanisms involved in the development of the neuronal circuit that regulates posteclosion behavior in Drosophila (Hari, 2008).

This study used a selective overexpression strategy to identify a novel function of Chip in a set of neurons that control a stereotyped behavioral program. Chip is a widely expressed multidomain cofactor molecule that interacts with many transcription factors. It can function both as a transcriptional coactivator and a bridging factor between proteins that bind to distal enhancers and the core transcription machinery. Identifying specific functions of such a protein is confounded by the superposition of a multitude of effects. The mutations in Chip cause early lethality precluding the examination of later functions. Because the molecule exists as a part of multiple complexes, even simultaneously within the same cell, altering the level of one class of interactors can potentially disrupt several functions. This study has elucidated a highly specific role of Chip in a particular class of neurons in Drosophila and implicated a known LIM-HD partner of Chip, Islet, in this function (Hari, 2008).

It is proposed that the defect is attributable to a failure in the release rather than in the production or the responsiveness to the neurohormone Bursicon. How might Bursicon release be controlled as a result of Chip function in development? The hemolymph transfer experiments provide a unique insight into this puzzle. The literature describes a model wherein posteclosion wing expansion requires a combination of a neural signal from the brain as well as Bursicon release. The results extend the understanding of how this interplay of activity and secreted factors is set up in development. It appears that, several days before eclosion, Chip is able to regulate an as-yet-unidentified event in the CCAP neurons, such that the hemolymph contains adequate levels of Bursicon after eclosion. The CCAP-expressing neurons are divided into at least two interacting subpopulations, only one of which secretes Bursicon. The other subpopulation does not secrete Bursicon but is implicated in regulating its release. Chip may therefore mediate the formation of proper connectivity among CCAP neurons, which eventually ensures timely Bursicon release several days later. Supporting this scenario, Chip has been reported to regulate axon pathfinding and proper innervation of targets in other systems. The data are suggestive of Chip requirement in the early period of puparium formation, which fits well with a report that CCAP neurons undergo extensive remodeling in this period of metamorphosis. The importance of this connectivity is underscored by the identification of several other genes in a gain-of-function screen, which displayed a simultaneous disruption of both posteclosion wing expansion and the pattern of CCAP neuron innervation. Therefore, thes findings motivate an examination of Chip function in regulating the connectivity of CCAP neurons, a role that directly links this key aspect of neuronal development with the control of posteclosion behavior in Drosophila (Hari, 2008).

In the course of a genetic screen for flies that failed to expand their wings, five mutants were identified that formed a single complementation group that mapped to the vicinity of CG13419. Genomic sequencing of these mutants show that each has an independent single base pair change at CG13419. All have wing spreading and sclerotization defects when either homozygous or heterozygous over deficiencies for the region. Four mutants have sequence changes that are predicted to cause significant alterations of the mature protein structure. The burs^Z4410 mutation is a G to A conversion at the splice acceptor site for the second exon, which likely prevents normal splicing. This mutation would lead to read-through of the first intron, resulting in premature termination due to the presence of two consecutive stop codons. Since the mutation is located 194 bp from the translation initiation site, the resulting protein product would be comprised of the signal sequence and only 12 additional amino acids (one mutated) and is likely nonfunctional. Three other alleles have mutations within the coding region and either remove or introduce a cysteine residue and thus are likely to disrupt disulfide bridge formation, a key structural feature of cystine knot proteins. The burs^Z1091 allele results in a conversion of cysteine residue 82 to tyrosine, removing a cysteine that is conserved among all members of the mucin subfamily of cystine knot proteins. The burs^Z1140 mutation results in a conversion of threonine residue 97 to cysteine. The burs^Z5569 mutation results in a conversion of glycine residue 115 to cysteine. The fifth mutation, burs^Z2803, appears to be a mutation within the regulatory region. It results in a C to T change at -203 base pairs from the translation start site (Dewey, 2004).

The phenotypes of two bursicon mutants, burs^Z1091 and burs^Z5569, were examined in detail. Flies homozygous for either mutation, heteroallelic burs^Z5569/burs^Z1091 or heterozygous over deficiencies for the region, fail to spread their wings following eclosion and show a prolonged retention of the elongate abdomen shape characteristic of a newly eclosed fly. By the day after eclosion, their abdomens shorten but fail to taper into the normal adult shape, consistent with a failure to sclerotize properly. The two alleles differ in their effects on the timing of cuticle pigmentation and the expansion of the thoracic cuticle. The burs^Z5569 mutants are delayed in melanization, with only 40% completing it during the 3 hr following eclosion, but they inflate their thoracic cuticle normally. In contrast, burs^Z1091 mutants pigment normally but fail to complete thorax expansion, resulting in the postscutellar bristles remaining crossed. Flies that are heteroallelic for the two mutations (burs^Z5569/burs^Z1091) show characteristics of both mutants. They resemble burs^Z5569 mutants in being slow to pigment, with only 18% completing pigmentation within 3 hr of eclosion. However, they have crossed postscutellar bristles like burs^Z1091 homozygotes. Both bursicon mutations are recessive, displaying no wing expansion or sclerotization defects when in heterozygous combinations with complementing deficiencies, nonallelic mutants, or balancer chromosomes (Dewey, 2004).

The levels of bursicon produced by mutant larvae were determined using bursicon bioassays based on neck-ligated Sarcophaga bullata (Fraenkel, 1965; Kostron, 1995) or Drosophila (Baker, 2002). In the Sarcophaga bioassay, extracts of CNSs from control bw; st larvae stimulated tanning with an average score of 2.50 ± 0.18. By contrast, CNS extracts from burs^Z1091 mutant larvae yielded an average score of 0.27 ± 0.08, similar to that obtained with phosphate buffer controls. (In the Sarcophaga bioassay, bursicon activity below 10% of maximum activity cannot be detected. Thus, the level of bursicon in burs^Z1091 flies is less than 10% that of wild-type.) burs^Z5569 flies gave a low but measurable average score of 0.85 ± 0.63. Extracts from burs^Z5569/burs^Z1091 and burs^Z5569/Df (3R) e-GC3 yielded low scores (0.81 and 0.75, respectively), consistent with the hypothesis that the mutations in CG13419 are solely responsible for decreased bursicon bioactivity. Extracts from burs^Z5569/TM6B Tb (essentially burs^Z5569/+) larvae gave scores of 2.15 and 2.20; these are similar to those of the bw; st control (Dewey, 2004).

The blood of newly emerged flies was assayed for bursicon activity using a Drosophila test system (Baker, 2002). Injection of hemolymph from control bw; st flies into neck-ligated control bw; st or bursicon mutant flies resulted in tanning. In contrast, the hemolymph from homozygous burs^Z5569 or burs^Z1091 mutants of the same age failed to cause neck-ligated control flies to tan. These results are consistent with decreased bursicon activity in the mutants. It is not currently understood why burs^Z1091 fails to cause pigmentation in these bioassays despite the fact that when homozygous or in heterozygous combinations with deficiencies, burs^Z1091 mutants pigment normally. The amino acid changes in the two mutants may result in differences of their interaction with the receptor and/or in aspects of transport, metabolism, and stability of the mutant peptides (Dewey, 2004).

Reference names in red indicate recommended papers.

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Arakane, Y., Li, B., Muthukrishnan, S., Beeman, R. W., Kramer, K. J. and Park, Y. (2008). Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum. Mech. Dev. 125(11-12): 984-95. PubMed Citation: 18835439

Baker, J. D. and Truman, J. W. (2002). Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. J. Exp. Biol. 205: 2555-2565. 12151362

Cottrell, C. B. (1962). The imaginal ecdysis of blowflies. Detection of the blood-borne darkening factor and determination of some of its properties. J. Exp. Biol. 39: 413-430

Davis, M. M., et al. (2007). A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster. Development 134: 4395-4404. PubMed Citation: 18003740

Dewey, E. M., McNabb, S. L., Ewer, J., Kuo, G. R., Takanishi, C. L. Truman, J. W. and Honegger, H.-W. (2004). Identification of the gene encoding Bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading. Curr. Biol. 14: 1208-1213. 1524261

Edmondson, M. E. (1948). Drosophila Inf. Serv. 22: 53

Fraenkel, G. and Hsiao, C. (1962). Hormonal and nervous control of tanning in the fly. Science 138: 27-29

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