Loss of RK function prevents cuticle sclerotization and delays the melanization of the adult fly but eventually results in a darker than normal exoskeleton
Flies trans-heterozygous for loss-of-function rk mutations (rk1/rk4) do not expand their wings, present an abnormal body shape and fail to melanize properly [9]. A comparable result was obtained when a rk RNAi or a membrane tethered bursicon hormone transgene (here called tBur), which acts as a dominant negative rk allele [16, 17], was expressed in all rk-expressing cells using a rk-GAL4 driver (Fig. 1a,b; results shown for female flies; similar results were obtained with adult male flies, Additional file 1: Figure S1). In all cases, the resulting adult flies did not expand their wings and their bodies did not rapidly pigment after adult emergence. Indeed, at 3 h posteclosion, median cuticle pigmentation was around 150 for control flies whereas it was around 100 for flies expressing tBur under the control of the rk-GAL4 driver (− 33%)(see “Methods” section for a description of the method used here to quantify cuticle pigmentation). Interestingly, at 48 h, the flies with impaired RK function showed a significantly darker pigmentation than their respective controls. Indeed, by 2 days posteclosion, the median score for these flies was around 250, whereas it was around 200 for controls (+ 25%). Importantly, these experiments revealed that driving the tBur transgene using the rk-GAL4 driver (abbreviated here rk>tBur) phenocopied the pigmentation defects expressed by rk1/rk4, and by rk1/rk1 and rk4/rk4 (not shown), mutant flies. For this reason, we chose to use the tBur transgene to interfere with RK function for most experiments reported here.
In addition to defects in the timecourse of melanization, we noticed that, at 48 h posteclosion, both the cuticle shape and its appearance were abnormal in rk1/rk4 flies and when tBur was expressed under the control of rk-GAL4 driver. Indeed, the abdominal cuticle of these flies showed abnormal folds (Additional file 1: Figure S2A) and also appeared matte compared to that of control flies, which always looked shiny by this time. Since these phenotypes could be caused by defects in sclerotization, we developed a semi-quantitative assay to measure the extent of cuticle hardening, based on the levels of soluble proteins that could be extracted from either the abdominal or the wing cuticle, and visualized in silver-stained protein gels (Fig. 2 and Additional file 1: Figure S2B). This assay showed that the levels of soluble proteins that could be extracted from the cuticle of control flies was maximal at 0 h post eclosion, decreased slightly at 3 h and was almost undetectable at 48 h, indicating that by 2 days posteclosion the protein crosslinking reaction that underlies the sclerotization process had rendered insoluble most cuticular proteins. By contrast, the levels of soluble proteins that could be extracted from rk1/rk4 and rk>tBur flies showed a twofold increase at 3 h compared to those of their respective control and many proteins remained detectable even 48 h after emergence.
Bursicon does not act directly on the epidermis to induce cuticle tanning
To determine if bursicon acts directly on the epidermis to cause melanization, we created mosaic flies bearing marked patches of homozygous rk mutant (rk4/rk4) epidermal cells in an otherwise normal (rk4/rk+) animal. Surprisingly, the cuticle overlying the patches of homozygous rk mutant epidermis did not show any pigmentation defects at 3 h or 48 h post emergence (Fig. 3a,b); this result was observed regardless of patch size, consistent with the cell-autonomous property expected of the rk GPCR. To confirm this result using a separate approach, we expressed tBur in the epidermis using two different epidermal GAL4 driver lines: Tyrosine Hydroxylase-GAL4 (TH-GAL4) and Dopa-Decarobylase-GAL4 (DDC-GAL4), both used in combination with elav-GAL80 to prevent knockdown of RK expression in the nervous system. Consistent with the results obtained using mosaic animals, we found that knockdown of RK function in the epidermis produced flies with normal levels of pigmentation at 3 h and 48 h post emergence (Fig. 3c; results shown for female flies; similar results were obtained in males, Additional file 1: Figure S1). Similar results were obtained using rk RNAi transgenes driven by TH-GAL4 (Additional file 1: Figure S3). Taken together, these results show that RK function is not required in the epidermis to regulate postecdysial melanization.
We noticed that the cuticle of flies in which RK function had been knocked down in the epidermis showed abnormal folds and a matte appearance, suggesting that in the epidermis RK may be involved in the process of sclerotization. To address this hypothesis, we estimated the quantity of soluble protein present in abdominal or wing cuticle of flies in which RK function was knocked down in the epidermis. Surprisingly, neither wings nor abdomens showed any increase in the levels of soluble proteins in these flies compared to those of their respective control (Fig. 3d). In parallel experiments, we used the engrailed-GAL4 (en-GAL4) driver to express tBur in the posterior half of the adult wing. Although the posterior part of the wings appeared misfolded and more matte than its anterior counterpart (Fig. 3e, left panel), we found no differences in the amounts of soluble protein that could be extracted from the anterior vs. the posterior half of the wing at either 3 h or 48 h post emergence (Fig. 3e, right panel). Taken together, these results show that RK function is not required in the epidermis to regulate postecdysial melanization or sclerotization. Nevertheless, the expression of rk in the epidermis and the morphological defects observed when RK function is disabled in the epidermis (e.g., Fig. 3e, left panel; Additional file 1: Figure S2A) suggest that rk may play an additional, if currently unknown, role in this tissue.
rickets function is required in the CNS to regulate postecdysial cuticle maturation
The rk gene is widely expressed in the CNS (see Additional file 1: Figures S9 and S10, below), and rk-expressing neurons have recently been shown to play a critical role during pupal ecdysis [18]. To investigate a potential role for rk in the CNS for cuticle darkening, we first examined the pigmentation of flies in which rk was knocked down in the CNS by driving tBur using the GAL4 drivers, elav-GAL4 and nsyb-GAL4 (which are known to drive gene expression in the CNS and not in the epidermis). As shown in Fig. 4a, these flies showed pigmentation defects at both 3 h and 48 h postemergence, which were similar to those expressed by rk1/rk4 mutant flies. Comparable results were obtained using these GAL4 lines to drive rk RNAi transgenes (Fig. 4a). In addition, the wings of these flies failed to expand (Fig. 5b), which is expected because wing expansion requires the nervous system to cause the abdomen to contract and pump hemolymph into the wings [9]. Importantly, the pigmentation (Fig. 4b) and wing expansion defects (not shown) expressed by rk>tBur flies were rescued when combined with elav-GAL80, confirming that cuticle melanization and wing expansion require RK function in the CNS. Conversely, driving a rk cDNA using the neuron-specific driver, elav-GAL4, in rk1/rk4 mutant flies rescued pigmentation at 3 h (Fig. 4c, top), indicating that restoring rk function in the CNS is sufficient to cause normal cuticle melanization at this time. This contrasts to the results obtained using the (primarily) epidermal driver, TH-GAL4, for which no such rescue was obtained. Intriguingly, no rescue was obtained at 48 h post emergence using the elav-GAL4 driver (Fig. 4c, bottom). Nevertheless, only partial rescue was obtained at this time using the rk-GAL4 driver (which does rescue wing expansion ([19], and data not shown), suggesting that the UAS-rk construct may not provide wildtype levels of RK function regardless of the GAL4 driver used.
Interestingly, elav>tBur flies exhibited postemergence sclerotization defects in both wings and abdomens, thereby also implicating RK function in neurons in this process (Fig. 4d). Surprisingly, and in contrast to what occurred for melanization, including the elav-GAL80 transgene in rk>tBur flies was not sufficient to rescue the sclerotization defect except in 48 h wings. Thus, these results suggest that RK function in the CNS is necessary but not sufficient to control cuticle sclerotization.
rickets function is required in the ventral nervous system to regulate postecdysial pigmentation
We then used different GAL4 drivers to pinpoint the rk neurons that could be the direct targets of bursicon involved in the control of cuticle melanization. To this end, we first knocked down RK function only in brain neurons by driving tBur expression using the pan-neuronal elav-GAL4 driver in combination with tsh-GAL80, which drives GAL80 (thereby inhibiting GAL4) expression in the trunk [20]. As shown in Fig. 5a, these flies did not exhibit pigmentation defects at either 3 h or 48 h post emergence, and wing expansion was also normal in these flies (Fig. 5b). Conversely, when we then drove tBur only in the VNS using a tsh-GAL4 driver most the flies died at the start of metamorphosis yet the rare escapers exhibited a rk mutant phenotype (not shown). Taken together, these results show that RK function is primarily required in the VNS for the rapid postemergence melanization of the cuticle. Interestingly, we were able to rescue the pupal lethality observed in rk>tBur animals by including the elav-GAL80 transgene, consistent with the known role for rk signaling during pupal ecdysis [18, 21].
rickets function is required for cuticle melanization in peptidergic neurons that are not the neurons that produce bursicon
Our results suggest that rk is required in the VNS to cause cuticle melanization and wing expansion. A previous study showed that bursicon release is delayed in rk4/rk4 mutant flies [11], suggesting that cuticle melanization might require RK function in the bursicon-secreting neurons themselves. To test this hypothesis, we expressed the tBur transgene in bursicon neurons using two different GAL4 drivers: CCAP-GAL4, which drives expression in all CCAP neurons (including all bursicon neurons in the adult) and burs-GAL4, which drives expression in the BURS-expressing neurons [10, 12]. As shown in Fig. 6a, these flies were entirely normal, indicating that postemergence melanization does not require an autocrine RK function in the bursicon neurons.
Since ecdysis involves a number of neuropeptides acting on downstream peptidergic neurons [22, 23], we then considered the possibility that cuticle melanization involved bursicon action on other peptidergic neurons. To address this possibility, we used the tBur transgene to knock down RK function in large ensembles of peptidergic neurons using the drivers, dimm-GAL4 (dimmed-GAL4) and amon-GAL4 (amontillado-GAL4) which reflect the expression of the transcription factor DIMMED, which is required for peptidergic neuron maturation [24], and of the AMONTILLADO proprotein processing enzyme, PC2 [25], respectively. (Both of these drivers are expressed in peptidergic neurons and we confirmed that they are not expressed in the adult epidermis; Additional file 1: Figure S4). As shown in Fig. 6b, expression of tBur using dimm-GAL4 caused the melanization defects characteristic of rk mutant animals, with lighter and darker pigmentation than normal in 3-h- and 48-h-old flies, respectively. Interestingly, these flies were normal in terms of wing expansion. Expression of tBur using amon-GAL4 caused pigmentation defects similar to those of rk mutants at 3 h and 48 h post emergence but, interestingly, also caused wing expansion failures (not shown), and resulted in flies with matte cuticle similar to that observed in elav>tBur and rk>tBur flies. Importantly, the defects observed when RK function was knocked down using these drivers were rescued by including the elav-GAL80 (Additional file 1: Figure S9A) and tsh-GAL80 (Fig. 6b) transgenes, confirming the localization of the rk requirement to the CNS. However, they were not rescued by including the CCAP-GAL80 transgene (Fig. 6a), consistent with the results obtained using the CCAP-GAL4 driver to knock down RK function, and the lack of co-expression of CCAP and rk-GAL4 in the VNS (Additional file 1: Figure S9B). Taken together, these results suggest that bursicon acts in paracrine rather than an autocrine manner to control cuticle pigmentation.
Downregulation of the insulin receptor has been shown to severely decrease cuticle pigmentation in Drosophila [26] suggesting that some insulin-like peptides (ilp) could participate in the regulation of this process. The only ilp known to be expressed in the VNS is ilp7 [27, 28], and we observed that at least two ilp7-immunoreactive neurons coexpress rk (Additional file 1: Figure S10). Thus, in a final attempt to identify peptidergic neurons in the VNS that could play a direct role in pigmentation, we expressed tBur using an ilp7-GAL4 driver and observed significant pigmentation defects at both 3 h and 48 h of age (Fig. 6c). Nevertheless, we did not observe any pigmentation defects in a null mutant allele for ilp7 or in flies simultaneously mutant for ilp genes ilp2, ilp3, ilp5, and ilp7 (Fig. 6c). These results suggest that some of the 20 ILP7-secreting neurons from the VNS may be direct targets of bursicon and participate in the regulation of cuticle pigmentation but that this role is not mediated by the ILP7 hormone itself, similarly to what has been reported for fertility regulation [29].