Functional dissection of a neuronal network required for cuticle tanning and wing expansion in Drosophila

Abstract

A subset of Drosophila neurons that expresses crustacean cardioactive peptide (CCAP) has been shown previously to make the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Here we present evidence that CCAP-expressing neurons (NCCAP) 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 we call NCCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion that lie within the expression pattern of the enhancer-trap line c929-Gal4. We show that suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which we call NCCAP-R, consists of NCCAP neurons outside the c929-Gal4 pattern. Because suppression of synaptic transmission and protein kinase A (PKA) activity throughout NCCAP, but not in NCCAP-c929, also blocks tanning and wing expansion, we conclude that neurotransmission and PKA are required in NCCAP-R to regulate bursicon secretion from NCCAP-c929. Enhancement of electrical activity in NCCAP-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 NCCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. Our results suggest that NCCAP 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.

Figure 1.

Expression of bursiconα- and β-subunits is restricted to specific N_CCAP neurons in the pharate adult nervous system. A-D, Nervous systems excised from pharate adults expressing EGFP in N_CCAP (A) were double labeled with antibodies to both the bursicon α-subunit (B) and β-subunit (C). In the merged image (D), the green, red, and blue channels represent EGFP, α-subunit, and β-subunit labeling in CCAP-Gal4>UAS-EGFP animals, respectively. Strong overlap of all labels appears as white. The images are maximal projections of volume rendered z-stacks of confocal sections taken through the entire nervous system. Anatomical designations are as follows: SEG, subesophageal ganglia (D, M, and V refer to dorsal, middle, and ventral dispositions of the neurons within the SEG); T1-T3, thoracic ganglia 1-3, respectively; AG, abdominal ganglion. E, Consensus patterns of labeling of each N_CCAP neuron were established by analyzing 11 CCAP-Gal4>UAS-EGFP preparations double labeled with both anti-bursicon subunit antibodies. The intensity (I) of labeling was scored as described in Materials and Methods following a scale of 0 (no labeling) to 3 (intense labeling), and the frequency (ν) of labeling of each neuron was determined, with values of 0-3 indicating that a given neuron was labeled in 0, <33, 33-67, and >67% of the preparations. The N_CCAP neurons in the abdominal ganglion are shown in lateral and medial columns to indicate the generally observed presence of two pairs of CCAP-expressing neurons in each segment, but we have intentionally omitted labels because of ambiguities in assigning anatomical positions to some of these neurons. Segmental identities of the labeled neurons could typically be established unambiguously for A1-A4, but the identities of presumptive A5-A8 neurons were often not resolved. Also, although neurons were generally paired (with only 1 neuron in each pair immunopositive for the bursicon subunits), the relative positions of the neurons in a pair with respect to the midline varied considerably. For simplicity, we have idealized this pattern by labeling the neurons of the lateral column, without intending to denote anatomical position. Some preparations analyzed had two copies of the CCAP-Gal4 driver. These preparations differed only in labeling two additional midline neurons (asterisk in D). Because all crosses to UAS-effector transgenes used a single copy of CCAP-Gal4, these neurons were omitted from the consensus pattern.

Figure 2.

Suppression or enhancement of neuronal function in CCAP-expressing neurons blocks tanning and wing expansion. A, Bar graph showing the frequency of wing expansion deficits in the progeny of representative crosses between parental UAS-effector lines and the CCAP-Gal4 driver line. Progeny were heterozygous for the chromosomes bearing effector and driver transgenes, but, in the case of the EKO suppressor, up to three copies of the transgene were introduced by using chromosomes with multiple inserts and/or multiple chromosomes as described in Materials and Methods. The UAS-effector lines were designed to suppress excitability (EKO and Kir2.1), synaptic transmission (TNT and Shi^ts1), PKA activity (PKA^inh), and cell viability (reaper), or to enhance excitability (NaChBac). Animals in all cases except the crosses to UAS-Shi^ts1 were examined at least 24 h after eclosion, and wings were scored according to the criteria described in Materials and Methods as unexpanded (UEW, black), partially expanded (PEW, gray), or expanded (EW, white). An asterisk indicates that the cross was developmentally lethal, yielding ≤1% viable adult progeny. Flies expressing UAS-Shi^ts1 in N_CCAP were raised at 18°C, isolated within 5 min of eclosion, and transferred to the restrictive temperature (34°C) for 1 h before being placed back at 18°C for at least 48 h before scoring. B, Examples of the wing phenotypes seen in crosses using EKO; designations are as in A. Arrows indicate the wings; arrowheads show the unfolded costal elbow, typical of the PEW phenotype. C, Photograph taken 3 h after eclosion of age-matched, CCAP-Gal4 (left; crossed to Canton-S), or CCAP-Gal4>2× UAS-EKO flies (right) shows the characteristic inhibition of cuticle tanning in animals with N_CCAP suppression. D, Fluorescence micrograph of the excised nervous system of a pharate adult expressing 3× EKO under CCAP-Gal4. The intrinsic GFP fluorescence of the EKO channel shows that the CCAP-expressing neurons are still present. E, Electroretinograms from control animals (top) in response to a 4 s light stimulus compared with that of animals expressing NaChBac-EGFP in photoreceptors using the GMR-Gal4 driver (bottom). Photoreceptor depolarization in NaChBac-EGFP-expressing animals results in a strong, inactivating negative potential not seen in control animals, as expected for NaChBac-mediated currents. F, Experimental (CCAP-Gal4>UAS-Shi^ts1) and control flies lacking the driver were raised at 18°C and subjected to 1-h-long temperature jumps to 34°C at variable times before eclosion. Flies were returned to 18°C after the temperature jump and allowed to eclose and develop for at least 48 h before scoring the wing phenotype. The graph shows the frequency of unexpanded wing flies for each time point, taken as the time of onset of the temperature jump. Because development is typically observed at 25°C, the actual times (in hours at 18°C) were divided in half to obtain “Developmental time” at 25°C.

Figure 3.

The expression pattern of the c929-Gal4 enhancer-trap line includes most bursicon-expressing neurons of N_CCAP. A-F, The excised CNS of a pharate adult expressing UAS-EGFP in the c929-Gal4 expression pattern was double labeled with antibodies to CCAP (A, D, magenta) and bursiconβ-subunit (B, E, magenta). The c929-Gal4>UAS-EGFP pattern (C) is overlaid for each antibody (D, E, green) to show double-labeled cells, which appear as white, as do triple-labeled cells in the merged image (F). All bursicon-positive N_CCAP cells in the abdominal (AG) and thoracic (T3) ganglia were within the c929-Gal4 expression pattern. Two cells outside the c929-Gal4 pattern in the SEG also expressed bursicon (asterisks, E-G). This pair presumably corresponds to the normally CCAP-positive neurons of the ventral SEG (compare with Fig. 1E), but intriguingly these cells do not express CCAP in c929-Gal4 animals. Arrowheads (D, F) indicate a pair of CCAP-positive neurons that are also consistently in the c929-Gal4 pattern but that do not express bursicon. Images are volume-rendered confocal z-stacks as in Figure 1. G-I, Single confocal sections through the abdominal (G-I) or subesophageal (J-L) ganglion of a c929-Gal4>UAS-EGFP animal double labeled with antibodies to the bursicon α-subunit. The overlap of c929-Gal4 driven EGFP (G, J) and bursicon (H, K) is evident in the merged images (I, L) in which EGFP appears as green, bursicon as magenta, and double labeling as white. The B_AG and B_SEG (asterisks) are as indicated.

Figure 4.

Consensus expression patterns of c929-Gal4 within N_CCAP and overlap with bursicon. Consensus labeling patterns, derived from analysis of nine triple-labeled preparations, showing the average frequencies and intensities of expression (compare with Fig. 1) of c929-Gal4 (visualized with UAS-EGFP) within N_CCAP (left) and bursicon β-subunit (right). Note that the neurons designated B_SEG (asterisks) are not CCAP immunopositive in c929-Gal4 animals and are not included in the c929-Gal4 expression pattern. D, M, and V refer to dorsal, middle, and ventral dispositions of the neurons within the SEG.

Figure 5.

Suppression of excitability in the N_CCAP component of the c929-Gal4 pattern blocks wing expansion. The c929-Gal4 driver was used to express the suppressors and enhancers of activity with (bottom) or without (top) coexpression of Gal80 in N_CCAP as described in Materials and Methods. The frequencies of wing phenotypes in the progeny of the crosses are represented as in Figure 2A. Progeny from the c929-Gal4>UAS-Shi^ts1 cross were raised at 18°C and transferred to 34°C immediately after eclosion for 1 h before being returned to 18°C. Only suppression of excitability by EKO caused wing expansion deficits, an effect that was clearly exerted by suppression of neurons within N_CCAP.

Figure 6.

Suppression of excitability with both CCAP-Gal4 and c929-Gal4 blocks bursicon secretion into the hemolymph. Western blots of hemolymph extracted from flies in which CCAP-Gal4 (left) or c929-Gal4 (right) was used to drive 3× EKO, PKA^inh, NaChBac-EGFP, or nothing (Canton-S) probed with antibodies to the bursiconα-subunit. The positions of selected molecular weight markers are shown. The bursicon α-subunit runs at ∼16 kDa (arrow). The ∼75 kDa band of unknown identity (see Results) serves as a useful control for the amount of sample loaded.

Figure 7.

NaChBac-EGFP expression with CCAP-Gal4, but not c929-Gal4, depletes bursicon immunoreactivity in neuronal processes. A-H, Representative patterns of bursicon α-subunit immunoreactivity in processes of the first and second thoracic ganglia (T1 and T2) of CNS from control pharate adults (A, E) or pharate adults expressing NaChBac-EGFP (B, F), 3× EKO (C, G), or PKA^inh (D, H) in either N_CCAP, using the CCAP-Gal4 driver (B-D), or in the c929-Gal4 pattern (F-H). The two arrowheads in each panel indicate identified fibers present in all preparations for comparison. These are the paired fibers of the medial tract (bottom arrow) and an anterior process characteristic of T1 (top right arrow). α-Subunit immunoreactivity in the central neuronal processes of up to seven preparations from each cross was scored blind, as described in Materials and Methods. Average scores from crosses to CCAP-Gal4 (I) and c929-Gal4 (J) were analyzed by the Kruskal-Wallis test for deviations from a uniform distribution. CS, Canton-S. Only the CCAP-Gal4 crosses had scores that were significantly different from random (p < 0.001). For these crosses, bursicon α-subunit process labeling in animals expressing NaChBac-EGFP was 2.5- to 4-fold lower than that observed in other preparations. SEM are indicated.

Figure 8.

A model for the regulation of bursicon release by N_CCAP neurons. A, Manipulations of neuronal activity made with c929-Gal4 and CCAP-Gal4 distinguish two subsets of N_CCAP, one consisting of neurons always (black circles) or sometimes (gray circles) within the c929-Gal4 expression pattern (N_CCAP-c929), the other consisting of the rest of N_CCAP (N_CCAP-R, blue circles). N_CCAP-c929 includes most of the bursicon-expressing neurons, which are shown in red. Dark red indicates neurons that consistently expressed bursicon and consist primarily of neurons in the abdominal ganglion (B_AG), all of which lie within N_CCAP-c929 and which secrete bursicon into the hemolymph. The two bursicon-expressing subesophageal neurons within N_CCAP-R are designated B_SEG. Red stripes indicate neurons that expressed bursicon at low frequency and, in the case of the brain and subesophageal neurons, not at all in c929-Gal4 animals. B, A model for the regulation of bursicon secretion from the B_AG by N_CCAP-R. In the absence of a positive signal from N_CCAP-R, the B_AG are electrically silent and do not secrete bursicon (top). However, stimulation of PKA in N_CCAP-R (presumably in the period around eclosion) causes release of a positive (possibly synaptic) signal (S) from these neurons, which results in the activation of the B_AG and the secretion of bursicon (bottom). D, M, and V refer to dorsal, middle, and ventral dispositions of the neurons within the SEG.