Retrograde bone morphogenetic protein signaling shapes a key circadian pacemaker circuit

Abstract

The neuropeptide pigment-dispersing factor (PDF) synchronizes molecular oscillations within circadian pacemakers in the Drosophila brain. It is expressed in the small ventral lateral neurons (sLNvs) and large ventral lateral neurons, the former being indispensable for maintaining behavioral rhythmicity under free-running conditions. How PDF circuits develop the specific connectivity traits that endow such global behavioral control remains unknown. Here, we show that mature sLNv circuits require PDF signaling during early development, acting through its cognate receptor PDFR at postsynaptic targets. Yet, axonal defects by PDF knockdown are presynaptic and become apparent only after metamorphosis, highlighting a delayed response to a signal released early on. Presynaptic expression of constitutively active bone morphogenetic protein (BMP) receptors prevents pdfr mutants misrouting phenotype, while sLNv-restricted downregulation of BMP signaling components phenocopied pdf(01). Thus, we have uncovered a novel mechanism that provides an early "tagging" of synaptic targets that will guide circuit refinement later in development.

Figure 1.

A subset of the sLNv projections displays an aberrant morphology in pdf⁰¹. A, B, Representative confocal images of the sLNv dorsal projections in control (pdf>GFP; A) and mutant (pdf>GFP;pdf⁰¹; B) brains. A subset of the sLNvs projections (black arrow) continues growing toward the posterior optic tract (POT). Brains were stained with anti-GFP (shown in black) and anti-PDF (shown in red) antibodies. The average length of the abnormal axons was 106.7 μm with a SD of 33.5 μm. C, Quantitation of the misrouting phenotype. The percentage of brains displaying aberrant fibers was significantly different between pdf>GFP and pdf>GFP;pdf⁰¹ (one-way ANOVA, p < 0.001). Approximately 80% of the mutant brains show a misrouting defect in contrast to 7% in controls. Similar results were obtained employing different markers. Although quantitation was performed on a single brain hemisphere, the defect is largely present in both. Experiments were repeated three times independently. In each experiment, 10 flies were analyzed per genotype, and a mean value was obtained. Error bars indicate SEM. The asterisk indicates a significant difference between genotypes. D, Representative confocal images of sLNvs dorsal projections from late third-instar larvae from pdf>RFP (top panel) and pdf>RFP;pdf⁰¹ (bottom panel). No differences were detected. The defect was only present in a small proportion of the brains (pdf>RFP, 8%; pdf>RFP;pdf⁰¹, 0%; Kruskal–Wallis, p = 0.40). Scale bars: A, B, D, 20 μm.

Figure 2.

PDF is required early on during circuit establishment. A, Outline of the specific treatments. Control (pdf-GS>+) and pdf^RNAi-4380GD (pdf-GS>pdf^RNAi-4380GD) flies were employed. Six stages were selected to evaluate PDF requirement. Activation of pdf^RNAi-4380GD expression during larval stages was accomplished after a 2 min dip in RU486. Delivering RU486 to pupae required a small anterior incision in the pupal case. For acute activation of pdf^RNAi-4380GD in the adult, flies were transferred to standard vials supplemented with RU486. To mimic pdf⁰¹ conditions, pdf-GS>pdf^RNAi-4380GD flies were raised throughout development in food supplemented with RU486. B, C, PDF is required in L1. B, Representative confocal images of the projections from pdf-GS>pdf^RNAi-4380GD adult brains treated with RU486 during adulthood (left) or L1 (right). Brains were stained with anti-GFP (black) and anti-PDF (red) antibodies. PDF downregulation during adulthood did not trigger any misrouting defect. Scale bar, 10 μm. C, Summary of the results obtained. Statistical analysis included a two-way ANOVA. The interaction was significant (p < 0.001). Only treatments involving downregulation in L1 reproduced the sLNv aberrant morphology. Interestingly, downregulation of PDF levels in l-L3 and pupal stages gave rise to abnormal large LNvs. Experiments were repeated three times independently; 10 brains were analyzed per genotype/experiment; the mean value is reported. D, The misrouting phenotype does not affect locomotor behavior. Representative actograms of selected genotypes are shown. PDF downregulation in L1 does not affect period or rhythmicity; adult-specific PDF silencing phenocopied the locomotor defects observed in pdf⁰¹. Note that RU486 administration extends the free-running period during administration. Period analysis included a one-way ANOVA (p = 0.0001) with Tukey's post hoc test (p < 0.05; least-significant difference, 0.44 h). Rhythmicity analysis included a one-way ANOVA (p < 0.0001, Tukey, p < 0.05; least-significant difference, 17.27%). Experiments were independently repeated three times, with ∼32 flies analyzed per genotype/experiment.

Figure 3.

PDFR knockdown in distinct subsets of PDFR+ neurons is sufficient to reproduce the misrouting phenotype. A, B, PDFR mutant han⁵³⁰⁴ phenocopies pdf⁰¹ aberrant morphology. A, Representative confocal images of the dorsal projections in han⁵³⁰⁴;pdf>GFP brains. A subset of the sLNv projections continue their growth toward the POT (marked with an arrow). Brains were stained with anti-GFP (black) and anti-PDF (red) antibodies. Scale bar, 20 μm. B, Quantification of the misrouting phenotype. The percentage of brains displaying morphological aberrations was significantly different between pdf>GFP and han⁵³⁰⁴;pdf>GFP, but no difference was detected between han⁵³⁰⁴;pdf>GFP and pdf>GFP/pdf⁰¹ (one-way ANOVA, p < 0.001; Tukey, p < 0.05; least-significant difference, 27.39%). Error bars indicate SEM. The asterisk indicates a significant difference between genotypes. C, PDFR downregulation in Tim+PDF− neurons or C232+ neurons is sufficient to trigger the defect. Different GAL4 lines were employed to downregulate PDFR in specific neuronal clusters. pdfr>pdfr^RNAi-42724GD and tim>pdfr^RNAi-42724GD reproduce the abnormal phenotype (not significantly different from han⁵³⁰⁴;pdf>GFP). pdf>pdfr^RNAi-42724GD was indistinguishable from pdf>GFP. pdfr silencing in clock neurons excluding the PDF+ cells (tim.pdf80>pdfr^RNAi-42724GD) phenocopied han⁵³⁰⁴ defects. Downregulation of pdfr in the central complex (c232>pdfr^RNAi-42724GD) partially reproduced the han⁵³⁰⁴ results. On the contrary, when pdfr^RNAi was expressed in mushroom bodies (ok107>pdfr^RNAi-42724GD) or in the pars intercerebralis (kurs58>pdfr^RNAi-42724GD, mai301>pdfr^RNAi-42724GD), the sLNvs dorsal arborization was indistinguishable from the control (one-way ANOVA, p < 0.0001; Duncan, p < 0.05). ^φSame letter indicates no significant differences. Three independent experiments were carried out; each experiment included 10 flies per genotype; the mean value is reported.

Figure 4.

Activation of the BMP signaling pathway rescues the han⁵³⁰⁴ misrouting phenotype. Ligands from different signaling pathways were expressed in PDFR+ cells in the context of a loss-of-function PDFR mutant. A, B, GBB but not Wg partially rescues the han⁵³⁰⁴ phenotype. Representative confocal images of projections from whole brains from han⁵³⁰⁴;pdfr>GFP (A) and han⁵³⁰⁴;pdfr>GBB (B). While most han⁵³⁰⁴;pdfr>GFP brains showed defects in both hemispheres, a small proportion of han⁵³⁰⁴;pdfr>GBB brains had defects on both (>70 and <20%, respectively). Adult brains were dissected and incubated with anti-PDF antiserum to reveal the PDF circuitry. Scale bar, 20 μm. C, D, Constitutively active forms of Tkv and Sax receptors restore wild-type morphology. Representative confocal images of projections from han⁵³⁰⁴;pdf>Tkv^A (C) and han⁵³⁰⁴;pdf>Sax^A (D) brains. Expression of either type I receptor (Tkv or Sax) within the PDF circuit significantly reduced the percentage of pdfr mutant brains displaying a misrouting defect. E, Summary of the results obtained. Statistical analysis included one-way ANOVA (“at least one side affected,” p = 0.0006; “both sides affected,” p < 0.0001) and Tukey (p < 0.05 in both cases; “at least one side affected” least significant difference, 33.46%; “both sides affected” least significant difference, 26.72%). ^φSame letter indicates no significant differences. Three independent experiments were carried out. Approximately 10 flies were analyzed per genotype/experiment, and a mean value is reported.

Figure 5.

Med is required from L3 to pupa for proper development of the sLNvs. med was downregulated using a specific RNAi. A similar protocol to that in Figure 2A was employed (Dev + Ad and Ad were not evaluated). A–F, Representative confocal images of projections from whole adult brains from pdf-GS>+ and pdf-GS>med^{RNAi-106767KK} flies soaked in RU at different developmental stages. Only those in which med was downregulated in e-L3, l-L3, or pupa resulted in abnormal projections in the adulthood. Brains were stained with anti-PDF antibody (black). A, Control pdf-GS>+. RU was administrated at l-L3. B–F, pdf-GS>med^{RNAi-106767KK}. RU was administrated at L1 (B), L2 (C), e-L3 (D), l-L3 (E), and pupa (F). G, Summary of the result obtained. Statistical analysis included a two-way ANOVA. The interaction was significant (p = 0.0009). Only treatments involving downregulation in e-L3, l-L3, and pupa produced an aberrant morphology. Defects included misrouting (arrows) and defasciculation (arrowheads). As seen in F, in some cases projections from the large ventral lateral neurons were also affected. Experiments were repeated three times independently; ∼10 brains were analyzed per genotype/experiment; the mean value is reported.

Figure 6.

The ecdysone receptor is implicated in sLNv development. A specific RNAi directed toward the EcR was expressed within PDF+ neurons. A, B, Representative confocal images of the adult sLNv dorsal projections in pdf.Dicer>+ (A) and pdf.Dicer>EcR^RNAi-37059GD (B) brains. When EcR was downregulated, a subset of the sLNvs resulted in an extreme misrouting phenotype. In other examples, the dorsal arborizations of the four sLNvs were affected more severely (data not shown). C, Quantitation of the aberrant phenotype. Significant differences in brains displaying aberrant sLNvs were observed between flies expressing EcR^RNAi-37059GD and controls (one-way ANOVA, p = 0.011). Two independent experiments were carried out. Approximately 10 flies were analyzed per genotype/experiment, and a mean value is reported. Error bars indicate SEM. The asterisk indicates a significant difference between genotypes.