Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophila vesicular monoamine transporter gene

Abstract

Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling.

Fig. 1

CRISPR-Cas9 based mutagenesis and phenotypic screen for mutants. A. The neuronal isoform of dVMAT (dVMAT-A) contains at least two trafficking motifs (yellow rectangles) within the C-terminal cytoplastic domain, which allow sorting to both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs). B. Ablation of this domain disrupts dVMAT-A trafficking to SVs and increases trafficking to LDCVs. C. dVMAT gene expresses two splice variants. D. The splice acceptor site for dVMAT-A is proximal to a dileucine motif. E. The ssDNA repair construct mutated four base pairs (magenta). F. The BgllI site allows cleavage of a PCR product in dVMAT^Δ3 that is uncut in WT.G. A western probed for DVMAT. H. Quantitation of the DVMAT bands normalized to a CSP loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Phenotypic analysis. Fertility (A), fecundity (B) and larval locomotion (C) were assayed for four genotypically verified dVMAT^Δ3 mutants, a WT control line genotypically wild type at the dVMAT locus and wild type Canton S flies (CS). One-way ANOVA with Bonferroni's multiple comparisons test, p ≤ 0.0001****, mean indicated as horizontal lines. No significant differences in locomotion (C) were detected between mutant and wildtype larva (One way ANOVA). D. Adult survival on standard food was quantified for one of dVMAT^Δ3 alleles (Δ3A) and a WT control with Kaplan Meier analysis by Mantel-Cox Test, p ≤ 0.0001****, N = 200 per group, median survival WT = 32.5 days, Δ3A = 22.0 days. E) Quantification via spectrophotometry of ingested blue dye (n = 27–28 groups of 10 flies), two-tailed unpaired t-test (p ≤ 0.0332*). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

dVMAT^Δ3mutants retain mature eggs in their ovaries. A. Quantification of mature follicles in female ovaries post egg-laying in controls expressing a WT dVMAT allele (grey) and the Δ3A dVMAT^Δ3 mutant (blue) derived from the same genetic background. Graphs show individual datapoints and group means, (WT n = 19 flies and Δ3A n = 20 flies, two-tailed unpaired t-test, p ≤ 0.0001****). B. Location of the egg within the reproductive tract (RT) after 6 h of mating. Graphs show mean ± 95% confidence interval, (n = 54 flies for WT control and 60 flies for Δ3A), Fisher' exact test (p ≤ 0.001***). C. Optogenetic stimulation of Tdc2-LexA » LexAop-ChR2-XXL in flies harboring dVMAT^Δ3 (Δ3A) mutant (blue) or a WT dVMAT allele derived from w¹¹¹⁸ (black, Control/“Cont.”) induces similar numbers of lateral oviduct contractions. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)