Transposition-driven genomic heterogeneity in the Drosophila brain

Abstract

Recent studies in mammals have documented the neural expression and mobility of retrotransposons and have suggested that neural genomes are diverse mosaics. We found that transposition occurs among memory-relevant neurons in the Drosophila brain. Cell type-specific gene expression profiling revealed that transposon expression is more abundant in mushroom body (MB) αβ neurons than in neighboring MB neurons. The Piwi-interacting RNA (piRNA) proteins Aubergine and Argonaute 3, known to suppress transposons in the fly germline, are expressed in the brain and appear less abundant in αβ MB neurons. Loss of piRNA proteins correlates with elevated transposon expression in the brain. Paired-end deep sequencing identified more than 200 de novo transposon insertions in αβ neurons, including insertions into memory-relevant loci. Our observations indicate that genomic heterogeneity is a conserved feature of the brain.

Fig. 1. Exclusive labeling of MB neuron subsets in the fly brain.

(A) MB-expressing GAL4s with expression elsewhere in the brain were intersected with an almost pan MB-expressing LexA. See Supporting Online Material for detail of genetic approach. Confocal projection of individual brains from flies labeling (B) α′β′ (C) γ and (D) αβ neurons. (E) all neurons in the brain, except MB neurons, no MB. Scale bar 40 μm.

Fig. 2. Gene expression profiling MB neurons.

(A) Microarray data emphasizing elevated expression in αβ neurons. Each signal column per category represents an independent replicate. Fold change (FC) and t-test P values are shown for αβ versus other MB neurons (average value for α′β′ and γ) and αβ versus the no MB group. Transposons denoted in red. Scale bars linear for fold change, −log10 for P-value and log₂ of the signal level. (B) QRT-PCR validation of increased transposon mRNA levels in αβ neurons. * denotes αβ signal significantly different to all other populations, all P≤0.05, T-Test. ** denotes αβ signal significantly different to MB but not no MB group for R2, and αβ significantly different to all except γ for invader3.

Fig. 3. Aub and Ago3 are not abundant in αβ neurons.

(A) Aub immunostaining (magenta) labels the ellipsoid body (EB) and MB subdivision in the peduncle (ped). Single confocal section at the level of the MB peduncle. Dotted box denotes area in panels (B-D). (B) Aub labels α′β′ and γ neurons in the peduncle more than αβ. Aub staining is mutually exclusive to GFP (green) expressing αβ neurons but overlaps with (C) α′β′ and (D) γ neurons. (B-D) left panels Aub staining; middle GFP; right panel merge. (E) Ago3 immunostaining (magenta) labels neurons throughout the brain and MB subdivision in the peduncle. Single confocal section at the level of the MB peduncle. Dotted box denotes area in panels (F-H). (F) Ago3 staining is prominent in the αβ core (closed triangle) and does not overlap with outer GFP labeled αβ neurons (open triangle) nor (G) α′β′ but overlaps with (H) γ neurons. Left panels Ago3 staining; middle GFP; right panel merge. Scale bar 10μm. (I) Several transposon transcripts are elevated in ago3, aub and armi mutant fly brains. QRT-PCR analysis from wild-type, aub^HN2/aub^QC42, ago3^t2/ago3^t3 and armi¹/armi^27.1 mutant heads. Values normalized to wild-type heads. * denotes significant increase, P<0.05 (T-test).

Fig. 4. Identification of de novo transposition events in αβ neurons.

(A) Venn diagram of transposon insertions identified in this study. Direct genome sequencing identified 215 insertion sites in αβ neuron DNA and 200 in DNA from other brain cells that were not present in embryo DNA from genetically identical sibling flies. (B) Chromosomal distribution of new transposon insertions in αβ neurons. Bar height indicates number of insertions in each location. piRNA clusters are shown. (C) The proportion of new insertions found for each transposon class in αβ neurons. (D) Proportion of insertions per transposon class in the inherited fly genome from our sequencing of embryo DNA. (E) Distribution of transposons in the annotated genome significantly differs from de novo insertions in αβ neurons, the rest of brain and ovary DNA with respect to neighboring genes (p-value <2.2E-16 for αβ neurons, the rest of brain and ovary; χ² test).