The RNA-binding protein caper is required for sensory neuron development in Drosophila melanogaster

Abstract

Background: Alternative splicing mediated by RNA-binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue-specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail.

Results: We find that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons and neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, we find that Caper dysfunction results in aberrant locomotor behavior in adult flies. Transcriptome-wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis.

Conclusions: Our results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper-mediated RNA regulation. Developmental Dynamics 246:610-624, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: Drosophila; RNA-binding proteins; alternative splicing; dendrite morphogenesis; locomotor behavior; mechanosensory neurons; neurogenesis; post-transcriptional gene behavior.

Figure 1. caper regulates dendrite morphogenesis of Class IV da neurons

(A) Caper:GFP truncated protein (70 kDa) is detected in the larvae caper^CC01391. Coomassie brilliant blue-stained membrane (top panel) and immunoblot (bottom panel) of cell lysates from larvae and adult D. melanogaster caper^CC01391 and oreR control lines. While Western blot analysis of the caper^CC01391 GFP protein trap allele shows a 70 kDA truncated Caper:GFP fusion product in larval lysates, no GFP fusion protein is detected in adult lysates. Three immunoblots were performed, one representative image is shown. (B) Control Class IV ddaC neurons expressing td-Tomato driven by ppkGal4 elaborate hundreds of dendritic branches to cover their sensory field. (C) caper^CC01391 (designated by caper ^−/−) da neurons show defects in field coverage and dendrite morphogenesis. (D) Quantification of dendritic termini reveals that caper dysfunction leads to the formation of supernumerary dendritic termini, compared to controls (p=0.03). (E) However, individual branches are significantly shorter in caper^CC01391 da neurons as compared to controls (p=0.0068). (F) Thus caper^CC01391 da neurons form significantly more branch points per dendrite length but fail to cover their field, as compared to controls (p=0.0068). Scale bar = 50 μm.

Figure 2. Caper is widely expressed throughout embryogenesis

Caper expression was visualized by confocal microscopy by performing anti-GFP immunofluorescence in the caper^CC01391 GFP protein trap genetic background. Caper is expressed early in embryogenesis prior to gastrulation (A) and is seen throughout all cells of stage 4 blastoderm embryos including within the pole cells. (B) In stage 9 embryos, Caper is seen throughout the mesoderm and ventral ectoderm as neurogenesis begins. In later embryonic stages, (C, stage 13; D, stage 15) Caper is enriched within the ventral nerve cord (vnc) and central brain lobes (cb). Scale bar = 100 μm. (E,G) Higher magnification images reveal that Caper is localized to the nucleus during embryogenesis as determined by co-immunofluorescence with (F,G) DAPI. Scale bar = 10 μm.

Figure 3. caper regulates development and positioning of the lch5 organ

Caper:GFP (B,E) is expressed in da neurons (A) and lch5 organs (D) of the embryonic PNS. Overlays of the PNS (marked by DHSB m22C10 and m21A6 antibody stains) and Caper:GFP are shown in (C,F). (G,I) Control lch5 organs form a stereotyped array in the abdominal segments. (H,I) caper^CC01391 embryos exhibit dorsal mispositioning and disorganization of lch5 clusters, as marked by an asterisk. (I) A schematic of the lch5 dorsal mispositioning phenotype as compared to control lch5 organs. Caper:GFP is expressed in the embryonic ventral midline (K), but is excluded from the CNS axons (marked by DHSB BP102 antibody stain; J). Overlay is shown in (L). Scale bar = 25 μm.

Figure 4. caper is required for bristle development and locomotor behavior in adults

(A) yw and (C) RNAi control flies exhibit a stereotyped pattern of macrochaete bristle organs. On day one post-eclosion (B) caper^CC01391 and (D) caper RNAi knockdown adults are often missing bristle organs (arrows). (E) This missing bristle phenotype is significant among both caper^CC01391 males and females, but males are more affected than females (p= 0.013). (F) caper RNAi knockdown specifically affects males bristle development but not female bristle development. (G) caper^CC01391 adults are slower to climb up a cylinder after being startled, as compared to control flies; males are significantly more affected than females (ANOVA: sex, p= 0.002; genotype, p< 0.0001; sex × genotype interaction, p= 0.007). P-values reported on the figure are from post-hoc pairwise comparisons using Sidak’s adjustment for multiple testing. (H) caper RNAi knockdown also results in slower climbing speeds after startling as compared to controls but there was no difference in the magnitude of the effect in males versus females (ANOVA: sex, p= 0.617; genotype, p< 0.0001; sex x genotype, p= 0.366). Means and 95% confidence intervals (error bars) are back-transformed.

Figure 5. Caper dysfunction results in segmentation defects

(B) caper^CC01391 adults exhibit abdominal segmentation and (C) thoracic segmentation defects in an incompletely penetrant manner (D) as compared to control adults (A). (D) Segmentation defects are significantly affected in both sexes in a caper^CC01391 background, as compared to controls.

Figure 6. Transcriptome-wide bioinformatics analyses implicate caper in the regulation of neurogenesis, endocytosis, RNA processing and cytoskeletal organization

caper^CC01391 mutant embryos show significant differences in the expression of genes involved in many developmental and cellular processes. A subset of the significant GO terms are specifically relevant to neurogenesis and include (A) endocytosis, (B) nervous system development, (C) RNA processing and (D) cytoskeletal organization. Genes were clustered into functional groups based on GO terms for biological process and molecular function. Each node represents the most significantly enriched GO term for that cluster (FDR cut-off = 0.01). Green nodes represent clusters in which the majority of genes are significantly upregulated in caper^CC01391 mutant embryos, while red nodes represent clusters in which the majority of genes are significantly downregulated in caper^CC01391 mutant embryos. Color intensity is positively correlated with the proportion of genes in the cluster that are up- or downregulated. Grey nodes represent cases where upregulated and downregulated genes occurred in equal proportions. Node size correlates with statistical significance, where larger nodes are more significant than smaller nodes. (A,B) The GO term for each node is numbered and listed in the panel legend.

Figure 7. JunctionSeq analysis in caper^CC01391 mutant animals compared to controls reveals differences in alternative isoform regulation of genes that are known regulators in da neuron development dendrite

caper^CC01391 mutant embryos use alternative first exons and alternative stop sites for (A) cut mRNA. (B) fry mRNA also shows alternative first exon usage in caper^CC01391 mutant embryos. (C) shep mRNA shows alternative usage of internal exons in caper^CC01391 mutant embryos (D) trio mRNA shows alternative start and alternative internal exon usage in caper^CC01391 mutant embryos. Exons and splice junctions (brackets) colored in pink are those in which the relative level of expression between caper^CC01391 and control embryos differs from the levels observed for the gene overall (depicted in the right panel). Exon expression levels are plotted above the gene model with control reads in blue and caper^CC01391 reads in red. Alternative start and stop sites are depicted by a curved line underneath the alternative first exon or alternative stop site.