Analysis of Drosophila melanogaster testis transcriptome

Abstract

Background: The formation of matured and individual sperm involves a series of molecular and spectacular morphological changes of the developing cysts in Drosophila melanogaster testis. Recent advances in RNA Sequencing (RNA-Seq) technology help us to understand the complexity of eukaryotic transcriptomes by dissecting different tissues and developmental stages of organisms. To gain a better understanding of cellular differentiation of spermatogenesis, we applied RNA-Seq to analyse the testis-specific transcriptome, including coding and non-coding genes.

Results: We isolated three different parts of the wild-type testis by dissecting and cutting the different regions: 1.) the apical region, which contains stem cells and developing spermatocytes 2.) the middle region, with enrichment of meiotic cysts 3.) the basal region, which contains elongated post-meiotic cysts with spermatids. Total RNA was isolated from each region and analysed by next-generation sequencing. We collected data from the annotated 17412 Drosophila genes and identified 5381 genes with significant transcript accumulation differences between the regions, representing the main stages of spermatogenesis. We demonstrated for the first time the presence and region specific distribution of 2061 lncRNAs in testis, with 203 significant differences. Using the available modENCODE RNA-Seq data, we determined the tissue specificity indices of Drosophila genes. Combining the indices with our results, we identified genes with region-specific enrichment in testis.

Conclusion: By multiple analyses of our results and integrating existing knowledge about Drosophila melanogaster spermatogenesis to our dataset, we were able to describe transcript composition of different regions of Drosophila testis, including several stage-specific transcripts. We present searchable visualizations that can facilitate the identification of new components that play role in the organisation and composition of different stages of spermatogenesis, including the less known, but complex regulation of post-meiotic stages.

Keywords: Drosophila; RNA sequencing; Spermatogenesis; Testis; Transcriptome.

Fig. 1

Distribution of transcripts in different region of testis and analysis of the RNA-Seq results. a Distribution of Drosophila melanogaster transcripts by RNA-Seq. b Number of genes showing significant differences (FDR-corrected p-value< 0.05) in transcript level between different region of testis. c The combined number of transcripts and the significant differences (FDR-corrected p-value< 0.05) between different region of testis. d Comparison of the previous microarray data [7] with present RNA-seq data. e Distribution of genes with lower or higher transcript levels in the basal testicular regions compared to the apical region. The comparison was based on the tissue where their expression maximum is defined by modENCODE database [56]. f Distribution of transcript level differences in basal region compared to the apical region as a function of gene expression levels. g Distribution of differences in transcript levels along the apical-basal axis of the testis as a function of tissue specificity index. h Distribution of genes with statistically significant transcript levels along the apical-basal axis of the testis as a function of tissue specificity index

Fig. 2

In situ hybridization and reporter genes. a Positive control (CycB) and negative control (sense probe) of the in situ hybridization. b-g Transcript distribution of genes from different gene groups by DIG-RNA in situ hybridization. Insets show the RNA-seq data of the genes visualized by Cytoscape. h Expression pattern of the testis-specific malate dehydrogenases, CG10748-GFP and CG10749-mCherry in testis. Arrow points to the nebenkern. Scale bars represent 200 μm

Fig. 3

Transcript distribution of the ubiquitin activating E1 and ubiquitin conjugating E2 genes. Transcript differences between apical and post-meiotic regions were visualized by Cytoscape software platform. Bottom part of the figure contains the description of the symbols

Fig. 4

Distribution of transcripts of the deubiquitinating enzymes visualized by Cytoscape

Fig. 5

Distribution of transcripts of the 26S proteasome visualized by Cytoscape. The 20S core proteasome is composed of core Alpha and core Beta subunits and the 19S regulatory proteasome are built from Rpn and Rpt subunits

Fig. 6

Distribution of transcripts of main cytoskeletal genes visualized by Cytoscape

Fig. 7

Distribution of transcripts of genes of the Hsp family visualized by Cytoscape

Fig. 8

Distribution of transcripts of genes of the citrate cycle visualized by Cytoscape