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. 2024 Mar 2;16(3):evae059.
doi: 10.1093/gbe/evae059.

scRNA-seq Reveals Novel Genetic Pathways and Sex Chromosome Regulation in Tribolium Spermatogenesis

Michael Robben  1 , Balan Ramesh  2 , Shana Pau  2 , Demetra Meletis  2 , Jacob Luber  1 , Jeffery Demuth  2
Affiliations

scRNA-seq Reveals Novel Genetic Pathways and Sex Chromosome Regulation in Tribolium Spermatogenesis

Michael Robben et al. Genome Biol Evol. .

Abstract

Spermatogenesis is critical to sexual reproduction yet evolves rapidly in many organisms. High-throughput single-cell transcriptomics promises unparalleled insight into this important process but understanding can be impeded in nonmodel systems by a lack of known genes that can reliably demarcate biologically meaningful cell populations. Tribolium castaneum, the red flour beetle, lacks known markers for spermatogenesis found in insect species like Drosophila melanogaster. Using single-cell sequencing data collected from adult beetle testes, we implement a strategy for elucidating biologically meaningful cell populations by using transient expression stage identification markers, weighted principal component clustering, and SNP-based haploid/diploid phasing. We identify populations that correspond to observable points in sperm differentiation and find species specific markers for each stage. Our results indicate that molecular pathways underlying spermatogenesis in Coleoptera are substantially diverged from those in Diptera. We also show that most genes on the X chromosome experience meiotic sex chromosome inactivation. Temporal expression of Drosophila MSL complex homologs coupled with spatial analysis of potential chromatin entry sites further suggests that the dosage compensation machinery may mediate escape from meiotic sex chromosome inactivation and postmeiotic reactivation of the X chromosome.

Keywords: Tribolium castaneum; computational biology; meiotic sex chromosome inactivation; scRNA-seq; spermatogenesis.

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Conflict of interest statement

Conflict of Interest The authors report no competing interests.

Figures

Fig. 1.
Fig. 1.
Extraction and single-cell sequencing of testes from four adult beetles. a and b) Microscope pictures of adult beetle testes dissected. a) Stereomicroscope image of dissected beetle testes in water. b) DAPI stained slide squash of adult beetle testes. I, Distal end of one teste organ. II, Proximal end of same teste organ; and III, Seminary tubule. c) Magnified views of cell populations for GSC (upper panel) and spermatocytes and spermatids (lower panel). d) Diagram of beetle testes with estimated expression levels of previously validated markers; β2-tubulin (TC009035), Rad50 (TC006703), and Enolase (TC011729). Estimated expression patterns were derived through examination of promoter driven GFP expression in Khan et al., (2021). Numbers represent the number of cells at each stage of mitotic/meiotic division. Spermatids are represented with 29 cells even though there is no division due to each cyst encompassing 2 bundles of antiparallel sperm. The distal end of testes is at the bottom of diagram and proximal end is at the top. e) Expression of previously validated markers for spermatogenic staging across all cells. f) Expression of homologs to tissue markers used to stage spermatogenesis in D. melanogaster and A. gambiae (Table 1). Expression of genes displayed as rows of heatmap with cell expression in each column. g) UMAP projection of weighted principal components showing stage specific clusters. h–j) Expression of previously validated stage specific markers in cells displayed as UMAP projection. k) Switch-like mechanics of β2-tubulin, Rad50, and Enolase for determining pseudotime status of each cell.
Fig. 2.
Fig. 2.
Identification of cluster cell types using phasing evidence. a and b) Expression of a) G2M and b) S phase specific expression markers as rows across cell clusters in columns. c) Estimated mitotic phase of each cell displayed in UMAP projection from homologous Drosophila markers. d) Histogram showing the distribution of cells as a function of their percent heterozygosity. Heterozygosity estimated from SNPs in sequenced reads may not represent actual allele status due to low sequencing coverage and variable gene expression. e) Ploidy status of each cell as inferred from cells < 5% heterozygous across alleles. f) Inferred cell type using multiple evidences. Parallel clusters independently separated during UMAP construction represent 2 distinct groups labeled group 1 and group 2. g and h) Stacked barplots showing the total numbers of g) G1, G2M, and S and h) haploid and diploid phased cells in each cluster.
Fig. 3.
Fig. 3.
Differentiation trajectory and marker expression of beetle testes. a and b) The linear development of sperm cells as inferred by ouija pseudotime analysis of β2-tubulin, Rad50, and Enolase is consistent with labeled clusters. Reported as per cell expression on a) UMAP projection and b) per cluster boxplot. c and d) Trajectories are also reported as determined by c) monocle v3 and d) SCvelo RNA velocity. Cells are colored by their cluster identification. e) Markers that show some differentiation and linear relationships between or within clusters are reported as expressions of each cell in UMAP projection. f) Markers identified from monocle-derived gene expression markers and represented as normalized average expression values across clusters on a heatmap.
Fig. 4.
Fig. 4.
Pathway analysis of cluster expressions. a and b) Average enrichment scores per cluster for GO biological process terms related to a) meiosis and b) chromosome. Reported values are averaged across per cell enrichment for each term. c) Average expression of genes contained within relevant terms. d) Expression of female bias genes by cluster by chromosome.
Fig. 5.
Fig. 5.
X inactivation and MSCI of adult beetle testes. a) Boxplot showing average expression of each gene across all chromosomes, the X chromosome, and autosomes by cluster. b) Percent of cells expressing X chromosome genes above an average of 0.25 normalized expression. c) Expression of Unr (TC002472) per cell displayed on UMAP projection. d) Expression of msl1-3 per cluster. e) Density plot showing motif enrichment of DCC CES sites on the X chromosome plotted over mean expression data of X chromosome genes in “Secondary Spermatocyte 1” cluster cells. Dashed line represents density of highly expressed X-chromosomal genes.
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