Uncoupling of transcriptomic and cytological differentiation in mouse spermatocytes with impaired meiosis

Abstract

Cell differentiation is driven by changes in gene expression that manifest as changes in cellular phenotype or function. Altered cellular phenotypes, stemming from genetic mutations or other perturbations, are widely assumed to directly correspond to changes in the transcriptome and vice versa. Here, we exploited the cytologically well-defined Prdm9 mutant mouse as a model of developmental arrest to test whether parallel programs of cellular differentiation and gene expression are tightly coordinated, or can be disassociated. By comparing cytological phenotype markers and transcriptomes in wild-type and mutant spermatocytes, we identified multiple instances of cellular and molecular uncoupling in Prdm9^-/- mutants. Most notably, although Prdm9^-/- germ cells undergo cytological arrest in a late-leptotene/zygotene stage, they nevertheless develop gene expression signatures characteristic of later developmental substages. These findings suggest that transcriptomic changes may not reliably map to cellular phenotypes in developmentally perturbed systems.

FIGURE 1:

Cytological phenotypes of Prdm9^–/– spermatocytes reflect meiotic arrest. (A) Cytological analyses and RNA sequencing were performed on germ cells enriched from testes at 8, 12, and 16 dpp. (B) Representative images of spermatocytes in each meiotic substage across Prdm9^+/+ and Prdm9^–/– samples. Germ cells were immunostained for combinatorial arrays of marker proteins that are well established for cytological characterization of meiotic prophase substages in spermatocytes. (C) Quantification of average frequencies of spermatogenic and meiotic prophase substages represented at each time point in the samples of germ cells retrieved from Prdm9^+/+ and Prdm9^–/– testes.

FIGURE 2:

ComBat-adjusted data of gene expression across genotype and age conditions shows differential expression between Prdm9^+/+ and Prdm9^–/– samples. (A) Principal component 1 (PC1) vs. principal component 2 (PC2) from PCA of all ComBat-adjusted samples. Colors denote genotype, and shapes denote sample age, as indicated. (B, C) Shared and unique differentially expressed transcripts with decreased or increased abundance in Prdm9^–/– samples compared with Prdm9^+/+. FDR < 0.01 and LFC > 0.5 for B and C.

FIGURE 3:

Changes in expression of specific meiotic gene reflect abnormalities and meiotic arrest in Prdm9^–/– germ cells. Colors denote genotypes, as indicated. (A) Log₂(TPM+1) expression of Morc2b, Hspa2, and Piwil1 at 8, 12, and 16 dpp. (B) Log₂(TPM+1) expression of Dmc1, Spo11, and Stra8 at 8, 12, and 16 dpp. (C) Relative expression of piRNA precursors at 8, 12, and 16 dpp. *** represents FDR < 0.0001.

FIGURE 4:

Substage specificity of transcripts determined from PMCA is different in Prdm9^–/– germ cells than in WT germ cells. (A) Relative expression of transcripts assigned to LP/D substage based on their WT expression patterns. (B) Relative number of genes shared among substages assigned in WT and Prdm9^–/– samples. The size of the circle represents the relative number of genes shared between two substage assignments. (C) Relative expression of transcripts assigned to LP/D in WT samples but to LL/Z in Prdm9^–/– samples.

FIGURE 5:

Summary model of cellular and molecular progression in Prdm9^+/+ and Prdm9^–/– germ cells. Arrows represent meiotic progression, colored by genotype as indicated. Text labels adjacent to the arrows indicate the transcriptional regulators of the genes expressed in the cell substage following the arrow.

FIGURE 6:

Upstream regulators of LP/D-specific genes show divergent expression changes in Prdm9^–/– germ cells. (A) Log₂(TPM+1) expression of activating transcriptional regulator Rfx4 at 8, 12, and 16 dpp. (B, C) Relative expression of RFX4 targets in Prdm9^+/+ and Prdm9^–/– samples, respectively. (D) Log₂(TPM+1) expression of repressive transcriptional regulator Etv4 at 8, 12, and 16 dpp. (E, F) Relative expression of ETV4 targets in Prdm9^+/+ and Prdm9^–/– samples, respectively. *** represents FDR < 0.0001.