Spatial Transcriptomic Profiling of Male Germ Cells from Control and Cbfb cKO mice

Much of the framework for spermatogenesis is established postnatally before sexual maturation. To evaluate the role of core-binding factor subunit β (CBFβ) in prepubertal male germline development, we generated male mice with conditional knockout of Cbfb using Blimp1-Cre (denoted Blimp1-Cbfb cKO). Outcomes from phenotype analysis revealed that Blimp1-Cbfb cKO males were completely infertile and experienced rapid germline degeneration prior to puberty. To evaluate the pathways downstream of Cbfb that coincide with this degeneration, we conducted spatial transcriptomics of heterozygous control and Blimp1-Cbfb cKO testes at postnatal days 6 (P6) and 21 (P21) and used fluorescence immunostaining to identify and capture cell-type specific transcriptomic signatures. Collectively, our analysis revealed central roles of CBFβ in maintenance, differentiation, and cell cycle checkpoint regulation within the prepubertal male germline. Spatial transcriptomic signatures were obtained from both heterozygous control and Blimp1-Cbfb cKO male mice at P6 and P21 using NanoString whole-transcriptome analysis (WTA). Briefly, tissue cross-sections were generated from paraformaldehyde fixed, paraffin embedded testes. For P6, triplicate tissue cross-sections were generated from three males of each genotype to yield triplicate technical and n=3 biological replication per genotype for a total of 18 sections evaluated. For P21, triplicate cross-sections were generated from duplicate males of each genotype to yield triplicate technical and n=2 biological replication per genotype for a total of 12 sections evaluated. Tissue sections were placed on glass slides and processed for immunofluorescence staining using antibodies recognizing the undifferentiated spermatogonia marker LIN28A and the pan-germ cell marker DDX4 alongside the nuclear stain SYTO13. One circular region of interest (ROI) was randomly placed on each tissue section and segmented using florescent signal to capture cell populations corresponding to undifferentiated spermatogonia (LIN28A+, DDX4+, SYTO13+) and other germ cells (LIN28A-, DDX4+, SYTO13+). Resulting transcriptomic signatures were captured using photo-cleavable probes and analyzed following the manufacturers protocol.