81 ANKRD49 is required for preimplantation development of mouse embryos

While spermatogenic regeneration is essential for male fertility, the mechanisms that govern this process are largely unknown. We previously utilised RNAi to conduct a high-throughput screening of transcription factors, which identified the gene ankyrin repeat domain 49 (ANKRD49) as a novel candidate gene with potential regulatory roles in spermatogenesis. Real-time PCR analysis revealed testis-specific expression in adult mice. To examine the functional importance of ANKRD49, we used CRISPR/Cas9 methodology to engineer mouse embryos with inactivated alleles. The top ranking sgRNAs with target predictor scores of >90 were selected and generated by a commercial source (Synthego Inc.), then introduced into donor embryos for transfer. Live animals were born that possessed monoallelic edits of 1.7 kb and 1 kb deletions in ANKRD49, but no biallelically edited offspring were born. In addition, the building of lines from founders of either deletion allele yielded heterozygous ANKRD49+/− offspring at expected Mendelian ratios, but homozygous ANKRD49−/− offspring were never produced. Both heterozygous and wild-type embryos could be detected at E7.5, E9.5, and E12.5 from timed mating of ANKRD49+/− mice, but knockout embryos were absent, suggesting that loss of ANKRD49 function leads to preimplantation death. In corroboration, an expected Mendelian ratio of 22.5% of blastocyst stage embryos at E3.5 were found to be ANRKD49−/− in heterozygous intercrosses by genotyping analysis. Interestingly, an abnormally high percentage (12.5–37.5%) of implantation sites at E12.5 of ANKRD49+/− intercrosses were found to be empty of the embryo proper but have persistent placental tissue. Outgrowth assays revealed that ANKRD49−/− blastocysts hatch and form typical inner cell mass (ICM) and trophoblast derived colonies in vitro. Additionally, apoptotic blastomeres were detected via TUNEL analysis in late blastocyst- but not morula-stage embryos from ANKRD49+/− intercrosses, whereas no apoptotic cells were detected in embryos from wild-type control mice at either stage. Collectively, these findings demonstrate that, although ANKRD49 expression is restricted to testes in adult mice, expression also occurs during early embryogenesis and is critical for development of the ICM and gastrulation; evident as genetic inactivation of ANKRD49 resulted in embryonic death between E4.5 and E6.5. Embryonic lethality at this timepoint implicates a role for ANKRD49 in the regulation of genes involved in lineage specification, embryonic polarity, the patterning of tissue progenitors, and the morphogenetic movement of cells and tissues. Though further studies are needed to elucidate the role of ANKRD49 in spermatogenesis as a gene required for ICM development and gastrulation, ANKRD49 joins the list of nearly 700 characterised mammalian genes that are essential for the beginnings of life.

This research was supported by NICHD R01HD101223.