Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival.

Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

A Single-Cell Transcriptomics CRISPR-Activation Screen Identifies Epigenetic Regulators of the Zygotic Genome Activation Program.

Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex manipulation techniques and maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early embryos. Here, we combined pooled CRISPR activation (CRISPRa) with single-cell transcriptomics to identify regulators of ZGA-like transcription in mouse embryonic stem cells, which serve as a tractable, in vitro proxy of early mouse embryos. Using multi-omics factor analysis (MOFA+) applied to ∼200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. Follow-up assays validated top screen hits, including the DNA-binding protein Dppa2, the chromatin remodeler Smarca5, and the transcription factor Patz1, and functional experiments revealed that Smarca5's regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides both system-level and molecular insights into the mechanisms that orchestrate ZGA.

The Jeff Mouse Mutant Model for Chronic Otitis Media Manifests Gain-of-Function as Well as Loss-of-Function Effects.

Chronic otitis media (OM) is the most common cause of hearing loss worldwide, yet the underlying genetics and molecular pathology are poorly understood. The mouse mutant Jeff is a single gene mouse model for OM identified from a deafness screen as part of an ENU mutagenesis program at MRC Harwell. Jeff carries a missense mutation in the Fbxo11 gene. Jeff heterozygotes (Fbxo11 Jf/+ ) develop chronic OM at weaning and have reduced hearing. Homozygotes (Fbxo11 Jf/Jf ) display perinatal lethality due to developmental epithelial abnormalities. In order to investigate the role of FBXO11 and the type of mutation responsible for the phenotype of the Jeff mice, a knock-out mouse model was created and compared to Jeff. Surprisingly, the heterozygote knock-outs (Fbxo11 tm2b/+ ) show a much milder phenotype: they do not display any auditory deficit and only some of them have thickened middle ear epithelial lining with no fluid in the ear. In addition, the knock-out homozygote embryos (Fbxo11 tm2b/tm2b ), as well as the compound heterozygotes (Fbxo11 tm2b/Jf ) show only mild abnormalities compared to Jeff homozygotes (Fbxo11 Jf/Jf ). Interestingly, 3 days after intranasal inoculation of the Fbxo11 tm2b/+ mice with non-typeable Haemophilus influenzae (NTHi) a proportion of them have inflamed middle ear mucosa and fluid accumulation in the ear suggesting that the Fbxo11 knock-out mice are predisposed to NTHi induced middle ear inflammation. In conclusion, the finding that the phenotype of the Jeff mutant is much more severe than the knock-out indicates that the mutation in Jeff manifests gain-of-function as well as loss-of-function effects at both embryonic and adult stages.