The molecular etiology of idiopathic pulmonary fibrosis (IPF) has been extensively investigated to identify new therapeutic targets. Although anti-inflammatory treatments are not effective for patients with IPF, damaged alveolar epithelial cells play a critical role in lung fibrogenesis. Here, we establish an organoid-based lung fibrosis model using mouse and human lung tissues to assess the direct communication between damaged alveolar type II (AT2)-lineage cells and lung fibroblasts by excluding immune cells. Using this in vitro model and mouse genetics, we demonstrate that bleomycin causes DNA damage and activates p53 signaling in AT2-lineage cells, leading to AT2-to-AT1 transition-like state with a senescence-associated secretory phenotype (SASP). Among SASP-related factors, TGF-β plays an exclusive role in promoting lung fibroblast-to-myofibroblast differentiation. Moreover, the autocrine TGF-β-positive feedback loop in AT2-lineage cells is a critical cellular system in non-inflammatory lung fibrogenesis. These findings provide insights into the mechanism of IPF and potential therapeutic targets.

In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.

Significance: In specific tissues, cell death does not represent the end of cell function. The death of the uppermost stratum granulosum keratinocytes (SG1 cells) facilitates their conversion into stratum corneum (SC) corneocytes. Such cells are not removed by efferocytosis as would occur after apoptosis or necrosis; instead, nonviable cell bodies contribute to the protective barrier function of the SC. The present study demonstrates that SG1 cell death is initiated via a single episode of prolonged intracellular Ca ²⁺ elevation, followed by rapid acidification. Such intracellular ionic changes facilitate organellar degradation events specific to SC corneocyte formation. These findings further expand the current knowledge on cell death modes and highlight that nonviable cells contribute to physiological functions in specific contexts.

The cerebral cortex is formed by diverse neurons generated sequentially from neural stem cells (NSCs). A clock mechanism has been suggested to underlie the temporal progression of NSCs, which is mainly defined by the transcriptome and the epigenetic state. However, what drives such a developmental clock remains elusive. We show that translational control of histone H3 trimethylation in Lys27 (H3K27me3) modifiers is part of this clock. We find that depletion ofFbl, an rRNA methyltransferase, reduces translation of both Ezh2 methyltransferase and Kdm6b demethylase of H3K27me3 and delays the progression of the NSC state. These defects are partially phenocopied by simultaneous inhibition of H3K27me3 methyltransferase and demethylase, indicating the role of Fbl in the genome-wide H3K27me3 pattern. Therefore, we propose that Fbl drives the intrinsic clock through the translational enhancement of the H3K27me3 modifiers that predominantly define the NSC state.