Idiopathic pulmonary fibrosis (IPF) consists of fibrotic alveolar remodeling and progressive loss of pulmonary function. Genetic and experimental evidence indicates that chronic alveolar injury and failure to properly repair the respiratory epithelium are intrinsic to IPF pathogenesis. Loss of alveolar type 2 (AT2) stem cells or mutations that either impair their self-renewal and/or impair their differentiation into AT1 cells can serve as a trigger of pulmonary fibrosis. Recent reports indicate increased YAP activity in respiratory epithelial cells in IPF lungs. Individual IPF epithelial cells with aberrant YAP activation in bronchiolized regions frequently co-express AT1, AT2, conducting airway selective markers and even mesenchymal or EMT markers, demonstrating ‘indeterminate’ states of differentiation and suggesting that aberrant YAP signaling might promote pulmonary fibrosis. Yet, Yap and Taz have recently also been shown to be important for AT1 cell maintenance and alveolar epithelial regeneration after Streptococcus pneumoniae-induced injury. To investigate how epithelial Yap/Taz might promote pulmonary fibrosis or drive alveolar epithelial regeneration, we inactivated the Hippo pathway in AT2 stem cells resulting in increased nuclear Yap/Taz, and found that this promotes their alveolar regenerative capacity and reduces pulmonary fibrosis following bleomycin injury by pushing them along the AT1 cell lineage. Vice versa, inactivation of both Yap1 and Wwtr1 (encoding Taz) or Wwtr1 alone in AT2 cell stem cells impaired alveolar epithelial regeneration and resulted in increased pulmonary fibrosis upon bleomycin injury. Interestingly, the inactivation of only Yap1 in AT2 stem cells promoted alveolar epithelial regeneration and reduced pulmonary fibrosis. Together, these data suggest that epithelial Yap promotes, and epithelial Taz reduces pulmonary fibrosis suggesting that targeting Yap but not Taz-mediated transcription might help promote AT1 cell regeneration and treat pulmonary fibrosis.

Alveolar Type 1 and Type 2 cells are vital for lung gas exchange and become compromised in several diseases. While key differentiation signals are known, their emergence and fate plasticity are unclear. Here we show in the embryonic lung that single AT2s emerge at intermediate zones, extrude, and connect with nearby epithelium via interlumenal junctioning. We observe AT2s retain fate plasticity until the bZIP transcription factor C/EBPα suppresses Notch signaling at a novel Dlk1 enhancer. Both Dlk1 and Cebpa are regulated by the polycomb repressive complex (PRC2), which together form a “pulse generator” circuit that times Dlk1 expression and thus Notch activation, resulting in a “salt and pepper” pattern of AT1 and AT2 fate. In injured adult lungs, C/EBPα downregulation is required to re-access AT2 fate plasticity and is mediated by the dominant negative C/EBP family member CHOP. Finally, Cebpa loss also activates a “defender” AT2 state, distinct from its reparative state, and we propose AT2s toggle between either state following infection to protect and repair alveoli.

The coordination between epithelial progenitors and their mesenchymal niche is critical for organogenesis and repair, yet the mechanisms governing their competitive interactions remain unclear. Here, we reveal a paradigm of tissue-scale fitness sensing in the lung, where mesenchymal Yap levels antagonize epithelial Yap levels to dictate epithelial stem cell fate. We show that reduced fitness in alveolar fibroblasts (AF1s) via Yap/Taz or Myc deletion leads to their apoptotic elimination and a collapse of the alveolar stem cell niche. This niche collapse triggers a pathological competitive response from the epithelium, which undergoes aberrant bronchiolization that phenocopies human pulmonary fibrosis. Mechanistically, we uncovered a molecular switch that controls mesenchymal fate. During development and fibrosis resolution, Snail1/2 sequesters Yap/Taz to drive an adipogenic program, generating niche-supportive AF1s. Conversely, Yap/Taz-TEAD-Myc binding instructs a myogenic, pro-fibrotic program. Our findings demonstrate that inter-tissue cell competition, governed by a Snail/Yap rheostat, orchestrates lung architecture and provides a framework for targeting the mesenchymal niche to treat fibrotic disease.

The mammalian lung operates under a biological paradox, requiring architectural fragility for gas exchange while maintaining robust regenerative plasticity to withstand injury. The Hippo signaling pathway has emerged as a central “rheostat” in orchestrating these opposing needs, yet the distinct roles of its downstream effectors remain underappreciated. This review synthesizes recent genetic and mechanobiological advances to propose a “Tale of Two Effectors” model, arguing for the functional non-redundancy of YAP and TAZ. We posit that YAP functions to drive airway progenitor expansion, mechanical force generation, and maladaptive remodeling. Conversely, TAZ—regulated uniquely via transcriptional mechanisms and mechanotransduction—acts as an obligate driver of alveolar differentiation and adaptive repair through an NKX2-1 feed-forward loop. Furthermore, we introduce the “See-Saw” model of tissue fitness, where mesenchymal niche collapse releases the mechanical brake on the epithelium, triggering the bronchiolization characteristic of pulmonary fibrosis. Finally, we extend this framework to malignancy, illustrating how Small Cell Lung Cancer (SCLC) subtypes mirror these developmental and regenerative states. This integrated framework offers new therapeutic distinct targets for modulating tissue fitness and resolving fibrosis.

Fluorescence in situ hybridization (FISH) is a useful tool for analyzing RNA expression, but difficulties arise with low-abundance RNA and in tissues that are formalin-fixed paraffin-embedded (FFPE) because reagents can be expensive. In this protocol, we adapt a previously designed FISH amplification protocol (SABER [signal amplification by exchange reaction]) for adult mouse FFPE lung sections by using probes that are extended and branched to amplify the signal. We combine FISH and immunostaining to identify cell-specific RNA. For complete details on the use and execution of this protocol, please refer to Kishi et al.¹ and Lyu et al.².

Idiopathic pulmonary fibrosis (IPF) consists of fibrotic alveolar remodeling and progressive loss of pulmonary function. Genetic and experimental evidence indicate that chronic alveolar injury and failure to properly repair the respiratory epithelium are intrinsic to IPF pathogenesis. Loss of alveolar type 2 (AT2) stem cells or mutations that either impair their self-renewal and/or impair their differentiation into AT1 cells can serve as a trigger of pulmonary fibrosis. Recent reports indicate increased YAP activity in respiratory epithelial cells in IPF lungs. Individual IPF epithelial cells with aberrant Yap activation in bronchiolized regions frequently co-express AT1, AT2, conducting airway selective markers and even mesenchymal or EMT markers, demonstrating “indeterminate” states of differentiation and suggesting that aberrant Yap signaling might promote pulmonary fibrosis. Yet, Yap and Taz have recently also been shown to be important for AT1 cell maintenance and alveolar epithelial regeneration after Streptococcus pneumoniae induced injury. To investigate how epithelial Yap/Taz might promote pulmonary fibrosis or drive alveolar epithelial regeneration, we inactivated the Hippo pathway in AT2 stem cells resulting in increased nuclear Yap/Taz and found that this promotes their alveolar regenerative capacity and reduces pulmonary fibrosis following bleomycin injury by pushing them along the AT1 cell lineage. Vice versa, inactivation of both Yap and Taz or Taz alone in AT2 cells stem cells impaired alveolar epithelial regeneration and resulted in increased pulmonary fibrosis upon bleomycin injury. Interestingly, inactivation of only Yap in AT2 stem cells promoted alveolar epithelial regeneration and reduced pulmonary fibrosis. Together, these data suggest that epithelial Yap promotes, and epithelial Taz reduces pulmonary fibrosis suggesting that targeting Yap but not Taz mediated transcription might help promote AT1 cell regeneration and treat pulmonary fibrosis.