SUMMARY After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5(Krt5) remodeling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, via hypoxia inducible factor (HIF1α), drives Notch signaling and Krt5pos basal-like cell expansion. Single cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and trans-differentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2+ LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.

Basal cells (BCs) are p63-expressing multipotent progenitors of skin, tracheoesophageal and urinary tracts. p63 is abundant in developing airways; however, it remains largely unclear how embryonic p63 cells contribute to the developing and postnatal respiratory tract epithelium, and ultimately how they relate to adult BCs. Using lineage-tracing and functional approaches in vivo, we show that p63 cells arising from the lung primordium are initially multipotent progenitors of airway and alveolar lineages but later become restricted proximally to generate the tracheal adult stem cell pool. In intrapulmonary airways, these cells are maintained immature to adulthood in bronchi, establishing a rare p63Krt5 progenitor cell population that responds to H1N1 virus-induced severe injury. Intriguingly, this pool includes a CC10 lineage-labeled p63Krt5 cell subpopulation required for a full H1N1-response. These data elucidate key aspects in the establishment of regionally distinct adult stem cell pools in the respiratory system, potentially with relevance to other organs.

Twist1 is an epithelial-mesenchymal transition (EMT)-inducing transcription factor (TF) that promotes cell migration and invasion. To determine the intrinsic role of Twist1 in EMT and breast cancer initiation, growth, and metastasis, we developed mouse models with an oncogene-induced mammary tumor containing wild-type (WT) or tumor cell-specific knockout (Twist1). knockout showed no effects on tumor initiation and growth. In both models with early-stage tumor cells, Twist1, and mesenchymal markers were not expressed, and lung metastasis was absent. Twist1 expression was detected in ∼6% of the advanced WT tumor cells. Most of these Twist1 cells coexpressed several other EMT-inducing TFs (Snail, Slug, Zeb2), lost ERα and luminal marker K8, acquired basal cell markers (K5, p63), and exhibited a partial EMT plasticity (E-cadherin/vimentin). In advanced Twist1 tumor cells, knockout largely diminished the expression of the aforementioned EMT-inducing TFs and basal and mesenchymal markers, but maintained the expression of the luminal markers. Circulating tumor cells (CTCs) were commonly detected in mice with advanced WT tumors, but not in mice with advanced Twist1 tumors. Nearly all WT CTCs coexpressed Twist1 with other EMT-inducing TFs and both epithelial and mesenchymal markers. Mice with advanced WT tumors developed extensive lung metastasis consisting of luminal tumor cells with silenced Twist1 and mesenchymal marker expression. Mice with advanced Twist1 tumors developed very little lung metastasis. Therefore, Twist1 is required for the expression of other EMT-inducing TFs in a small subset of tumor cells. Together, they induce partial EMT, basal-like tumor progression, intravasation, and metastasis.

The prostate epithelium is composed of basal (BC), luminal (LEC), and neuroendocrine (NEC) cells. It is unclear how many subtypes of BCs in the prostate and which subtype of BCs contains the main stem cell niche in the adult prostate. Here we report seven BC subpopulations according to their p63, cytokeratin 14 (K14) and K5 expression patterns, including p63-positive/K14-negative/K5-negative (p63+/K14-/K5-), p63-/K14+/K5-, p63-/K14-/K5+, p63+/K14+/K5-, p63+/K14-/K5+, p63-/K14+/K5+, and p63+/K14+/K5+ BCs. We generated a p63-CreERT2 knock-in mouse line that expresses tamoxifen-inducible Cre activity in the p63-expressing cells, including the prostate BCs. We then crossbred this line with ROSA26R mice, and generated p63-CreERT2×ROSA26R bi-genic mice harboring the Cre-activated β-galactosidase reporter gene. We treated these bi-genic mice with tamoxifen to mark the p63+ BCs at different ages or under different hormonal conditions, and then traced the lineage differentiation of these genetically labeled BCs. We discovered that these p63+ BCs contain self-renewable stem cells in culture and efficiently differentiated into LECs, NECs and BCs in the postnatal, adult and re-generating mouse prostates. Therefore, BC population contains heterogeneous BCs that express different combinations of the p63, K14 and K5 differentiation markers. Because K14+ and K5+ BCs were previously shown to be extremely inefficient to produce LECs in adulthood, we propose that the p63+/K5-/K14- subpopulation of BCs contains most stem-like cells, especially in adult animals.

Nuclear receptors and coregulators orchestrate diverse aspects of biological functions and inappropriate expression of these factors often associates with human diseases. The present study describes a conditional overexpression system consisting of a minigene located at the Rosa26 locus in the genome of mouse embryonic stem (ES) cells. Before activation, the minigene is silent due to a floxed STOP cassette inserted between the promoter and the transgene. Upon cre-mediated excision of the STOP cassette, the minigene constitutively expresses the tagged transgene driven by the ubiquitous CAGGS promoter. Thus, this system can be used to express target gene in any tissue in a spatial and/or temporal manner if respective cre mouse lines are available. Serving as proof of principle, the CAG-S-hCOUP-TFI allele was generated in ES cells and subsequently in mice. This allele was capable of conditionally overexpressing human chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in all tissues tested upon activation by cre drivers. This allele was further subjected to address functionality of expressed COUP-TFI and the functional similarity between COUP-TFI and COUP-TFII. Expression of COUP-TFI in COUP-TFII-ablated uterus suppressed aberrant estrogen receptor-alpha activities and rescued implantation and decidualization defects of COUP-TFII mutants, suggesting that COUP-TFI and COUP-TFII are able to functionally compensate for each other in the uterus. A toolbox currently under construction will contain ES cell lines for overexpressing all 48 nuclear receptors and selected 10 coregulators. Upon completion, it will be a very valuable resource for the scientific community. Several ES cells are currently available for distribution.