Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha–positive (PDGFRA ⁺ ) fibroblasts are critical for alveolarization and that PDGFRA ⁺ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population–targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O ₂ postnatal days 0–7, PN0–PN7) and performed studies on sorted PDGFRA ⁺ cells during injury and room air recovery. After hyperoxia injury, PDGFRA ⁺ matrix and myofibroblasts decreased and PDGFRA ⁺ lipofibroblasts increased by transcriptional signature and population size. PDGFRA ⁺ matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA ⁺ sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA ⁺ fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA ⁺ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.

Tracheobronchomalacia and complete tracheal rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea’s cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/β-catenin target genes, including targets of WNT signaling: Notum and Axin2. In vitro, recombinant (r)BMP4 rescued the expression of Notum in Bmp4-deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4-deficient tracheas identified genes essential for chondrogenesis and muscle development coregulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

The most common mutation in surfactant protein C gene ( SFTPC ), SFTPC ^I73T , causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPC ^I73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity purification-mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including valosin containing protein (VCP) and its interactors. Treatment of Sftpc ^I73T knock-in mice and SFTPC ^I73T -expressing iAT2 cells derived from SFTPC ^I73T patient-specific iPSCs with the VCP inhibitor CB5083 restored alveolar structure and SFTPC ^I73T trafficking, respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPC ^I73T mutant, providing therapeutical targets for SFTPC ^I73T -associated interstitial lung disease.

The occurrence and impact of cellular senescence on skin aging and hyperpigmentation is an ongoing area of exploration, encompassing both intrinsic and extrinsic stressors. Traditionally, research has focused on melanocyte and fibroblast senescence due to their slower turnover compared to keratinocytes. In this study, we identified the accumulation of p16, a senescence marker, in keratinocytes from biopsies of multiple spot types. We explored their impact using doxorubicin-induced senescent keratinocytes in vitro. Conditioned media from these senescent keratinocytes stimulated melanocyte dendricity, a hallmark of hyperpigmented spots. Transcriptomic analysis of senescent keratinocytes identified two key senescence-induced factors: Insulin-like Growth Factor-Binding Protein 3 (IGFBP3) and Nerve Growth Factor (NGF). IGFBP3 and NGF ligand treatment enhanced melanin synthesis by 33% and 17%, and dendricity by 23% and 14%, respectively, in human melanocyte cultures. These findings suggest that keratinocyte senescence contributes to spot formation by mediating melanocyte activation through IGFBP3 and NGF. Furthermore, we evaluated skincare ingredients such as sucrose dilaurate, glabridin, and niacinamide in neutral and low pH solutions, demonstrating their efficacy in reducing the secretion of these ligands, thereby offering potential cosmetic benefits. This study provides insights into the mechanisms of spot formation and highlights promising strategies for managing pigmentation disorders.

Lung epithelial regeneration after acute injury requires coordination of extensive cellular and molecular processes controlling proliferation and differentiation of specialized alveolar cells to pattern the morphologically complex alveolar gas exchange surface. During regeneration, specialized Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells, though the precise molecular and epigenetic determinants of these processes remain unclear. Here, we report a refined primary murine alveolar organoid assay which recapitulates critical aspects of in vivo regeneration, providing a tractable model to dissect these regenerative processes. Clonal expansion of single AEPs generate complex alveolar organoids with extensive structural maturation and organization. These organoids contain properly patterned AT1 and AT2 cells surrounding numerous alveolar-like cavities with minimal structural contribution from mesenchymal cells, implying extensive cell autonomous regenerative function encoded in adult AEPs. Leveraging a time series of paired scRNAseq and scATACseq, we identified the AEP state at single cell resolution and described two distinct AEP to AT1 intermediate states: a widely reported Krt8 ⁺ transitional state defined by cell stress markers and a second state defined by differential activation of signaling pathways mediating AT1 cell differentiation. Transcriptional regulatory network (TRN) analysis demonstrated that these AT1 transition states were driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8 ⁺ state characterized by disorganized, uncontrolled growth. Finally, AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of AEP state, clonal expansion, and disorganization of alveolar structure, implying a continuous requirement for Nkx2-1 in maintenance and function of adult lung progenitors. Together, these data provide new insight into cellular hierarchies in lung regeneration and implicate dynamic epigenetic maintenance via lineage transcription factors as central to control of facultative progenitor activity in AEPs.