The respiratory system undergoes a diversity of structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify the heterogeneity of pulmonary cell types and dynamic changes in gene expression mediating adaptation to respiration, here we perform single cell RNA analyses of mouse lung on postnatal day 1. Using an iterative cell type identification strategy we unbiasedly identify the heterogeneity of murine pulmonary cell types. We identify distinct populations of epithelial, endothelial, mesenchymal, and immune cells, each containing distinct subpopulations. Furthermore we compare temporal changes in RNA expression patterns before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, including activation of cell stress and the unfolded protein response during perinatal adaptation of the lung. The present data provide a single cell view of the adaptation to air breathing after birth.

SummaryRecent studies have implicated keratin 5 (KRT5)+ cells in repopulation of damaged lung tissue following severe H1N1 influenza virus infection. However, the origins of the cells repopulating the injured alveolar region remain controversial. We sought to determine the cellular dynamics of lung repair following influenza infection and define whether nascent KRT5+ cells repopulating alveolar epithelium were derived from pre-existing alveolar or airway progenitor cells. We found that the wound-healing response begins with proliferation of SOX2+ SCGB1A1− KRT5− progenitor cells in airways. These cells generate nascent KRT5+ cells as an early response to airway injury and yield progeny that colonize damaged alveolar parenchyma. Moreover, we show that local alveolar progenitors do not contribute to nascent KRT5+ cells after injury. Repopulation of injured airway and alveolar regions leads to proximalization of distal airways by pseudostratified epithelium and of alveoli by airway-derived epithelial cells that lack the normal characteristics of mature airway or alveolar epithelium.

The SIN pathway blocks inappropriate actin rearrangements during cytokinesis by preventing activation of the MOR pathway component Orb6.

The epidermis provides an essential seal from the external environment and retains fluids within the body. To form an effective barrier, cells in the epidermis must form tight junctions and terminally differentiate into cornified envelopes. Here, we demonstrate that the branched actin nucleator, the actin-related protein (Arp)2/3 complex, is unexpectedly required for both these activities. Loss of the ArpC3 subunit of the Arp2/3 complex resulted in minimal changes in the morphogenesis and architecture of this stratified squamous epithelium, but resulted in profound defects in its physiology. Mutant embryos did not develop an effective barrier to the external environment and died within hours of birth. We discovered two underlying causes for these effects. First, ArpC3 was essential for robust assembly and function of tight junctions, specialized cell-cell adhesions that restrict water loss in the epidermis. Second, there were defects in differentiation of the epidermis and the production of cornified envelopes, structures essential for barrier activity. Underlying this defect, we found that YAP was inappropriately active not only in the ArpC3 mutant tissue, but also in cultured cells. Inhibition of YAP activity rescued the differentiation and barrier defects caused by loss of ArpC3. These results demonstrate previously unappreciated roles for the Arp2/3 complex and highlight the functions of branched actin networks in a complex tissue.