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.

Summary The Septum Initiation Network (SIN) regulates multiple functions during late mitosis to ensure successful completion of cytokinesis in S. pombe. One mechanism by which the SIN promotes cytokinesis is by inhibiting a competing polarity pathway called the MOR [1], which is required for initiation of polarized growth following completion of cytokinesis [2]. Mutual antagonism between the two NDR kinase pathways, SIN and MOR, is required to coordinate cytoskeletal rearrangements during the mitosis-interphase transition. To determine how the SIN regulates the MOR pathway, we developed a proteomics approach that allowed us to identify multiple substrates of the SIN effector kinase, Sid2, including the MOR pathway components Nak1 kinase and an associated protein Sog2. We show that Sid2 phosphorylation of Nak1 causes removal of Nak1 from the SPBs, which may both relieve Nak1 inhibition of the SIN, and block MOR signaling by preventing interaction of Nak1 with the scaffold protein Mor2. Because the SIN and MOR are conserved in mammalian cells (Hippo and Ndr1/2 pathways respectively), this work may provide important insight into how the activities of these essential pathways are coordinated.

β-Catenin provides stability to epithelia exposed to mechanical stress in part by strengthening adherens junctions in response to tension.

No abstract

The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe, and Saccharomyces cerevisiae, respectively. One function of these pathways is to keep the Cdc14-family phosphatase, called Clp1 in S. pombe, from being sequestered and inhibited in the nucleolus. In S. pombe, the SIN and Clp1 act as part of a cytokinesis checkpoint that allows cells to cope with cytokinesis defects. The SIN promotes checkpoint function by 1) keeping Clp1 out of the nucleolus, 2) maintaining the cytokinetic apparatus, and 3) halting the cell cycle until cytokinesis is completed. In a screen for suppressors of the SIN mutant cytokinesis checkpoint defect, we identified a novel nucleolar protein called Dnt1 and other nucleolar proteins, including Rrn5 and Nuc1, which are known to be required for rDNA transcription. Dnt1 shows sequence homology to Net1/Cfi1, which encodes the nucleolar inhibitor of Cdc14 in budding yeast. Like Net1/Cfi1, Dnt1 is required for rDNA silencing and minichromosome maintenance, and both Dnt1 and Net1/Cfi1 negatively regulate the homologous SIN and MEN pathways. Unlike Net1/Cfi1, which regulates the MEN through the Cdc14 phosphatase, Dnt1 can inhibit SIN signaling independently of Clp1, suggesting a novel connection between the nucleolus and the SIN pathway.