Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration.

Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus such as injury. Although previous studies have demonstrated that certain embryonic developmental programs are reactivated aberrantly in adult organs to drive repair and regeneration, it is not well understood how quiescence is maintained in organs such as the lung, which displays a remarkably low level of cellular turnover. Here we demonstrate that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signalling. Epithelial-specific deletion of sonic hedgehog (Shh) during postnatal homeostasis in the murine lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signalling is initially downregulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signalling prevents the restoration of quiescence during injury resolution. Finally, we show that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. These results demonstrate that epithelial-mesenchymal interactions coordinated by hedgehog actively maintain postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.

Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor.

Co-development of the cardiovascular and pulmonary systems is a recent evolutionary adaption to terrestrial life that couples cardiac output with the gas exchange function of the lung. Here we show that the murine pulmonary vasculature develops even in the absence of lung development. We have identified a population of multipotent cardiopulmonary mesoderm progenitors (CPPs) within the posterior pole of the heart that are marked by the expression of Wnt2, Gli1 and Isl1. We show that CPPs arise from cardiac progenitors before lung development. Lineage tracing and clonal analysis demonstrates that CPPs generate the mesoderm lineages within the cardiac inflow tract and lung including cardiomyocytes, pulmonary vascular and airway smooth muscle, proximal vascular endothelium, and pericyte-like cells. CPPs are regulated by hedgehog expression from the foregut endoderm, which is required for connection of the pulmonary vasculature to the heart. Together, these studies identify a novel population of multipotent cardiopulmonary progenitors that coordinates heart and lung co-development that is required for adaptation to terrestrial existence.

Ezh2 represses the basal cell lineage during lung endoderm development.

The development of the lung epithelium is regulated in a stepwise fashion to generate numerous differentiated and stem cell lineages in the adult lung. How these different lineages are generated in a spatially and temporally restricted fashion remains poorly understood, although epigenetic regulation probably plays an important role. We show that the Polycomb repressive complex 2 component Ezh2 is highly expressed in early lung development but is gradually downregulated by late gestation. Deletion of Ezh2 in early lung endoderm progenitors leads to the ectopic and premature appearance of Trp63+ basal cells that extend the entire length of the airway. Loss of Ezh2 also leads to reduced secretory cell differentiation. In their place, morphologically similar cells develop that express a subset of basal cell genes, including keratin 5, but no longer express high levels of either Trp63 or of standard secretory cell markers. This suggests that Ezh2 regulates the phenotypic switch between basal cells and secretory cells. Together, these findings show that Ezh2 restricts the basal cell lineage during normal lung endoderm development to allow the proper patterning of epithelial lineages during lung formation.

Wnt ligand/Frizzled 2 receptor signaling regulates tube shape and branch-point formation in the lung through control of epithelial cell shape.

Changing the morphology of a simple epithelial tube to form a highly ramified branching network requires changes in cell behavior that lead to tissue-wide changes in organ shape. How epithelial cells in branched organs modulate their shape and behavior to promote bending and sculpting of the epithelial sheet is not well understood, and the mechanisms underlying this process remain obscure. We show that the Wnt receptor Frizzled 2 (Fzd2) is required for domain branch formation during the initial establishment of the respiratory tree. Live imaging and transcriptome analysis of lung-branching morphogenesis demonstrate that Fzd2 promotes changes in epithelial cell length and shape. These changes in cell morphology deform the developing epithelial tube to generate and maintain new domain branches. Fzd2 controls branch formation and the shape of the epithelial tube by regulating Rho signaling and by the localization of phospho-myosin light chain 2, in turn controlling the changes in the shape of epithelial cells during morphogenesis. This study demonstrates the importance of Wnt/Fzd2 signaling in promoting and maintaining changes in epithelial cell shape that affect development of a branching network.

Hopx expression defines a subset of multipotent hair follicle stem cells and a progenitor population primed to give rise to K6+ niche cells.

The mammalian hair follicle relies on adult resident stem cells and their progeny to fuel and maintain hair growth throughout the life of an organism. The cyclical and initially synchronous nature of hair growth makes the hair follicle an ideal system with which to define homeostatic mechanisms of an adult stem cell population. Recently, we demonstrated that Hopx is a specific marker of intestinal stem cells. Here, we show that Hopx specifically labels long-lived hair follicle stem cells residing in the telogen basal bulge. Hopx(+) cells contribute to all lineages of the mature hair follicle and to the interfollicular epidermis upon epidermal wounding. Unexpectedly, our analysis identifies a previously unappreciated progenitor population that resides in the lower hair bulb of anagen-phase follicles and expresses Hopx. These cells co-express Lgr5, do not express Shh and escape catagen-induced apoptosis. They ultimately differentiate into the cytokeratin 6-positive (K6) inner bulge cells in telogen, which regulate the quiescence of adjacent hair follicle stem cells. Although previous studies have suggested that K6(+) cells arise from Lgr5-expressing lower outer root sheath cells in anagen, our studies indicate an alternative origin, and a novel role for Hopx-expressing lower hair bulb progenitor cells in contributing to stem cell homeostasis.

Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.

The causes of amyotrophic lateral sclerosis (ALS), a devastating human neurodegenerative disease, are poorly understood, although the protein TDP-43 has been suggested to have a critical role in disease pathogenesis. Here we show that ataxin 2 (ATXN2), a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2, is a potent modifier of TDP-43 toxicity in animal and cellular models. ATXN2 and TDP-43 associate in a complex that depends on RNA. In spinal cord neurons of ALS patients, ATXN2 is abnormally localized; likewise, TDP-43 shows mislocalization in spinocerebellar ataxia type 2. To assess the involvement of ATXN2 in ALS, we analysed the length of the polyQ repeat in the ATXN2 gene in 915 ALS patients. We found that intermediate-length polyQ expansions (27-33 glutamines) in ATXN2 were significantly associated with ALS. These data establish ATXN2 as a relatively common ALS susceptibility gene. Furthermore, these findings indicate that the TDP-43-ATXN2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

Foxp1/4 control epithelial cell fate during lung development and regeneration through regulation of anterior gradient 2.

The molecular pathways regulating cell lineage determination and regeneration in epithelial tissues are poorly understood. The secretory epithelium of the lung is required for production of mucus to help protect the lung against environmental insults, including pathogens and pollution, that can lead to debilitating diseases such as asthma and chronic obstructive pulmonary disease. We show that the transcription factors Foxp1 and Foxp4 act cooperatively to regulate lung secretory epithelial cell fate and regeneration by directly restricting the goblet cell lineage program. Loss of Foxp1/4 in the developing lung and in postnatal secretory epithelium leads to ectopic activation of the goblet cell fate program, in part, through de-repression of the protein disulfide isomerase anterior gradient 2 (Agr2). Forced expression of Agr2 is sufficient to promote the goblet cell fate in the developing airway epithelium. Finally, in a model of lung secretory cell injury and regeneration, we show that loss of Foxp1/4 leads to catastrophic loss of airway epithelial regeneration due to default differentiation of secretory cells into the goblet cell lineage. These data demonstrate the importance of Foxp1/4 in restricting cell fate choices during development and regeneration, thereby providing the proper balance of functional epithelial lineages in the lung.

Wnt ligands signal in a cooperative manner to promote foregut organogenesis.

Endoderm-mesenchyme cross-talk is a central process in the development of foregut-derived organs. How signaling pathways integrate the activity of multiple ligands to guide organ development is poorly understood. We show that two Wnt ligands, Wnt2 and Wnt7b, cooperatively induce Wnt signaling without affecting the stabilization of the Wnt canonical effector ß-catenin despite it being necessary for Wnt2-Wnt7b cooperativity. Wnt2-Wnt7b cooperation is specific for mesenchymal cell lineages and the combined loss of Wnt2 and Wnt7b leads to more severe developmental defects in the lung than loss of Wnt2 or Wnt7b alone. High-throughput small-molecule screens and biochemical assays reveal that the Pdgf pathway is required for cooperative Wnt2-Wnt7b signaling. Inhibition of Pdgf signaling in cell culture reduces Wnt2-Wnt7b cooperative signaling. Moreover, inhibition of Pdgf signaling in lung explant cultures results in decreased Wnt signaling and lung smooth-muscle development. These data suggest a model in which Pdgf signaling potentiates Wnt2-Wnt7b signaling to promote high levels of Wnt activity in mesenchymal progenitors that is required for proper development of endoderm-derived organs, such as the lung.

Regulation of lung endoderm progenitor cell behavior by miR302/367.

The temporal and spatial control of organ-specific endoderm progenitor development is poorly understood. miRNAs affect cell function by regulating programmatic changes in protein expression levels. We show that the miR302/367 cluster is a target of the transcription factor Gata6 in mouse lung endoderm and regulates multiple aspects of early lung endoderm progenitor development. miR302/367 is expressed at early stages of lung development, but its levels decline rapidly as development proceeds. Gain- and loss-of-function studies show that altering miR302/367 expression disrupts the balance of lung endoderm progenitor proliferation and differentiation, as well as apical-basal polarity. Increased miR302/367 expression results in the formation of an undifferentiated multi-layered lung endoderm, whereas loss of miR302/367 activity results in decreased proliferation and enhanced lung endoderm differentiation. miR302/367 coordinates the balance between proliferation and differentiation, in part, through direct regulation of Rbl2 and Cdkn1a, whereas apical-basal polarity is controlled by regulation of Tiam1 and Lis1. Thus, miR302/367 directs lung endoderm development by coordinating multiple aspects of progenitor cell behavior, including proliferation, differentiation and apical-basal polarity.

The association of Chinese hospital work environment with nurse burnout, job satisfaction, and intention to leave.

The purpose of this study was to describe nurse burnout, job satisfaction, and intention to leave and to explore the relationship of work environment to nursing outcomes in a sample of 9,698 nurses from 181 hospitals in China. Nurses reported moderate levels of emotional exhaustion and depersonalization and high levels of reduced personal accomplishment. Nearly one-fifth of the nurses reported high levels of burnout on all three dimensions. Forty-five percent of the nurses were dissatisfied with their current job; these nurses were most dissatisfied with their salary. Five percent of nurses reported an intention to leave. Nurses reporting mixed and good work environments were less likely to report high burnout, job dissatisfaction, and intention to leave compared with those in poor work environments. The results suggest that high burnout and low job satisfaction are prominent problems for Chinese nurses, and improving work environment might be an effective strategy for better nursing outcomes in Chinese hospitals.

Myocardial Notch signaling reprograms cardiomyocytes to a conduction-like phenotype.

BACKGROUND: Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus, and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. METHODS AND RESULTS: In hearts where Notch signaling is activated within the myocardium from early development onward, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system-specific genes and cell autonomous changes in electrophysiology. With the use of an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome, and in action potential characteristics, consistent with reprogramming to a conduction-like phenotype. CONCLUSIONS: Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.

Nf1 has an essential role in endothelial cells.

Neurofibromatosis type 1 (NF1) or von Recklinghausen neurofibromatosis is a genetic disorder that occurs in 1 of 4000 births and is characterized by benign and malignant tumors. Cardiovascular defects also contribute to NF1, though the pathogenesis is still unclear. Deficiency in neurofibromin (encoded by Nf1) in mice results in mid-embryonic lethality owing to cardiac abnormalities previously thought to be secondary to cardiac neural-crest defects. Using tissue-specific gene inactivation, we show that endothelial-specific inactivation of Nf1 recapitulates key aspects of the complete null phenotype, including multiple cardiovascular abnormalities involving the endocardial cushions and myocardium. This phenotype is associated with an elevated level of ras signaling in Nf1(-/-) endothelial cells and greater nuclear localization of the transcription factor Nfatc1. Inactivation of Nf1 in the neural crest does not cause cardiac defects but results in tumors of neural-crest origin resembling those seen in humans with NF1. These results establish a new and essential role for Nf1 in endothelial cells and confirm the requirement for neurofibromin in the neural crest.

MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.

The epicardium is a sheet of epithelial cells covering the heart during early cardiac development. In recent years, the epicardium has been identified as an important contributor to cardiovascular development, and epicardium-derived cells have the potential to differentiate into multiple cardiac cell lineages. Some epicardium-derived cells that undergo epithelial-to-mesenchymal transition and delaminate from the surface of the developing heart subsequently invade the myocardium and differentiate into vascular smooth muscle of the developing coronary vasculature. MicroRNAs (miRNAs) have been implicated broadly in tissue patterning and development, including in the heart, but a role in epicardium is unknown. To examine the role of miRNAs during epicardial development, we conditionally deleted the miRNA-processing enzyme Dicer in the proepicardium using Gata5-Cre mice. Epicardial Dicer mutant mice are born in expected Mendelian ratios but die immediately after birth with profound cardiac defects, including impaired coronary vessel development. We found that loss of Dicer leads to impaired epicardial epithelial-to-mesenchymal transition and a reduction in epicardial cell proliferation and differentiation into coronary smooth muscle cells. These results demonstrate a critical role for Dicer, and by implication miRNAs, in murine epicardial development.

Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

Cardiac and vascular functions of the zebrafish orthologues of the type I neurofibromatosis gene NFI.

Von Recklinghausen neurofibromatosis is a common autosomal dominant genetic disorder characterized by benign and malignant tumors of neural crest origin. Significant progress in understanding the pathophysiology of this disease has occurred in recent years, largely aided by the development of relevant animal models. Von Recklinghausen neurofibromatosis is caused by mutations in the NF1 gene, which encodes neurofibromin, a large protein that modulates the activity of Ras. Here, we describe the identification and characterization of zebrafish nf1a and nf1b, orthologues of NF1, and show neural crest and cardiovascular defects resulting from morpholino knockdown, including vascular and cardiac valvular abnormalities. Development of a zebrafish model of von Recklinghausen neurofibromatosis will allow for structure-function analysis and genetic screens in this tractable vertebrate system.

Cardiomyocyte cyclooxygenase-2 influences cardiac rhythm and function.

Nonsteroidal anti-inflammatory drugs selective for inhibition of COX-2 increase heart failure and elevate blood pressure. The COX-2 gene was floxed and crossed into merCremer mice under the alpha-myosin heavy-chain promoter. Tamoxifen induced selective deletion of COX-2 in cardiomyocytes depressed cardiac output, and resulted in weight loss, diminished exercise tolerance, and enhanced susceptibility to induced arrhythmogenesis. The cardiac dysfunction subsequent to pressure overload recovered progressively in the knockouts coincident with increasing cardiomyocyte hypertrophy and interstitial and perivascular fibrosis. Inhibition of COX-2 in cardiomyocytes may contribute to heart failure in patients receiving nonsteroidal anti-inflammatory drugs specific for inhibition of COX-2.

Nurse staffing levels make a difference on patient outcomes: a multisite study in Chinese hospitals.

PURPOSE: The purpose of this study is to examine the relationship between nurse staffing and patient outcomes in hospitals in mainland China. METHODS: The study was conducted in 181 hospitals across all of the eight economic zones in mainland China using a four-stage sampling design. Two instruments, the China Nurse Survey and the patient satisfaction measurement from the Hospital Consumer Assessment of Healthcare Providers and Systems, were employed in data collection. In this article, 7,802 nurse surveys and 5,430 patient surveys from 600 medical and surgical units were analyzed. RESULTS: The adjusted joint effects of nurse staffing on patient outcomes from logistic regression analyses showed that more nursing staff per patient had statistically significant positive effects on all necessary nursing care, nurses' reports of quality of care, their confidence on patients' self-care ability on discharge from the hospital, patient adverse events, as well as patients' report of satisfaction. When the nurse-to-patient ratio (total number of nurses on all shifts on the unit divided by total number of patients who stay on the unit) increased to the 0.5-<0.6 category, most patient outcomes were significantly improved, considering hospital and patient factors and nurse skill mix in the logistic regression models. CONCLUSIONS: The findings provide evidence on how inadequate nurse staffing might result in missed but needed nursing care and negative patient outcomes, while better staffing levels could be an effective strategy for improving patient outcomes. CLINICAL RELEVANCE: We recommend that the nurse-to-patient ratio on medical and surgical units in Chinese hospitals be increased to at least 0.5-0.6 so as to secure patient safety and the quality of health services.

Foxp1/2/4-NuRD interactions regulate gene expression and epithelial injury response in the lung via regulation of interleukin-6.

To determine the underlying mechanism of Foxp1/2/4-mediated transcriptional repression, a yeast two-hybrid screen was performed that identified p66beta, a transcriptional repressor and component of the NuRD chromatin-remodeling complex. We show that direct interactions between Foxp1/4 and p66beta are mediated by the CR2 domain within p66beta and the zinc finger/leucine zipper repression domain found in Foxp1/2/4. These direct interactions are functionally relevant as overexpression of p66beta in combination with Foxp factors cooperatively represses Foxp target gene expression, whereas loss of p66 and Foxp factors results in de-repression of endogenous Foxp target genes in lung epithelial cells. Moreover, the NuRD components HDAC1/2 associate in a macromolecular complex with Foxp proteins, and loss of expression or inhibition of HDAC1/2 activity leads to de-repression of Foxp target gene expression. Importantly, we show in vivo that Foxp1 and HDAC2 act cooperatively to regulate expression of the cytoprotective cytokine interleukin-6, which results in increased resistance to hyperoxic lung injury in Foxp1/HDAC2 compound mutant animals. These data reveal an important interaction between the Foxp transcription factors and the NuRD chromatin-remodeling complex that modulates transcriptional repression critical for the lung epithelial injury response.

Gata4 and Gata5 cooperatively regulate cardiac myocyte proliferation in mice.

GATA5 is a member of the zinc finger transcription factor GATA family (GATA1-6) that plays a wide variety of roles in embryonic and adult development. Experiments in multiple model systems have emphasized the importance of the GATA family members 4-6 in the development of the endoderm and mesoderm. Yet despite overlapping expression patterns, there is little evidence of an important role for GATA5 in mammalian cardiac development. We have generated a new Gata5 mutant allele lacking exons 2 and 3 that encodes both zinc finger domains (Gata5(tm)(2)(Eem)), and we show that although Gata5(-/-) mice are viable, Gata4(+/-)5(-/-) mutants die at mid-gestation and exhibit profound cardiovascular defects, including abnormalities of cardiomyocyte proliferation and cardiac chamber maturation. These results demonstrate functional redundancy between Gata4 and Gata5 during cardiac development and implicate Gata5 as a candidate modifier gene for congenital heart disease.

Persistent expression of Pax3 in the neural crest causes cleft palate and defective osteogenesis in mice.

Transcription factors regulate tissue patterning and cell fate determination during development; however, expression of early regulators frequently abates upon differentiation, suggesting that they may also play a role in maintaining an undifferentiated phenotype. The transcription factor paired box 3 (Pax3) is expressed by multipotent neural crest precursors and is implicated in neural crest disorders in humans such as Waardenburg syndrome. Pax3 is required for development of multiple neural crest lineages and for activation of lineage-specific programs, yet expression is generally extinguished once neural crest cells migrate from the dorsal neural tube and differentiate. Using a murine Cre-inducible system, we asked whether persistent Pax3 expression in neural crest derivatives would affect development or patterning. We found that persistent expression of Pax3 in cranial neural crest cells resulted in cleft palate, ocular defects, malformation of the sphenoid bone, and perinatal lethality. Furthermore, we demonstrated that Pax3 directly regulates expression of Sostdc1, a soluble inhibitor of bone morphogenetic protein (BMP) signaling. Persistent Pax3 expression renders the cranial crest resistant to BMP-induced osteogenesis. Thus, one mechanism by which Pax3 maintains the undifferentiated state of neural crest mesenchyme may be to block responsiveness to differentiation signals from the environment. These studies provide in vivo evidence for the importance of Pax3 downregulation during differentiation of multipotent neural crest precursors and cranial development.