Single cell RNA-seq study of wild type and Hox9,10,11 mutant developing uterus.

The uterus is a remarkable organ that must guard against infections while maintaining the ability to support growth of a fetus without rejection. The Hoxa10 and Hoxa11 genes have previously been shown to play essential roles in uterus development and function. In this report we show that the Hoxa9,10,11, Hoxc9,10,11, Hoxd9,10,11 genes play a redundant role in the formation of uterine glands. In addition, we use single cell RNA-seq to create a high resolution gene expression atlas of the developing wild type mouse uterus. Cell types and subtypes are defined, for example dividing endothelial cells into arterial, venous, capillary, and lymphatic, while epithelial cells separate into luminal and glandular subtypes. Further, a surprising heterogeneity of stromal and myocyte cell types are identified. Transcription factor codes and ligand/receptor interactions are characterized. We also used single cell RNA-seq to globally define the altered gene expression patterns in all developing uterus cell types for two Hox mutants, with 8 or 9 mutant Hox genes. The mutants show a striking disruption of Wnt signaling as well as the Cxcl12/Cxcr4 ligand/receptor axis.

Kras(G12D) and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung.

Mucinous adenocarcinoma of the lung is a subtype of highly invasive pulmonary tumors and is associated with decreased or absent expression of the transcription factor NK2 homeobox 1 (NKX2-1; also known as TTF-1). Here, we show that haploinsufficiency of Nkx2-1 in combination with oncogenic Kras(G12D), but not with oncogenic EGFR(L858R), caused pulmonary tumors in transgenic mice that were phenotypically similar to human mucinous adenocarcinomas. Gene expression patterns distinguished tumor goblet (mucous) cells from nontumorigenic airway and intestinal goblet cells. Expression of NKX2-1 inhibited urethane and oncogenic Kras(G12D)-induced tumorigenesis in vivo. Haploinsufficiency of Nkx2-1 enhanced Kras(G12D)-mediated tumor progression, but reduced EGFR(L858R)-mediated progression. Genome-wide analysis of gene expression demonstrated that a set of genes induced in mucinous tumors was shared with genes induced in a nontumorigenic chronic lung disease, while a distinct subset of genes was specific to mucinous tumors. ChIP with massively parallel DNA sequencing identified a direct association of NKX2-1 with the genes induced in mucinous tumors. NKX2-1 associated with the AP-1 binding element as well as the canonical NKX2-1 binding element. NKX2-1 inhibited both AP-1 activity and tumor colony formation in vitro. These data demonstrate that NKX2-1 functions in a context-dependent manner in lung tumorigenesis and inhibits Kras(G12D)-driven mucinous pulmonary adenocarcinoma.

GP130-STAT3 regulates epithelial cell migration and is required for repair of the bronchiolar epithelium.

Following injury, bronchiolar cells undergo rapid squamous metaplasia, followed by proliferation and re-establishment of the complex columnar epithelium that is characteristic of the normal airway. Mechanisms that regulate the repair of bronchiolar epithelium are of considerable relevance for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. This study was designed to identify the role of the GP130-STAT3 signaling pathway during repair of the bronchiolar epithelium. STAT3 (signal transducer and activator of transcription 3) and GP130 (glycoprotein 130) were each selectively deleted from the pulmonary epithelial cells of transgenic mice in vivo, producing Stat3(Delta/Delta) and Gp130(Delta/Delta) mice, respectively. Airway injury was induced in adult mice by administration of naphthalene, a toxicant of nonciliated respiratory epithelial cells (Clara cells). Nuclear STAT3 staining was induced in bronchiolar epithelial cells following naphthalene-mediated injury in control (Stat3(flox/flox)) mice. Whereas nearly complete repair of the bronchiolar epithelium was observed in control mice within 13 days, restoration of cell shape, cell density, and the pattern of ciliated and nonciliated cells did not occur in the peripheral bronchioles of either Stat3(Delta/Delta) or Gp130(Delta/Delta) mice. Expression of dominant-negative STAT3 inhibited airway epithelial cell migration during repair in vitro; wild-type STAT3 expression activated such migration. In the present study, we show that GP130-STAT3 signaling functions in a cell-autonomous manner to restore cell shape and numbers required for repair of the bronchiolar epithelium following injury.

R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis.

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2(Tg)). The predicted protein encoded by Rspo2(Tg) neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2(Tg/Tg) mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2(Tg/Tg) lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2(Tg) and Lrp6(-) alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.

Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells.

Pulmonary surfactant is a complex of lipids and proteins produced and secreted by alveolar type II cells that provides the low surface tension at the air-liquid interface. The phospholipid most responsible for providing the low surface tension in the lung is dipalmitoylphosphatidylcholine. Dipalmitoylphosphatidylcholine is synthesized in large part by phosphatidylcholine (PC) remodeling, and a lysophosphatidylcholine (lysoPC) acyltransferase is thought to play a critical role in its synthesis. However, this acyltransferase has not yet been identified. We have cloned full-length rat and mouse cDNAs coding for a lysoPC acyltransferase (LPCAT). LPCAT encodes a 535-aa protein of approximately 59 kDa that contains a transmembrane domain and a putative acyltransferase domain. When transfected into COS-7 cells and HEK293 cells, LPCAT significantly increased lysoPC acyltransferase activity. LPCAT preferred lysoPC as a substrate over lysoPA, lysoPI, lysoPS, lysoPE, or lysoPG and prefers palmitoyl-CoA to oleoyl-CoA as the acyl donor. This LPCAT was preferentially expressed in the lung, specifically within alveolar type II cells. Expression in the fetal lung and in rat type II cells correlated with the expression of the surfactant proteins. LPCAT expression in fetal lung explants was sensitive to dexamethasone and FGFs. KGF was a potent stimulator of LPCAT expression in cultured adult type II cells. We hypothesize that LPCAT plays a critical role in regulating surfactant phospholipid biosynthesis and suggest that understanding the regulation of LPCAT will offer important insight into surfactant phospholipid biosynthesis.

Beta-catenin regulates differentiation of respiratory epithelial cells in vivo.

An activated form of beta-catenin [Catnb(Delta(ex3))] was expressed in respiratory epithelial cells of the developing lung. Although morphogenesis was not altered at birth, air space enlargement and epithelial cell dysplasia were observed in the early postnatal period and persisted into adulthood. The Catnb(Delta(ex3)) protein caused squamous, cuboidal, and goblet cell dysplasia in intrapulmonary conducting airways. Atypical epithelial cells that stained for surfactant pro protein C (pro-SP-C) and had morphological characteristics of alveolar type II cells were observed in bronchioles of the transgenic mice. Catnb(Delta(ex3)) inhibited expression of Foxa2 and caused goblet cell hyperplasia associated with increased staining for mucins and the MUC5A/C protein. In vitro, both wild type and activated beta-catenin negatively regulated the expression of the Foxa2 promoter. Catnb(Delta(ex3)) also caused pulmonary tumors in adult mice. Activation of beta-catenin caused ectopic differentiation of alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, demonstrating a critical role for the Wnt/beta-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung.

The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)-associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80(-/-) mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80(-/-) APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80(-/-) mice generated an efferent CD8(+) T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80(-/-) mice by adoptive transfer of F4/80(+) APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8(+) T reg cells.

Oncogenic transcription factor Evi1 regulates hematopoietic stem cell proliferation through GATA-2 expression.

The ecotropic viral integration site-1 (Evi1) is an oncogenic transcription factor in murine and human myeloid leukemia. We herein show that Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, suggesting a physiological role of Evi1 in HSCs. We therefore investigate the role and authentic target genes of Evi1 in hematopoiesis using Evi1-/- mice, which die at embryonic day 10.5. HSCs in Evi1-/- embryos are markedly decreased in numbers in vivo with defective self-renewing proliferation and repopulating capacity. Notably, expression rate of GATA-2 mRNA, which is essential for proliferation of definitive HSCs, is profoundly reduced in HSCs of Evi1-/- embryos. Restoration of the Evi1 or GATA-2 expression in Evi1-/- HSCs could prevent the failure of in vitro maintenance and proliferation of HSC through upregulation of GATA-2 expression. An analysis of the GATA-2 promoter region revealed that Evi1 directly binds to GATA-2 promoter as an enhancer. Our results reveal that GATA-2 is presumably one of critical targets for Evi1 and that transcription factors regulate the HSC pool hierarchically.

c-myb Heterozygous mice are hypersensitive to 5-fluorouracil and ionizing radiation.

Hypersensitivity to chemo- and radiotherapy employed during cancer treatment complicates patient management. Identifying mutations in genes that compromise tissue recovery would rationalize treatment and may spare hypersensitive patients undue tissue damage. Genes that govern stem cell homeostasis, survival, and progenitor cell maintenance are of particular interest in this regard. We used wild-type and c-myb knock-out mice as model systems to explore stem and progenitor cell numbers and sensitivity to cytotoxic damage in two radiosensitive tissue compartments, the bone marrow and colon. Because c-myb null mice are not viable, we used c-myb heterozygous mice to test for defects in stem-progenitor cell pool recovery following gamma-radiation and 5-fluorouracil treatment, showing that c-myb(+/-) mice are hypersensitive to both agents. While apoptosis is comparable in mutant and wild-type mice following radiation exposure, the crypt beds of c-myb(+/-) mice are markedly depleted of proliferating cells. Extrapolating from these data, we speculate that acute responses to cytotoxic damage in some patients may also be attributed to compromised c-myb function.

Midkine is regulated by hypoxia and causes pulmonary vascular remodeling.

Midkine (MK) is expressed in a precise temporal-spatial pattern during lung morphogenesis; however, its role in pulmonary homeostasis is unknown. Increased MK staining and mRNA expression were observed in the lungs of hypoxia-susceptible CAST/eiJ mice during hypoxia. MK expression was induced by hypoxia in cell lines in vitro. Because the transcription factor hypoxiainducible factor-1alpha (HIF-1alpha) modulates cellular responses to hypoxia, we tested whether increased expression of MK in the lung was mediated by HIF-1alpha. HIF-1alpha enhanced the transcription of MK, acting on HIF-1alpha regulatory elements located in the MK gene promoter. Site-directed mutagenesis of the 3' HIF response element in the MK promoter blocked the stimulatory effects of HIF-1alpha. To directly assess the role of MK on lung morphogenesis, transgenic mice were generated in which MK was expressed in the respiratory epithelial cells of the developing lung. MK increased muscularization of small pulmonary arteries, increasing alpha-smooth muscle actin and caldesmon staining and the expression of myocardin. MK directly enhanced the expression of myocardin and the smooth muscle-specific genes alpha-smooth muscle actin, calponin, and SM-22 in vascular smooth muscle precursor cells. Expression of MK in the respiratory epithelium is regulated by hypoxia and HIF-1alpha. These data provide a model wherein the respiratory epithelium responds to hypoxia via HIF-1alpha-dependent regulation of MK, enhancing myocardin expression to influence pulmonary vascular gene expression.

beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis.

The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that beta-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for beta-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that beta-catenin is one of the essential components of this specification.

Thyroid transcription factor (TTF) -1 regulates the expression of midkine (MK) during lung morphogenesis.

Midkine (MK) is a 13-kDa heparin-binding growth factor that is thought to mediate developmental processes, including vasculogenesis, cell migration, and proliferation in various organs. To determine whether MK plays a role during lung morphogenesis, immunostaining for MK was assessed in mouse lung from embryonic day (E) 13 to postnatal day (PN) 24. MK was detected in mesenchymal and respiratory epithelial cells of the peripheral mouse lung from E13.0 to E15.5. From E18.5 to PN1, MK was observed primarily in epithelial cells lining conducting airways and peripheral lung saccules. By PN10, expression was no longer observed in respiratory epithelial cells but was readily detected in small blood vessels in the alveolar region of the lung. Although most respiratory epithelial cells uniformly expressed MK before E13.0, MK was restricted to subsets of cells by E18.5, colocalizing with the Clara cell secretory protein (CCSP) marker in conducting airways and with pro-SPC, a marker specific for alveolar type II pneumocytes. By PN10, MK was not detected in respiratory epithelial cells of the conducting airways and was closely associated with capillary networks. The sites of intense MK staining in the respiratory epithelial cells correlated with sites of expression of thyroid transcription factor (TTF) -1, a transcription factor regulating formation and gene expression in the lung parenchyma. TTF-1 enhanced transcription of the mouse MK gene promoter, acting on TTF-1 regulatory elements located in the 5'-region of the gene. Furthermore, MK expression was not detected in lungs of TTF-1 null mice. TTF-1 regulates expression of MK in the lung. The temporal/spatial distribution of midkine is consistent with a potential role in paracrine signaling during lung morphogenesis.