Ontology-guided segmentation and object identification for developmental mouse lung immunofluorescent images.

BACKGROUND: Immunofluorescent confocal microscopy uses labeled antibodies as probes against specific macromolecules to discriminate between multiple cell types. For images of the developmental mouse lung, these cells are themselves organized into densely packed higher-level anatomical structures. These types of images can be challenging to segment automatically for several reasons, including the relevance of biomedical context, dependence on the specific set of probes used, prohibitive cost of generating labeled training data, as well as the complexity and dense packing of anatomical structures in the image. The use of an application ontology helps surmount these challenges by combining image data with its metadata to provide a meaningful biological context, modeled after how a human expert would make use of contextual information to identify histological structures, that constrains and simplifies the process of segmentation and object identification. RESULTS: We propose an innovative approach for the semi-supervised analysis of complex and densely packed anatomical structures from immunofluorescent images that utilizes an application ontology to provide a simplified context for image segmentation and object identification. We describe how the logical organization of biological facts in the form of an ontology can provide useful constraints that facilitate automatic processing of complex images. We demonstrate the results of ontology-guided segmentation and object identification in mouse developmental lung images from the Bioinformatics REsource ATlas for the Healthy lung database of the Molecular Atlas of Lung Development (LungMAP1) program CONCLUSION: We describe a novel ontology-guided approach to segmentation and classification of complex immunofluorescence images of the developing mouse lung. The ontology is used to automatically generate constraints for each image based on its biomedical context, which facilitates image segmentation and classification.

Cell- and developmental stage-specific Dicer1 ablation in the lung epithelium models cystic pleuropulmonary blastoma.

Inherited syndromes provide unique opportunities to identify key regulatory mechanisms governing human disease. We previously identified germline loss-of-function DICER1 mutations in a human syndrome defined by the childhood lung neoplasm pleuropulmonary blastoma (PPB), which arises during lung development. DICER1 regulates many biological processes critical in development and disease pathogenesis. Significant challenges in defining the role of DICER1 in human disease are identifying cause-effect relationships and generating manipulatable systems that model the complexity of organ development and disease pathogenesis. Here we report the generation of a murine model for PPB and demonstrate that precise temporal and cell type-specific Dicer1 ablation is necessary and sufficient for the development of cystic lungs that histologically and phenotypically model PPB. Dicer1 ablation in the distal airway epithelium during early stages of lung development resulted in a cystic lung phenotype indistinguishable from PPB, whereas DICER1 function was not required for development of the proximal airway epithelium or during later stages of organogenesis. Mechanistic studies demonstrate that Dicer1 loss results in epithelial cell death, followed by cystic airway dilatation accompanied by epithelial and mesenchymal proliferation. These studies define precise temporal and epithelial cell type-specific DICER1 functions in the developing lung and demonstrate that loss of these DICER1 functions is sufficient for the development of cystic PPB. These results also provide evidence that PPB arise through a novel mechanism of non-cell-autonomous tumour initiation, in which the genetic abnormality initiating the neoplasm does not occur in the cells that ultimately transform, but rather occurs in a benign-appearing epithelial cell component that predisposes underlying mesenchymal cells to malignant transformation.

A Molecular atlas of Xenopus respiratory system development.

BACKGROUND: Respiratory system development is regulated by a complex series of endoderm-mesoderm interactions that are not fully understood. Recently Xenopus has emerged as an alternative model to investigate early respiratory system development, but the extent to which the morphogenesis and molecular pathways involved are conserved between Xenopus and mammals has not been systematically documented. RESULTS: In this study, we provide a histological and molecular atlas of Xenopus respiratory system development, focusing on Nkx2.1+ respiratory cell fate specification in the developing foregut. We document the expression patterns of Wnt/ß-catenin, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling components in the foregut and show that the molecular mechanisms of respiratory lineage induction are remarkably conserved between Xenopus and mice. Finally, using several functional experiments we refine the epistatic relationships among FGF, Wnt, and BMP signaling in early Xenopus respiratory system development. CONCLUSIONS: We demonstrate that Xenopus trachea and lung development, before metamorphosis, is comparable at the cellular and molecular levels to embryonic stages of mouse respiratory system development between embryonic days 8.5 and 10.5. This molecular atlas provides a fundamental starting point for further studies using Xenopus as a model to define the conserved genetic programs controlling early respiratory system development.

Sox17 is required for normal pulmonary vascular morphogenesis.

The SRY-box containing transcription factor Sox17 is required for endoderm formation and vascular morphogenesis during embryonic development. In the lung, Sox17 is expressed in mesenchymal progenitors of the embryonic pulmonary vasculature and is restricted to vascular endothelial cells in the mature lung. Conditional deletion of Sox17 in splanchnic mesenchyme-derivatives using Dermo1-Cre resulted in substantial loss of Sox17 from developing pulmonary vascular endothelial cells and caused pulmonary vascular abnormalities before birth, including pulmonary vein varices, enlarged arteries, and decreased perfusion of the microvasculature. While survival of Dermo1-Cre;Sox17Δ/Δ mice (herein termed Sox17Δ/Δ) was unaffected at E18.5, most Sox17Δ/Δ mice died by 3 weeks of age. After birth, the density of the pulmonary microvasculature was decreased in association with alveolar simplification, biventricular cardiac hypertrophy, and valvular regurgitation. The severity of the postnatal cardiac phenotype was correlated with the severity of pulmonary vasculature abnormalities. Sox17 is required for normal formation of the pulmonary vasculature and postnatal cardiovascular homeostasis.

Genotype-phenotype correlations for infants and children with ABCA3 deficiency.

RATIONALE: Recessive mutations in the ATP-binding cassette transporter A3 (ABCA3) cause lethal neonatal respiratory failure and childhood interstitial lung disease. Most ABCA3 mutations are private. OBJECTIVES: To determine genotype-phenotype correlations for recessive ABCA3 mutations. METHODS: We reviewed all published and unpublished ABCA3 sequence and phenotype data from our prospective genetic studies of symptomatic infants and children at Washington and Johns Hopkins Universities. Mutations were classified based on their predicted disruption of protein function: frameshift and nonsense mutations were classified as "null," whereas missense, predicted splice site mutations, and insertion/deletions were classified as "other." We compared age of presentation and outcomes for the three genotypes: null/null, null/other, and other/other. MEASUREMENTS AND MAIN RESULTS: We identified 185 infants and children with homozygous or compound heterozygous ABCA3 mutations and lung disease. All of the null/null infants presented with respiratory failure at birth compared with 75% of infants with null/other or other/other genotypes (P = 0.00011). By 1 year of age, all of the null/null infants had died or undergone lung transplantation compared with 62% of the null/other and other/other children (P < 0.0001). CONCLUSIONS: Genotype-phenotype correlations exist for homozygous or compound heterozygous mutations in ABCA3. Frameshift or nonsense ABCA3 mutations are predictive of neonatal presentation and poor outcome, whereas missense, splice site, and insertion/deletions are less reliably associated with age of presentation and prognosis. Counseling and clinical decision making should acknowledge these correlations.

Kruppel-like factor 5 controls villus formation and initiation of cytodifferentiation in the embryonic intestinal epithelium.

Kruppel-like factor 5 (Klf5) is a transcription factor expressed by embryonic endodermal progenitors that form the lining of the gastrointestinal tract. A Klf5 floxed allele was efficiently deleted from the intestinal epithelium by a Cre transgene under control of the Shh promoter resulting in the inhibition of villus morphogenesis and epithelial differentiation. Although proliferation of the intestinal epithelium was maintained, the expression of Elf3, Pparγ, Atoh1, Ascl2, Neurog3, Hnf4α, Cdx1, and other genes associated with epithelial cell differentiation was inhibited in the Klf5-deficient intestines. At E18.5, Klf5(Δ/Δ) fetuses lacked the apical brush border characteristic of enterocytes, and a loss of goblet and enteroendocrine cells was observed. The failure to form villi was not attributable to the absence of HH or PDGF signaling, known mediators of this developmental process. Klf5-deletion blocked the decrease in FoxA1 and Sox9 expression that accompanies normal villus morphogenesis. KLF5 directly inhibited activity of the FoxA1 promoter, and in turn FOXA1 inhibited Elf3 gene expression in vitro, linking the observed loss of Elf3 with the persistent expression of FoxA1 observed in Klf5-deficient mice. Genetic network analysis identified KLF5 as a key transcription factor regulating intestinal cell differentiation and cell adhesion. These studies indicate a novel requirement for KLF5 to initiate morphogenesis of the early endoderm into a compartmentalized intestinal epithelium comprised of villi and terminally differentiated cells.

CDC42 is required for structural patterning of the lung during development.

The formation of highly branched epithelial structures is critical for the development of many essential organs, including lung, liver, pancreas, kidney and mammary glands. Elongation and branching of these structures require precise control of complex morphogenetic processes that are dependent upon coordinate regulation of cell shape, apical-basal polarity, proliferation, migration, and interactions among multiple cell types. Herein, we demonstrate that temporal-spatial regulation of epithelial cell polarity by the small GTPase, CDC42, is essential for branching morphogenesis of the developing lung. Epithelial cell-specific deletion of CDC42 in fetal mice disrupted epithelial cell polarity, the actin cytoskeleton, intercellular contacts, directional trafficking of proteins, proliferation and mitotic spindle orientation, impairing the organization and patterning of the developing respiratory epithelium and adjacent mesenchyme. Transition from a pseudostratified to a simple columnar epithelium was impaired, consistent with coordinate dysregulation of epithelial cell polarity, mitotic spindle orientation, and repositioning of mitotic cells within the epithelium during cell cycle progression. Expression of sonic hedgehog and its receptor, patched-1, was decreased, while fibroblast growth factor 10 expression in the mesenchyme was expanded, resulting in disruption of branching morphogenesis and bronchiolar smooth muscle formation in this model. CDC42 is required for spatial positioning of proliferating epithelial cells, as well as signaling interactions between the epithelium and mesenchyme and is, therefore, essential for formation and maintenance of the respiratory tract during morphogenesis of the fetal lung.

Foxm1 transcription factor is critical for proliferation and differentiation of Clara cells during development of conducting airways.

Respiratory epithelial cells are derived from cell progenitors in the foregut endoderm that subsequently differentiate into the distinct cell types lining the conducting and alveolar regions of the lung. To identify transcriptional mechanisms regulating differentiation and maintenance of respiratory epithelial cells, we conditionally deleted Foxm1 transcription factor from the conducting airways of the developing mouse lung. Conditional deletion of Foxm1 from Clara cells, controlled by the Scgb1a1 promoter, dramatically altered airway structure and caused peribronchial fibrosis, resulting in airway hyperreactivity in adult mice. Deletion of Foxm1 inhibited proliferation of Clara cells and disrupted the normal patterning of epithelial cell differentiation in the bronchioles, causing squamous and goblet cell metaplasia, and the loss of Clara and ciliated cells. Surprisingly, conducting airways of Foxm1-deficient mice contained highly differentiated cuboidal type II epithelial cells that are normally restricted to the alveoli. Lineage tracing studies showed that the ectopic alveolar type II cells in Foxm1-deficient airways were derived from Clara cells. Deletion of Foxm1 inhibited Sox2 and Scgb1a1, both of which are critical for differentiation and function of Clara cells. In co-transfection experiments, Foxm1 directly bound to and induced transcriptional activity of Scgb1a1 and Sox2 promoters. Foxm1 is required for differentiation and maintenance of epithelial cells lining conducting airways.

Activation of sterol-response element-binding proteins (SREBP) in alveolar type II cells enhances lipogenesis causing pulmonary lipotoxicity.

Pulmonary inflammation is associated with altered lipid synthesis and clearance related to diabetes, obesity, and various inherited metabolic disorders. In many tissues, lipogenesis is regulated at the transcriptional level by the activity of sterol-response element-binding proteins (SREBP). The role of SREBP activation in the regulation of lipid metabolism in the lung was assessed in mice in which both Insig1 and Insig2 genes, encoding proteins that bind and inhibit SREBPs in the endoplasmic reticulum, were deleted in alveolar type 2 cells. Although deletion of either Insig1 or Insig2 did not alter SREBP activity or lipid homeostasis, deletion of both genes (Insig1/2(Δ/Δ) mice) activated SREBP1, causing marked accumulation of lipids that consisted primarily of cholesterol esters and triglycerides in type 2 epithelial cells and alveolar macrophages. Neutral lipids accumulated in type 2 cells in association with the increase in mRNAs regulating fatty acid, cholesterol synthesis, and inflammation. Although bronchoalveolar lavage fluid phosphatidylcholine was modestly decreased, lung phospholipid content and lung function were maintained. Insig1/2(Δ/Δ) mice developed lung inflammation and airspace abnormalities associated with the accumulation of lipids in alveolar type 2 cells, alveolar macrophages, and within alveolar spaces. Deletion of Insig1/2 activated SREBP-enhancing lipogenesis in respiratory epithelial cells resulting in lipotoxicity-related lung inflammation and tissue remodeling.

Genome-wide transcriptional profiling reveals connective tissue mast cell accumulation in bronchopulmonary dysplasia.

RATIONALE: Bronchopulmonary dysplasia (BPD) is a major complication of premature birth. Risk factors for BPD are complex and include prenatal infection and O(2) toxicity. BPD pathology is equally complex and characterized by inflammation and dysmorphic airspaces and vasculature. Due to the limited availability of clinical samples, an understanding of the molecular pathogenesis of this disease and its causal mechanisms and associated biomarkers is limited. OBJECTIVES: Apply genome-wide expression profiling to define pathways affected in BPD lungs. METHODS: Lung tissue was obtained at autopsy from 11 BPD cases and 17 age-matched control subjects without BPD. RNA isolated from these tissue samples was interrogated using microarrays. Standard gene selection and pathway analysis methods were applied to the data set. Abnormal expression patterns were validated by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: We identified 159 genes differentially expressed in BPD tissues. Pathway analysis indicated previously appreciated (e.g., DNA damage regulation of cell cycle) as well as novel (e.g., B-cell development) biological functions were affected. Three of the five most highly induced genes were mast cell (MC)-specific markers. We confirmed an increased accumulation of connective tissue MC(TC) (chymase expressing) mast cells in BPD tissues. Increased expression of MC(TC) markers was also demonstrated in an animal model of BPD-like pathology. CONCLUSIONS: We present a unique genome-wide expression data set from human BPD lung tissue. Our data provide information on gene expression patterns associated with BPD and facilitated the discovery that MC(TC) accumulation is a prominent feature of this disease. These observations have significant clinical and mechanistic implications.

Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease.

The alveolar region of the lung creates an extensive epithelial surface that mediates the transfer of oxygen and carbon dioxide required for respiration after birth. Maintenance of pulmonary function depends on the function of type II epithelial cells that synthesize and secrete pulmonary surfactant lipids and proteins, reducing the collapsing forces created at the air-liquid interface in the alveoli. Genetic and acquired disorders associated with the surfactant system cause both acute and chronic lung disease. Mutations in the ABCA3, SFTPA, SFTPB, SFTPC, SCL34A2, and TERT genes disrupt type II cell function and/or surfactant homeostasis, causing neonatal respiratory failure and chronic interstitial lung disease. Defects in GM-CSF receptor function disrupt surfactant clearance, causing pulmonary alveolar proteinosis. Abnormalities in the surfactant system and disruption of type II cell homeostasis underlie the pathogenesis of pulmonary disorders previously considered idiopathic, providing the basis for improved diagnosis and therapies of these rare lung diseases.

A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma.

BACKGROUND: Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined. OBJECTIVE: To evaluate the role of Serpin3a, the murine homolog of SERPINB3 and SERPINB4, in asthma. METHODS: We studied wild-type Balb/c and Serpinb3a-null mice in house dust mite or IL-13-induced asthma models and evaluated airway hyperresponsiveness, inflammation, and goblet cell hyperplasia. RESULTS: Airway hyperresponsiveness and goblet cell hyperplasia were markedly attenuated in the Serpinb3a-null mice compared with the wild-type mice after allergen challenge, with minimal effects on inflammation. Expression of sterile alpha motif pointed domain containing v-ets avian erythroblastosis virus E26 oncogene homolog transcription factor (SPDEF), a transcription factor that mediates goblet cell hyperplasia, was decreased in the absence of Serpinb3a. IL-13-treated Serpinb3a-null mice showed attenuated airway hyperresponsiveness, inflammation, and mucus production. CONCLUSION: Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production are not well understood. Our data reveal a novel nonredundant role for Serpinb3a in mediating mucus production through regulation of SPDEF expression. This pathway may be used to target mucus hypersecretion effectively.

Sox2 activates cell proliferation and differentiation in the respiratory epithelium.

Sox2, a transcription factor critical for the maintenance of embryonic stem cells and induction of pluripotent stem cells, is expressed exclusively in the conducting airway epithelium of the lung, where it is required for differentiation of nonciliated, goblet, and ciliated cells. To determine the role of Sox2 in respiratory epithelial cells, Sox2 was selectively and conditionally expressed in nonciliated airway epithelial cells and in alveolar type II cells in the adult mouse. Sox2 induced epithelial cell proliferation within 3 days of expression. Epithelial cell proliferation was associated with increased Ki-67 and cyclin D1 staining. Expression of cell cycle genes, including FoxM1, Ccna2 (Cyclin A2), Ccnb2 (Cyclin B2), and Ccnd1 (Cyclin D1), was increased. Consistent with a role in cell proliferation, Sox2 activated the transcription of FoxM1 in vitro. In alveoli, Sox2 caused hyperplasia and ectopic differentiation of epithelial cells to those with morphologic and molecular characteristics of conducting airway epithelium. Sox2 induced the expression of conducting airway epithelial specific genes, including Scgb1a1, Foxj1, Tubb3, and Cyp2f2. Although prolonged expression of Sox2 caused cell proliferation and epithelial hyperplasia, Sox2 did not induce pulmonary tumors. Sox2 induces proliferation of respiratory epithelial cells and, subsequently, partially reprograms alveolar epithelial cells into cells with characteristics of the conducting airways.

Ectopic respiratory epithelial cell differentiation in bronchiolised distal airspaces in idiopathic pulmonary fibrosis.

BACKGROUND: Bronchiolisation of distal airspaces is an unexplained feature of idiopathic pulmonary fibrosis (IPF). The authors sought to identify mechanisms driving the differentiation of mucus cells during the bronchiolisation process. METHODS: Pathways governing airway mucus cell differentiation include SRY (sex determining region Y)-box 2 (SOX2), Notch, forkhead box A3(FOXA3)/SAM pointed domain containing ETS transcription factor (SPDEF), epidermal growth factor (EGF) and the EGF-related neuregulins NRG1α and NRG1ß. Immunostaining for components of those pathways and mucins were performed on lung tissue obtained from patients with IPF (n=20), chronic obstructive pulmonary disease (n=13), idiopathic pulmonary artery hypertension (n=5) and from organ donors (n=6). NRG1α and NRG1ß were quantified in bronchoalveolar lavage fluid (BALF) of patients with early IPF (n=20), controls (n=9), and patients with other interstitial pneumonias (n=13). RESULTS: In IPF, the bronchiolised and enlarged distal airspaces stained for SOX2 are consistent with epithelial differentiation characteristic of conducting airway epithelium. IPF mucus cells expressed MUC5B but low levels of MUC5AC and MUC2, a profile typical of submucosal glands. Singularly, SPDEF, a transcription factor associated with mucus metaplasia, was rarely detected in mucus cells in IPF. The Notch target, HES1, was present in mucus cells from all groups. NRG1α was detected in serous cells within normal submucosal glands and in epithelial cells lining honeycombing areas in IPF, and was not detected in other patients. NRG1α concentrations were elevated in BALF from patients with early IPF. CONCLUSION: Expression of SOX2 and MUC5B and lack of SPDEF in atypically differentiated cells of bronchiolised distal airspaces are consistent with abnormal programming of airway epithelial cells in IPF. NRG1α may contribute to bronchiolisation of the distal lung seen in IPF.

Pulmonary pathology in thyroid transcription factor-1 deficiency syndrome.

Thyroid transcription factor-1 (TTF-1) deficiency syndrome is characterized by neurologic, thyroidal, and pulmonary dysfunction. Children usually have mild-to-severe respiratory symptoms and occasionally die of respiratory failure. Herein, we describe an infant with a constitutional 14q12-21.3 haploid deletion encompassing the TTF-1 gene locus who had cerebral dysgenesis, thyroidal dysfunction, and respiratory insufficiency. The clinical course was notable for mild hyaline membrane disease, continuous ventilatory support, and symmetrically distributed pulmonary cysts by imaging. He developed pneumonia and respiratory failure and died at 8 months. Pathologically, the lungs had grossly visible emphysematous changes with "cysts" up to 2 mm in diameter. The airway generations and radial alveolar count were diminished. In addition to acute bacterial pneumonia, there was focally alveolar septal fibrosis, pneumocyte hypertrophy, and clusters of airspace macrophages. Ultrastructurally, type II pneumocytes had numerous lamellar bodies, and alveolar spaces contained fragments of type II pneumocytes and extruded lamellar bodies. Although immunoreactivity for surfactant protein SP-A and ABCA3 was diminished, that for SP-B and proSP-C was robust, although irregularly distributed, corresponding to the distribution of type II pneumocytes. Immunoreactivity for TTF-1 protein was readily detected. In summation, we document abnormal airway and alveolar morphogenesis and altered expression of surfactant-associated proteins, which may explain the respiratory difficulties encountered in TTF-1 haploinsufficiency. These findings are consistent with experimental evidence documenting the important role of TTF-1 in pulmonary morphogenesis and surfactant metabolism.

Patient-derived granulocyte/macrophage colony-stimulating factor autoantibodies reproduce pulmonary alveolar proteinosis in nonhuman primates.

RATIONALE: Granulocyte/macrophage colony-stimulating factor (GM-CSF) autoantibodies (GMAb) are strongly associated with idiopathic pulmonary alveolar proteinosis (PAP) and are believed to be important in its pathogenesis. However, levels of GMAb do not correlate with disease severity and GMAb are also present at low levels in healthy individuals. OBJECTIVES: Our primary objective was to determine whether human GMAb would reproduce PAP in healthy primates. A secondary objective was to determine the concentration of GMAb resulting in loss of GM-CSF signaling in vivo (i.e., critical threshold). METHODS: Nonhuman primates (Macaca fascicularis) were injected with highly purified, PAP patient-derived GMAb in dose-ranging (2.2-50 mg) single and multiple administration studies, and after blocking antihuman immunoglobulin immune responses, in chronic administration studies maintaining serum levels greater than 40 microg/ml for up to 11 months. MEASUREMENTS AND MAIN RESULTS: GMAb blocked GM-CSF signaling causing (1) a milky-appearing bronchoalveolar lavage fluid containing increased surfactant lipids and proteins; (2) enlarged, foamy, surfactant-filled alveolar macrophages with reduced PU.1 and PPARgamma mRNA, and reduced tumor necrosis factor-alpha secretion; (3) pulmonary leukocytosis; (4) increased serum surfactant protein-D; and (5) impaired neutrophil functions. GM-CSF signaling varied inversely with GMAb concentration below a critical threshold of 5 microg/ml, which was similar in lungs and blood and to the value observed in patients with PAP. CONCLUSIONS: GMAb reproduced the molecular, cellular, and histopathologic features of PAP in healthy primates, demonstrating that GMAb directly cause PAP. These results have implications for therapy of PAP and help define the therapeutic window for potential use of GMAb to treat other disorders.

Hereditary pulmonary alveolar proteinosis: pathogenesis, presentation, diagnosis, and therapy.

RATIONALE: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor function, and increased GM-CSF. OBJECTIVES: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction. METHODS: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls. MEASUREMENT AND MAIN RESULTS: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor ß chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy. CONCLUSIONS: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.

Forkhead Box m1 transcription factor is required for perinatal lung function.

The Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is an important positive regulator of DNA replication and mitosis in a variety of cell types. Global deletion of Foxm1 in Foxm1(-/-) mice is lethal in the embryonic period, causing multiple abnormalities in the liver, heart, lung, and blood vessels. In the present study, Foxm1 was deleted conditionally in the respiratory epithelium (epFoxm1(-/-)). Surprisingly, deletion of Foxm1 did not alter lung growth, branching morphogenesis, or epithelial proliferation but inhibited lung maturation and caused respiratory failure after birth. Maturation defects in epFoxm1(-/-) lungs were associated with decreased expression of T1-alpha and aquaporin 5, consistent with a delay of type I cell differentiation. Expression of surfactant-associated proteins A, B, C, and D was decreased by deletion of Foxm1. Foxm1 directly bound and induced transcriptional activity of the mouse surfactant protein B and A (Sftpb and Sftpa) promoters in vitro, indicating that Foxm1 is a direct transcriptional activator of these genes. Foxm1 is critical for surfactant homeostasis and lung maturation before birth and is required for adaptation to air breathing.

Forkhead box M1 transcriptional factor is required for smooth muscle cells during embryonic development of blood vessels and esophagus.

The forkhead box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is expressed in a variety of tissues during embryogenesis, including vascular, airway, and intestinal smooth muscle cells (SMCs). Although global deletion of Foxm1 in Foxm1(-/-) mice is lethal in the embryonic period due to multiple abnormalities in the liver, heart, and lung, the specific role of Foxm1 in SMC remains unknown. In the present study, Foxm1 was deleted conditionally in the developing SMC (smFoxm1(-/-) mice). The majority of smFoxm1(-/-) mice died immediately after birth due to severe pulmonary hemorrhage and structural defects in arterial wall and esophagus. Although Foxm1 deletion did not influence SMC differentiation, decreased proliferation of SMC was found in smFoxm1(-/-) blood vessels and esophagus. Depletion of Foxm1 in cultured SMC caused G(2) arrest and decreased numbers of cells undergoing mitosis. Foxm1-deficiency in vitro and in vivo was associated with reduced expression of cell cycle regulatory genes, including cyclin B1, Cdk1-activator Cdc25b phosphatase, Polo-like 1 and JNK1 kinases, and cMyc transcription factor. Foxm1 is critical for proliferation of smooth muscle cells and is required for proper embryonic development of blood vessels and esophagus.

Conditional deletion of Abca3 in alveolar type II cells alters surfactant homeostasis in newborn and adult mice.

ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3(Δ/Δ)) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3(Δ/Δ) mice survived after birth. Surviving Abca3(Δ/Δ) mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ∼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3(Δ/Δ) mice. In adult Abca3(Δ/Δ) mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells.