FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2057700884> ?p ?o ?g. }

• W2057700884 endingPage "582" @default.
• W2057700884 startingPage "571" @default.
• W2057700884 abstract "Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure. Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure. Neonatal respiratory distress caused by immaturity of the fetal lung continues to be an important clinical problem. The molecular mechanism of lung development and its relationship to neonatal pulmonary diseases remains incompletely understood. Disruption of normal lung developmental processes can result in either neonatal respiratory failure if lung formation is severely affected, or susceptibility to lung diseases during later life if mild changes occur in the developing lung.1Warburton D Gauldie J Bellusci S Shi W Lung development and susceptibility to chronic obstructive pulmonary disease.Proc Am Thorac Soc. 2006; 3: 668-672Crossref PubMed Scopus (47) Google ScholarLung development is initiated by the formation of a pair of primary epithelial buds that evaginate from the laryngo-tracheal groove of endoderm into the surrounding splanchnic mesenchyme.2Ten Have-Opbroek AA Lung development in the mouse embryo.Exp Lung Res. 1991; 17: 111-130Crossref PubMed Scopus (219) Google Scholar, 3Hilfer SR Morphogenesis of the lung: control of embryonic and fetal branching.Annu Rev Physiol. 1996; 58: 93-113Crossref PubMed Scopus (73) Google Scholar The respiratory tree then develops by branching morphogenesis, in which reiterated outgrowth, elongation, and subdivision of epithelial buds occurs,4Hogan BL Morphogenesis.Cell. 1999; 96: 225-233Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 5Hogan BL Grindley J Bellusci S Dunn NR Emoto H Itoh N Branching morphogenesis of the lung: new models for a classical problem.Cold Spring Harb Symp Quant Biol. 1997; 62: 249-256Crossref PubMed Google Scholar followed later on by alveolarization to form a large gas-exchange surface. Because the lung developmental process is quite well conserved, mouse lung development is an ideal model for studying the mechanism of lung organogenesis and congenital respiratory diseases in man. In the mouse, lung development begins at embryonic day (E) 9.5, and is divided into pseudoglandular stage (E9.5 to E16.5), canalicular stage (E16.6 to E17.4), saccular stage (E17.5 to postnatal day 5 or P5), and alveolar stage (P5 to P30).2Ten Have-Opbroek AA Lung development in the mouse embryo.Exp Lung Res. 1991; 17: 111-130Crossref PubMed Scopus (219) Google Scholar Although the proximal-distal axis as seen by specific gene expression is already evident in the E10.5 mouse lung,6Wert SE Glasser SW Korfhagen TR Whitsett JA Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.Dev Biol. 1993; 156: 426-443Crossref PubMed Scopus (272) Google Scholar, 7Bellusci S Henderson R Winnier G Oikawa T Hogan BL Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis.Development. 1996; 122: 1693-1702Crossref PubMed Google Scholar functional distal lung epithelial cells are induced at late gestation stage (E17.5) with characteristic morphological changes from early columnar cells to late flat cuboidal and squamous cells, whereas the terminal differentiation of functional type I and type II alveolar epithelial cells (AECI and AECII) only occurs after birth in mice.8Warburton D Schwarz M Tefft D Flores-Delgado G Anderson KD Cardoso WV The molecular basis of lung morphogenesis.Mech Dev. 2000; 92: 55-81Crossref PubMed Scopus (621) Google Scholar Lung development is regulated by many growth factors, including bone morphogenetic proteins (BMPs).4Hogan BL Morphogenesis.Cell. 1999; 96: 225-233Abstract Full Text Full Text PDF PubMed Scopus (468) Google ScholarBMPs, with more than 20 family members, have been shown to regulate many fundamental biological processes including cell proliferation, differentiation, apoptosis, migration, and adhesion.9Sporn MB Roberts AB The transforming growth factor βs.in: Sporn MB Roberts AB Peptide Growth Factors and Their Receptor: Handbook of Experimental Pharmacology. Springer-Verlag, Heidelberg1991: 419-472Crossref Google Scholar Furthermore, they are involved in the development of almost all tissues and organs, as well as the specification of the basic embryonic body plan, such as dorso-ventral patterning, left-right asymmetric axis, and proximal-distal axis formation.10Hogan BL Bone morphogenetic proteins: multifunctional regulators of vertebrate development.Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1713) Google Scholar As extracellular growth factors, BMPs bind to heteromeric complexes of BMP serine/threonine kinase type I and type II receptors.11Shi Y Massague J Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4739) Google Scholar, 12Massagu é J TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar Upon ligand-induced aggregation of the receptors, constitutively activated BMP type II receptor kinase phosphorylates and activates the type I receptor, which subsequently recognizes and phosphorylates receptor-bound BMP-specific Smad proteins (Smad1, Smad5, and Smad8) on the carboxyl terminal SSXS motif. These Smads dissociate from the receptors, form complexes with a common partner Smad4, translocate into the nucleus, bind to BMP responsive element, and act as transcriptional co-modulators to induce or repress BMP target gene expression.13Attisano L Wrana JL Smads as transcriptional co-modulators.Curr Opin Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar The specificity of the biological response to BMP ligand is maintained by the utilization of specific type I receptors and Smad proteins. Three cognate BMP type I receptors (Alk2, Alk3, and Alk6) have been identified. Alk3, also called BMP receptor type IA (BMPR-IA), plays an essential role during early embryonic development, particularly in mesoderm formation and gastrulation. The conventional Alk3 gene null mutation is early embryonic lethal in mice (E7.5 to 9.5) before lung organogenesis.14Mishina Y Suzuki A Ueno N Behringer RR Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis.Genes Dev. 1995; 9: 3027-3037Crossref PubMed Scopus (636) Google ScholarBMP4 is an important BMP member that plays a key role in normal lung development.15Hogan BL Bone morphogenetic proteins in development.Curr Opin Genet Dev. 1996; 6: 432-438Crossref PubMed Scopus (659) Google Scholar Addition of exogenous BMP4 to intact embryonic lung explant culture stimulates lung branching, as reported by us and other groups.16Shi W Zhao J Anderson KD Warburton D Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis.Am J Physiol. 2001; 280: L1030-L1039Google Scholar, 17Bragg AD Moses HL Serra R Signaling to the epithelium is not sufficient to mediate all of the effects of transforming growth factor beta and bone morphogenetic protein 4 on murine embryonic lung development.Mech Dev. 2001; 109: 13-26Crossref PubMed Scopus (70) Google Scholar However, in isolated E11.5 mouse lung endoderm cultured in Matrigel, addition of BMP4 inhibited epithelial growth induced by the morphogen FGF10.18Weaver M Dunn NR Hogan BL Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.Development. 2000; 127: 2695-2704Crossref PubMed Google Scholar On the other hand, overexpression of BMP4 in the distal endoderm of fetal mouse lung, driven by a 3.7-kb human surfactant protein C (SP-C) promoter, causes abnormal lung morphogenesis with cystic terminal sacs.7Bellusci S Henderson R Winnier G Oikawa T Hogan BL Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis.Development. 1996; 122: 1693-1702Crossref PubMed Google Scholar In contrast, SP-C promoter-driven overexpression of either the BMP antagonist Xnoggin or Gremlin to block BMP signaling, results in severely reduced distal epithelial cell phenotypes and increased proximal cell phenotypes in the lungs of transgenic mice.19Weaver M Yingling JM Dunn NR Bellusci S Hogan BL Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development.Development. 1999; 126: 4005-4015Crossref PubMed Google Scholar, 20Lu MM Yang H Zhang L Shu W Blair DG Morrisey EE The bone morphogenic protein antagonist gremlin regulates proximal-distal patterning of the lung.Dev Dyn. 2001; 222: 667-680Crossref PubMed Scopus (85) Google Scholar Although these studies suggest that BMP4 signaling is essential for normal lung morphogenesis, the data obtained from in vitro and transgenic animal studies are confusing. In particular, the specific physiological functions in lung epithelia during different developmental stages have not been determined. Herein, we have used an inducible lung epithelial Alk3 conditional knockout mouse model to dissect Alk3-mediated BMP signaling in promoting lung development and its role in preventing neonatal respiratory diseases.Materials and MethodsMouse Strains and BreedingAlk3 heterozygous null mutant (Alk3+/−) and floxed Alk3 (Alk3fx/fx) mice were generated in Dr. Yuji Mishina's laboratory.21Mishina Y Hanks MC Miura S Tallquist MD Behringer RR Generation of Bmpr/Alk3 conditional knockout mice.Genesis. 2002; 32: 69-72Crossref PubMed Scopus (215) Google Scholar In Alk3fx/fx, the exon 2 of Alk3 gene was flanked with two loxP DNA elements. Deletion of exon 2 will cause frameshift and eliminate functional Alk3 protein expression. Inducible lung epithelial cell-specific Cre transgenic mice (SPC-rtTA/TetO-Cre) were generated in Dr. Jeffrey Whitsett's laboratory.22Perl AK Wert SE Nagy A Lobe CG Whitsett JA Early restriction of peripheral and proximal cell lineages during formation of the lung.Proc Natl Acad Sci USA. 2002; 99: 10482-10487Crossref PubMed Scopus (403) Google Scholar Timed mating between Alk3fx/fx and Alk3+/−/SPC-rtTA/tetO-Cre mice generated lung epithelial-specific Alk3 conditional knockout (CKO) mice (Alk3fx/−/SPC-rtTA/TetO-Cre), heterozygous Alk3 knockout (HT) mice (Alk3fx/−, or Alk3fx/−/SPC-rtTA, or Alk3fx/−/TetO-Cre, or Alk3fx/+/SPC-rtTA/TetO-Cre), and control mice (Alk3fx/+, or Alk3fx/+/SPC-rtTA, or Alk3fx/+/TetO-Cre) when inducing agent doxycycline (Dox) was present. Because lung development in the control mice is the same as in wild-type mice (Alk3+/+), they are all classified as wild-type (WT) group in this study. Administration of Dox started from different gestation stages (E7.5, or E17.5, or P1) to the end point of experiment by feeding the pregnant mice with Dox food (625 mg/kg; TestDiet, Richmond, IN) and drinking water (0.5 mg/ml; Sigma, St. Louis, MO). All mice were bred in C57BL/6 strain background, and genotyped by genomic DNA polymerase chain reaction (PCR). Mice used in this study were housed in pathogen-free conditions according to the protocol approved by Institutional Animal Care and Use Committee at Saban Research Institute of Childrens Hospital Los Angeles.Histology and Morphometric AnalysisEmbryonic lung was fixed with 4% buffered paraformaldehyde at 4°C overnight, dehydrated, and embedded in paraffin. Five-μm sections were stained with hematoxylin and eosin (H&E), as reported previously.23Chen H Sun J Buckley S Chen C Warburton D Wang XF Shi W Abnormal mouse lung alveolarization caused by Smad3 deficiency is a developmental antecedent of centrilobular emphysema.Am J Physiol. 2005; 288: L683-L691Google Scholar Quantification of air sac space in lung tissue was performed by measuring air space area in five different tissue sections of every 100-μm distance using MetaMorph software (Molecular Devices, Sunnyvale, CA). Elastin was stained using Hart's resorcin-fuchsin solution, and counterstained with 0.5% tartrazine.Immunohistochemistry and Immunofluorescence StainingAntibodies used in these studies: Alk3 goat polyclonal antibody (sc-5676), Clara cell-specific protein (CCSP) goat polyclonal antibody (sc-9772), and SP-C goat polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). β-Tubulin IV mouse monoclonal antibody was obtained from BioGenex (San Ramon, CA).Alk3 immunofluorescence staining was performed on paraffin sections from 4% paraformaldehyde-fixed lung tissue. After deparaffinizing and rehydration, sections were blocked in 2.5% bovine serum for 1 hour at room temperature, followed by incubation with primary antibody for 1 hour, and detected using Alexa Fluor 488-labeled donkey anti-goat IgG (Invitrogen, Carlsbad, CA). Immunohistochemical staining of SP-C, CCSP, and β-tubulin IV were performed using a HistoStain kit from Zymed Laboratories (South San Francisco, CA) according to the manufacturer's instructions. Either 3-amino-9-ethylcarbazole or 3,3′-diaminobenzidine was used as chromogenic substrate.Cell Proliferation and ApoptosisCell proliferation was analyzed by proliferating cell nuclear antigen (PCNA) staining using a Zymed PCNA staining kit, and by Ki-67 immunostaining (NeoMarkers, Fremont, CA). Cell apoptosis was evaluated by terminal dUTP nick-end labeling (TUNEL) staining using an ApopTag kit (Millipore, Billerica, MA), as published previously.24Shi W Chen H Sun J Buckley S Zhao J Anderson KD Williams RG Warburton D TACE is required for fetal murine cardiac development and modeling.Dev Biol. 2003; 261: 371-380Crossref PubMed Scopus (95) Google ScholarCellular Structure under Transmission Electron MicroscopyOne-mm-thick lung tissue was fixed in 2% glutaraldehyde/1% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 10 minutes at 37°C, followed by 4 hours at room temperature. Tissue blocks were then postfixed overnight at 4°C in 1.5% osmium tetroxide in veronal acetate buffer (pH 7.4). After rinsing the specimen, the blocks were stained in 1.5% uranyl acetate (pH 5.2) for 1 hour at room temperature. Tissue was then dehydrated in graded acetone, infiltrated with propylene and oxide-Epon mixture, embedded in Epon. Ultra thin sections were then cut and observed under transmission electron microscopy (JEOL-1200EX; JEOL Ltd., Tokyo, Japan).Western BlotDetection of lung proteins has been previously described.25Zhao J Shi W Chen H Warburton D Smad7 and Smad6 differentially modulate transforming growth factor beta-induced inhibition of embryonic lung morphogenesis.J Biol Chem. 2000; 275: 23992-23997Crossref PubMed Scopus (71) Google Scholar Briefly, fresh lung tissues were lysed on ice in RIPA buffer containing 1 mmol/L phenylmethyl sulfonyl fluoride, 0.2 U/ml aprotinin, and 1 mmol/L sodium orthovanadate. Protein concentration was measured by the Bradford method using reagents purchased from Bio-Rad Laboratories (Hercules, CA). Equal amounts (40 μg) of total tissue lysate proteins were separated in NuPAGE 4 to 12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels using a MOP buffering system (Invitrogen). After protein was transferred into polyvinylidene difluoride membrane, proteins of interest were detected by specific antibodies. Antibodies for Smad1, pSmad1, Smad2, and pSmad2 were purchased from Cell Signaling Technology (Danvers, MA). Active β-catenin antibody was purchased from Chemicon (Temecula, CA). pLRP6 antibody was kindly provided by Dr. Xi He at Harvard University, Boston, MA.26He XC Zhang J Tong WG Tawfik O Ross J Scoville DH Tian Q Zeng X He X Wiedemann LM Mishina Y Li L BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling.Nat Genet. 2004; 36: 1117-1121Crossref PubMed Scopus (850) Google Scholar Fibrillin-1, Patched, and Sprouty 2 antibodies were obtained from Santa Cruz Biotechnology.Real-Time PCR Analysis and PrimersTotal tissue RNAs were isolated from snap-frozen lung tissue using a RNeasy kit (Qiagen, Valencia, CA). The quality was checked by an Experion automated electrophoresis system using an Experion RNA HighSens analysis kit (Bio-Rad Laboratories). Synthesis of cDNA and quantitative reverse transcriptase (RT)-PCR analysis were performed using iScript cDNA synthesis kit and SYBR Green I dye on iCycler-iQ system (Bio-Rad), as reported previously.24Shi W Chen H Sun J Buckley S Zhao J Anderson KD Williams RG Warburton D TACE is required for fetal murine cardiac development and modeling.Dev Biol. 2003; 261: 371-380Crossref PubMed Scopus (95) Google Scholar The PCR primers for SP-C, CCSP, and GAPDH were previously published.27Chen C Chen H Sun J Bringas Jr, P Chen Y Warburton D Shi W Smad1 expression and function during mouse embryonic lung branching morphogenesis.Am J Physiol. 2005; 288: L1033-L1039Google Scholar Other PCR primer sequences are: FoxJ1 (5′-CCACCTGGCAGAATTCCAT-3′; 5′-CCTCCGCTTCTTGAAGGC-3′), SP-B (5′-CGCTTCTGGCTAGACAGGC-3′; 5′-GGAGCAGGCTGCTGGAGA-3′), AQP5 (5′-ATCTCTGAGGTCTGAGCTGTGG-3′; 5′-CATGCCGCACACGGGGAT-3′), Wnt inhibitory factor-1 (WIF-1, 5′-CACTGCAATAAGAGGTATGGAGC-3′; 5′-GGGTTCACCAGATGTAATTGGA-3′), respectively. GAPDH was used to normalize equal loading of template cDNA.Data Presentation and Statistical AnalysisAt least three pairs of Alk3 gene CKO and WT littermate control mice from different dams were analyzed in each experimental subgroup. All experiments were repeated three times, and data represent consistent results. All quantitative data were expressed as mean ± SD. A Student's t-test was used for comparison of statistical difference and P values <0.05 were considered as significant.ResultsConditional Abrogation of BMP Type I Receptor Alk3 in Mouse Lung Epithelial Cells during Fetal Lung DevelopmentMouse embryos with the conventional Alk3-null mutation died before lung organogenesis.21Mishina Y Hanks MC Miura S Tallquist MD Behringer RR Generation of Bmpr/Alk3 conditional knockout mice.Genesis. 2002; 32: 69-72Crossref PubMed Scopus (215) Google Scholar Thus, the conventional Alk3 knockout mouse model is not applicable for studying Alk3 function in lung biology, and a lung-specific conditional Alk3 knockout mouse model using a Cre-loxP system is required for this in vivo study. By immunofluorescence staining, endogenous Alk3 protein expression was found to be predominantly localized in fetal mouse lung airway epithelial cells, with high intensity in peripheral airways, at early gestation stage (E12.5; Figure 1A). This Alk3 epithelial expression pattern persists in fetal lungs at different developmental stages (E14.5 and E18.5 in Figure 1, B and C). Thus, lung-specific Alk3 conditional knockout mice were then generated by crossing floxed Alk3 mice with SPC-rtTA/TetO-Cre transgenic mice,22Perl AK Wert SE Nagy A Lobe CG Whitsett JA Early restriction of peripheral and proximal cell lineages during formation of the lung.Proc Natl Acad Sci USA. 2002; 99: 10482-10487Crossref PubMed Scopus (403) Google Scholar, 28Wert SE Dey CR Blair PA Kimura S Whitsett JA Increased expression of thyroid transcription factor-1 (TTF-1) in respiratory epithelial cells inhibits alveolarization and causes pulmonary inflammation.Dev Biol. 2002; 242: 75-87Crossref PubMed Scopus (76) Google Scholar in which Cre expression is induced in airway epithelial cells of lung and bronchus by a lung epithelia specifically expressed rtTA, driven by a 3.7-kb human surfactant protein C promoter (SPC), in combination with the inducing agent Dox.22Perl AK Wert SE Nagy A Lobe CG Whitsett JA Early restriction of peripheral and proximal cell lineages during formation of the lung.Proc Natl Acad Sci USA. 2002; 99: 10482-10487Crossref PubMed Scopus (403) Google Scholar Moreover, Cre-mediated floxed Alk3 gene deletion could be started at different developmental stages by controlling the time point of Dox administration. Abrogation of Alk3 protein expression early in lung organogenesis was achieved by feeding the pregnant mother with Dox food and water from E7.5. As a result of floxed-Alk3 exon 2 deletion, lack of functional Alk3 protein in Alk3 knockout lung was confirmed by immunofluorescence staining using an Alk3 antibody (Figure 1D). Furthermore, the WT version of Alk3 mRNA transcript in Alk3 conditional knockout (CKO) lung tissue was barely detected by a more sensitive RT-PCR using exon 2-specific primers, indicating a high efficiency of gene knockout in lung (Figure 1E).Abrogation of Alk3 Gene Expression in Developing Lung Epithelia Early in Embryonic Mouse Lung Organogenesis Results in Dysplastic Lung Formation and Neonatal Respiratory DistressThe Alk3 CKO mice died within a couple of hours after birth with severe gasping and cyanosis. Both gross view and H&E-stained neonatal lung tissue sections indicated that the terminal air sacs in Alk3 CKO lung failed to inflate with air during postnatal air breathing, accompanied with eosin-stained amorphous materials in terminal sacs (Figure 2, A and B), suggesting atelectasis and failure of postnatal lung fluid reabsorption. Under high magnification, instead of normal squamous alveolar cells lining the peripheral air sac, only columnar cells were observed in the peripheral lung of Alk3 CKO mice (Figure 2B, insets), accompanied with thick and edematous mesenchyme. Lack of lamellar body formation and secretion were further observed in Alk3 knockout P1 lung by transmission electron microscopy (Figure 2C).Figure 2Phenotypes of mouse lung epithelial-specific Alk3 conditional knockout induced from E7.5. A: Gross view of neonatal lung at P1. Inflation with air was clearly seen in WT, but not in Alk3 CKO lungs. B: Collapsed lung structure in P1 Alk3 CKO mice was shown by H&E-stained tissue section. The cell shape of lining epithelial cells inside air sacs was changed from normal squamous/flat cells in WT to round/cuboidal cells in Alk3 CKO (inset). C: Surfactant production and secretion was barely detected in P1 Alk3 CKO lung by transmission electron microscopy, as shown by lack of lamellar bodies both inside cells and in air spaces (arrows). D–F: H&E-stained embryonic lung sections at different developmental stages. D: Less epithelial tubules with dilated lumen were observed in E14.5 lung of Alk3 CKO mice. E: At E16.5, peripheral airway epithelial cells still have columnar shape in Alk3 CKO lung versus cuboidal shape in WT control. F: At late gestation stage E18.5, less saccular formation with thickened mesenchyme was evident in Alk3 CKO lung.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To understand the mechanisms of Alk3-mediated BMP signal regulation during mouse fetal lung development, the lung tissue structure of Alk3 CKO mice induced from E7.5 were then examined at a series of gestation stages. Compared to WT littermates, most of the E12.5 embryonic lung did not have observable changes of epithelial growth and branching morphogenesis. However, a small portion of E12.5 Alk3 knockout lungs (two of six) already displayed reduced size and epithelial branching, suggesting a low penetrance of this early embryonic lung branching morphogenesis phenotype. Significant retardation of lung growth was observed in all E14.5 Alk3 CKO lungs, as shown by reduced numbers of airway epithelial tubules, accompanied with dilated lumen and less compacted mesenchyme (Figure 2D). Then, retardation of distal lung epithelial cell proliferation and differentiation was clearly indicated by retention of a columnar shape in Alk3 CKO mice versus changing to a low cuboidal shape in WT control at the beginning of the canalicular stage (E16.5, Figure 2E). Furthermore, at the end of gestation (E18.5, Figure 2F), peripheral airway sac formation was significantly decreased, and mesenchymal septation between air sacs was relatively thick in Alk3 CKO lung compared to WT littermate control. Morphometric measurement of peripheral air spaces was then performed to quantify the changes of lung structure. The peripheral airspace in E18.5 Alk3 CKO lung was increased by 22% (51 ± 5% in WT versus 62 ± 5% in Alk3 CKO lung, P < 0.001).Conditional Knockout of Alk3 Function in Airway Epithelial Cells Early in Embryonic Lung Organogenesis Resulted in Abnormal Distal Epithelial Cell Proliferation, Apoptosis, and DifferentiationTo determine the mechanism of the above phenotypic changes in Alk3 CKO mouse lung, cell proliferation, apoptosis, and differentiation were then measured. By PCNA immunostaining (Figure 3A), the numbers of PCNA-positive cells continuously decreased in Alk3 CKO mouse lungs from the early to the end of gestation (E14.5 and E18.5). This reduced cell proliferation was also confirmed by Ki-67 immunostaining, a different marker for cell proliferation (Figure 3B). Moreover, cell apoptosis was also evaluated by TUNEL labeling. The number of apoptotic cells was not significantly changed at earlier stages of lung development (E14.5), but increased in Alk3 CKO lung later on at E18.5, particularly in epithelial cells (Figure 3C). Thus, less saccular formation accompanied with enlarged air spaces in E18.5 Alk3 CKO lung could be caused by both reduced cell proliferation and increased cell apoptosis. In addition, expression of selected molecular markers for differentiated lung epithelial cells, including SP-B, SP-C, aquaporin 5, CCSP, and FoxJ1, was evaluated at the mRNA level using quantitative real-time PCR (Figure 4A). Consistently, expression of distal conducting airway and lung epithelial cell differentiation markers CCSP, SP-C, and AQP5 was significantly reduced in Alk3 CKO lung tissues at various developmental stages (E14.5 to E18.5), whereas expression of proximal epithelial cell marker FoxJ1 was not changed. The RNA data were further confirmed at protein level by SP-C, CCSP, and β-tubulin IV immunostaining (Figure 4). At E14.5, the number of SP-C-positive peripheral epithelial cells in Alk3 CKO lung were less than WT control, accompanied by reduced branching morphogenesis. Also, the intensity of SP-C staining signal was significantly reduced in Alk3 CKO lung at early embryonic stage E14.5, when excessive cell apoptosis was not detected (Figure 4B), suggesting retarded peripheral epithelial cell differentiation and lineage expansion in early Alk3 CKO lung. Whereas, at late gestation (E18.5), both CCSP and SP-C were barely detected in Alk3 CKO peripheral lung, whereas the pattern of proximal conducting airway epithelial cell marker β-tubulin IV remained the same compared to WT control (Figure 4, B–D). Therefore, disruption of normal peripheral lung epithelial cell differentiation at early stage and increased apoptosis of differentiated cell lineages at late stage observed in the Alk3 CKO may directly contribute to abnormal lung structure and function.Figure 3Altered cell proliferation and apoptosis of Alk3 CKO lung induced from E7.5. A and B: Cell proliferation was measured by PCNA and Ki-67 immunostaining (brown color) for lungs at E14.5 and E18.5. C: Cell apoptosis was also examined by TUNEL assay (brown color). Only a" @default.
• W2057700884 created "2016-06-24" @default.
• W2057700884 creator A5001813162 @default.
• W2057700884 creator A5011090841 @default.
• W2057700884 creator A5023461520 @default.
• W2057700884 creator A5033704842 @default.
• W2057700884 creator A5042634877 @default.
• W2057700884 creator A5059882947 @default.
• W2057700884 creator A5064548129 @default.
• W2057700884 creator A5079530350 @default.
• W2057700884 creator A5079936447 @default.
• W2057700884 date "2008-03-01" @default.
• W2057700884 modified "2023-10-14" @default.
• W2057700884 title "Prenatal Lung Epithelial Cell-Specific Abrogation of Alk3-Bone Morphogenetic Protein Signaling Causes Neonatal Respiratory Distress by Disrupting Distal Airway Formation" @default.
• W2057700884 cites W1648092828 @default.
• W2057700884 cites W1922697055 @default.
• W2057700884 cites W1930907473 @default.
• W2057700884 cites W1963272169 @default.
• W2057700884 cites W1976213734 @default.
• W2057700884 cites W1991082072 @default.
• W2057700884 cites W2005204227 @default.
• W2057700884 cites W2013575704 @default.
• W2057700884 cites W2020319374 @default.
• W2057700884 cites W2026566753 @default.
• W2057700884 cites W2027416180 @default.
• W2057700884 cites W2032700434 @default.
• W2057700884 cites W2033887719 @default.
• W2057700884 cites W2055110202 @default.
• W2057700884 cites W2067553572 @default.
• W2057700884 cites W2067910888 @default.
• W2057700884 cites W2073771842 @default.
• W2057700884 cites W2074978112 @default.
• W2057700884 cites W2076087769 @default.
• W2057700884 cites W2086331774 @default.
• W2057700884 cites W2088305423 @default.
• W2057700884 cites W2098278242 @default.
• W2057700884 cites W2103031848 @default.
• W2057700884 cites W2126684945 @default.
• W2057700884 cites W2131062818 @default.
• W2057700884 cites W2141317338 @default.
• W2057700884 cites W2142325605 @default.
• W2057700884 cites W2147850863 @default.
• W2057700884 cites W2154294808 @default.
• W2057700884 cites W2154568166 @default.
• W2057700884 cites W2155507705 @default.
• W2057700884 cites W2161558436 @default.
• W2057700884 cites W2165695219 @default.
• W2057700884 cites W2407486189 @default.
• W2057700884 cites W326368786 @default.
• W2057700884 cites W4247817378 @default.
• W2057700884 doi "https://doi.org/10.2353/ajpath.2008.070286" @default.
• W2057700884 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2258256" @default.
• W2057700884 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18258849" @default.
• W2057700884 hasPublicationYear "2008" @default.
• W2057700884 type Work @default.
• W2057700884 sameAs 2057700884 @default.
• W2057700884 citedByCount "46" @default.
• W2057700884 countsByYear W20577008842012 @default.
• W2057700884 countsByYear W20577008842013 @default.
• W2057700884 countsByYear W20577008842014 @default.
• W2057700884 countsByYear W20577008842015 @default.
• W2057700884 countsByYear W20577008842016 @default.
• W2057700884 countsByYear W20577008842017 @default.
• W2057700884 countsByYear W20577008842019 @default.
• W2057700884 countsByYear W20577008842021 @default.
• W2057700884 countsByYear W20577008842022 @default.
• W2057700884 countsByYear W20577008842023 @default.
• W2057700884 crossrefType "journal-article" @default.
• W2057700884 hasAuthorship W2057700884A5001813162 @default.
• W2057700884 hasAuthorship W2057700884A5011090841 @default.
• W2057700884 hasAuthorship W2057700884A5023461520 @default.
• W2057700884 hasAuthorship W2057700884A5033704842 @default.
• W2057700884 hasAuthorship W2057700884A5042634877 @default.
• W2057700884 hasAuthorship W2057700884A5059882947 @default.
• W2057700884 hasAuthorship W2057700884A5064548129 @default.
• W2057700884 hasAuthorship W2057700884A5079530350 @default.
• W2057700884 hasAuthorship W2057700884A5079936447 @default.
• W2057700884 hasBestOaLocation W20577008841 @default.
• W2057700884 hasConcept C104317684 @default.
• W2057700884 hasConcept C105922876 @default.
• W2057700884 hasConcept C126322002 @default.
• W2057700884 hasConcept C141983124 @default.
• W2057700884 hasConcept C142724271 @default.
• W2057700884 hasConcept C172680121 @default.
• W2057700884 hasConcept C203014093 @default.
• W2057700884 hasConcept C2777714996 @default.
• W2057700884 hasConcept C2779234561 @default.
• W2057700884 hasConcept C31507581 @default.
• W2057700884 hasConcept C42219234 @default.
• W2057700884 hasConcept C529295009 @default.
• W2057700884 hasConcept C534529494 @default.
• W2057700884 hasConcept C54355233 @default.
• W2057700884 hasConcept C55493867 @default.
• W2057700884 hasConcept C62478195 @default.
• W2057700884 hasConcept C71924100 @default.
• W2057700884 hasConcept C86803240 @default.
• W2057700884 hasConcept C87568996 @default.
• W2057700884 hasConcept C95444343 @default.

• next