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Published in: Respiratory Research 1/2017

Open Access 01-12-2017 | Research

Selective gene expression analysis of the neuroepithelial body microenvironment in postnatal lungs with special interest for potential stem cell characteristics

Authors: Line Verckist, Robrecht Lembrechts, Sofie Thys, Isabel Pintelon, Jean-Pierre Timmermans, Inge Brouns, Dirk Adriaensen

Published in: Respiratory Research | Issue 1/2017

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Abstract

Background

The pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME.

Methods

A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data.

Results

Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes.

Conclusions

This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.
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Literature
1.
go back to reference Adriaensen D, Scheuermann DW. Neuroendocrine cells and nerves of the lung. Anat Rec. 1993;236:70–86.CrossRefPubMed Adriaensen D, Scheuermann DW. Neuroendocrine cells and nerves of the lung. Anat Rec. 1993;236:70–86.CrossRefPubMed
2.
go back to reference Stahlman MT, Gray ME. Ontogeny of neuroendocrine cells in human-fetal lung 1. An electron-microscopic study. Lab Invest. 1984;51:449–63.PubMed Stahlman MT, Gray ME. Ontogeny of neuroendocrine cells in human-fetal lung 1. An electron-microscopic study. Lab Invest. 1984;51:449–63.PubMed
3.
go back to reference Haller CJ. A scanning and transmission electron-microscopic study of the development of the surface-structure of neuroepithelial bodies in the mouse lung. Micron. 1994;25:527–38.CrossRefPubMed Haller CJ. A scanning and transmission electron-microscopic study of the development of the surface-structure of neuroepithelial bodies in the mouse lung. Micron. 1994;25:527–38.CrossRefPubMed
4.
go back to reference Reynolds SD, Giangreco A, Power JHT, Stripp BR. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol. 2000;156:269–78.CrossRefPubMedPubMedCentral Reynolds SD, Giangreco A, Power JHT, Stripp BR. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol. 2000;156:269–78.CrossRefPubMedPubMedCentral
5.
go back to reference Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest. 2006;86:425–44.CrossRefPubMed Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest. 2006;86:425–44.CrossRefPubMed
6.
go back to reference De Proost I, Pintelon I, Brouns I, Kroese AB, Riccardi D, Kemp PJ, Timmermans JP, Adriaensen D. Functional live cell imaging of the pulmonary neuroepithelial body microenvironment. Am J Respir Cell Mol Biol. 2008;39:180–9.CrossRefPubMedPubMedCentral De Proost I, Pintelon I, Brouns I, Kroese AB, Riccardi D, Kemp PJ, Timmermans JP, Adriaensen D. Functional live cell imaging of the pulmonary neuroepithelial body microenvironment. Am J Respir Cell Mol Biol. 2008;39:180–9.CrossRefPubMedPubMedCentral
7.
go back to reference Brouns I, Pintelon I, Timmermans JP, Adriaensen D. Novel insights in the neurochemistry and function of pulmonary sensory receptors. Adv Anat Embryol Cell Biol. 2012;211:1–115.CrossRefPubMed Brouns I, Pintelon I, Timmermans JP, Adriaensen D. Novel insights in the neurochemistry and function of pulmonary sensory receptors. Adv Anat Embryol Cell Biol. 2012;211:1–115.CrossRefPubMed
8.
go back to reference Schnorbusch K, Lembrechts R, Pintelon I, Timmermans JP, Brouns I, Adriaensen D. GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice. Histochem Cell Biol. 2013;140:549–66.CrossRefPubMed Schnorbusch K, Lembrechts R, Pintelon I, Timmermans JP, Brouns I, Adriaensen D. GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice. Histochem Cell Biol. 2013;140:549–66.CrossRefPubMed
9.
go back to reference Peake JL, Reynolds SD, Stripp BR, Stephens KE, Pinkerton KE. Alteration of pulmonary neuroendocrine cells during epithelial repair of naphthalene-induced airway injury. Am J Pathol. 2000;156:279–86.CrossRefPubMedPubMedCentral Peake JL, Reynolds SD, Stripp BR, Stephens KE, Pinkerton KE. Alteration of pulmonary neuroendocrine cells during epithelial repair of naphthalene-induced airway injury. Am J Pathol. 2000;156:279–86.CrossRefPubMedPubMedCentral
10.
go back to reference Fu XW, Wang DS, Nurse CA, Dinauer MC, Cutz E. NADPH oxidase is an O2 sensor in airway chemoreceptors: Evidence from K+ current modulation in wild-type and oxidase-deficient mice. Proc Natl Acad Sci U S A. 2000;97:4374–9.CrossRefPubMedPubMedCentral Fu XW, Wang DS, Nurse CA, Dinauer MC, Cutz E. NADPH oxidase is an O2 sensor in airway chemoreceptors: Evidence from K+ current modulation in wild-type and oxidase-deficient mice. Proc Natl Acad Sci U S A. 2000;97:4374–9.CrossRefPubMedPubMedCentral
11.
go back to reference Pintelon I, De Proost I, Brouns I, Van Herck H, Van Genechten J, Van Meir F, Timmermans JP, Adriaensen D. Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species. Cell Tissue Res. 2005;321:21–33.CrossRefPubMed Pintelon I, De Proost I, Brouns I, Van Herck H, Van Genechten J, Van Meir F, Timmermans JP, Adriaensen D. Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species. Cell Tissue Res. 2005;321:21–33.CrossRefPubMed
12.
go back to reference De Proost I, Pintelon I, Wilkinson WJ, Goethals S, Brouns I, Van Nassauw L, Riccardi D, Timmermans JP, Kemp PJ, Adriaensen D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging. FASEB J. 2009;23:1153–60.CrossRefPubMed De Proost I, Pintelon I, Wilkinson WJ, Goethals S, Brouns I, Van Nassauw L, Riccardi D, Timmermans JP, Kemp PJ, Adriaensen D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging. FASEB J. 2009;23:1153–60.CrossRefPubMed
13.
go back to reference Schnorbusch K, Lembrechts R, Brouns I, Pintelon I, Timmermans JP, Adriaensen D. Precision-cut vibratome slices allow functional live cell imaging of the pulmonary neuroepithelial body microenvironment in fetal mice. Adv Exp Med Biol. 2012;758:157–66.CrossRefPubMed Schnorbusch K, Lembrechts R, Brouns I, Pintelon I, Timmermans JP, Adriaensen D. Precision-cut vibratome slices allow functional live cell imaging of the pulmonary neuroepithelial body microenvironment in fetal mice. Adv Exp Med Biol. 2012;758:157–66.CrossRefPubMed
14.
go back to reference Lembrechts R, Brouns I, Schnorbusch K, Pintelon I, Kemp PJ, Timmermans JP, Riccardi D, Adriaensen D. Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells. J Cell Sci. 2013;126:4490–501.CrossRefPubMed Lembrechts R, Brouns I, Schnorbusch K, Pintelon I, Kemp PJ, Timmermans JP, Riccardi D, Adriaensen D. Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells. J Cell Sci. 2013;126:4490–501.CrossRefPubMed
15.
go back to reference Cutz E, Fu XW, Yeger H. Methods to study neuroepithelial bodies as airway oxygen sensors. Methods Enzymol. 2004;381:26–40.CrossRefPubMed Cutz E, Fu XW, Yeger H. Methods to study neuroepithelial bodies as airway oxygen sensors. Methods Enzymol. 2004;381:26–40.CrossRefPubMed
16.
go back to reference Cutz E. Introduction to pulmonary neuroendocrine cell system, structure-function correlations. Microsc Res Tech. 1997;37:1–3.CrossRefPubMed Cutz E. Introduction to pulmonary neuroendocrine cell system, structure-function correlations. Microsc Res Tech. 1997;37:1–3.CrossRefPubMed
17.
go back to reference Speirs V, Wang YY, Yeger H, Cutz E. Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques. Am J Resp Cell Mol. 1992;6:63–7.CrossRef Speirs V, Wang YY, Yeger H, Cutz E. Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques. Am J Resp Cell Mol. 1992;6:63–7.CrossRef
18.
go back to reference Carney DN, Gazdar AF, Bepler G, Guccion JG, Marangos PJ, Moody TW, Zweig MH, Minna JD. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 1985;45:2913–23.PubMed Carney DN, Gazdar AF, Bepler G, Guccion JG, Marangos PJ, Moody TW, Zweig MH, Minna JD. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 1985;45:2913–23.PubMed
19.
go back to reference Gazdar AF, Carney DN, Nau MM, Minna JD. Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties. Cancer Res. 1985;45:2924–30.PubMed Gazdar AF, Carney DN, Nau MM, Minna JD. Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties. Cancer Res. 1985;45:2924–30.PubMed
20.
go back to reference O’Kelly I, Peers C, Kemp PJ. O2-sensitive K+ channels in neuroepithelial body-derived small cell carcinoma cells of the human lung. Am J Physiol. 1998;275:709–16. O’Kelly I, Peers C, Kemp PJ. O2-sensitive K+ channels in neuroepithelial body-derived small cell carcinoma cells of the human lung. Am J Physiol. 1998;275:709–16.
21.
go back to reference Brouns I, Oztay F, Pintelon I, De Proost I, Lembrechts R, Timmermans JP, Adriaensen D. Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs. Histochem Cell Biol. 2009;131:55–74.CrossRefPubMed Brouns I, Oztay F, Pintelon I, De Proost I, Lembrechts R, Timmermans JP, Adriaensen D. Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs. Histochem Cell Biol. 2009;131:55–74.CrossRefPubMed
22.
go back to reference Uddman R, Luts A, Sundler F. Occurrence and distribution of calcitonin gene-related peptide in the mammalian respiratory tract and middle ear. Cell Tissue Res. 1985;241:551–5.CrossRefPubMed Uddman R, Luts A, Sundler F. Occurrence and distribution of calcitonin gene-related peptide in the mammalian respiratory tract and middle ear. Cell Tissue Res. 1985;241:551–5.CrossRefPubMed
23.
go back to reference Van Lommel A. Pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEB): chemoreceptors and regulators of lung development. Paediatr Respir Rev. 2001;2:171–6.PubMed Van Lommel A. Pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEB): chemoreceptors and regulators of lung development. Paediatr Respir Rev. 2001;2:171–6.PubMed
24.
go back to reference Reynolds SD, Hong KU, Giangreco A, Mango GW, Guron C, Morimoto Y, Stripp BR. Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells. Am J Physiol Lung Cell Mol Physiol. 2000;278:1256–63. Reynolds SD, Hong KU, Giangreco A, Mango GW, Guron C, Morimoto Y, Stripp BR. Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells. Am J Physiol Lung Cell Mol Physiol. 2000;278:1256–63.
25.
go back to reference Thebaud B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F, Hashimoto K, Harry G, Haromy A, Korbutt G, Archer SL. Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization. Circulation. 2005;112:2477–86.CrossRefPubMed Thebaud B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F, Hashimoto K, Harry G, Haromy A, Korbutt G, Archer SL. Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization. Circulation. 2005;112:2477–86.CrossRefPubMed
26.
go back to reference Adriaensen D, Brouns I, Van Genechten J, Timmermans JP. Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anat Rec. 2003;270:25–40.CrossRef Adriaensen D, Brouns I, Van Genechten J, Timmermans JP. Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anat Rec. 2003;270:25–40.CrossRef
27.
go back to reference Sorokin SP, Kobzik L, Hoyt Jr RF, Godleski JJ. Development of surface membrane characteristics of “premedullary” macrophages in organ cultures of embryonic rat and hamster lungs. J Histochem Cytochem. 1989;37:365–76.CrossRefPubMed Sorokin SP, Kobzik L, Hoyt Jr RF, Godleski JJ. Development of surface membrane characteristics of “premedullary” macrophages in organ cultures of embryonic rat and hamster lungs. J Histochem Cytochem. 1989;37:365–76.CrossRefPubMed
28.
go back to reference Hoyt Jr RF, McNelly NA, Sorokin SP. Dynamics of neuroepithelial body (NEB) formation in developing hamster lung: light microscopic autoradiography after 3H-thymidine labeling in vivo. Anat Rec. 1990;227:340–50.CrossRefPubMed Hoyt Jr RF, McNelly NA, Sorokin SP. Dynamics of neuroepithelial body (NEB) formation in developing hamster lung: light microscopic autoradiography after 3H-thymidine labeling in vivo. Anat Rec. 1990;227:340–50.CrossRefPubMed
29.
go back to reference Sorokin SP, Hoyt Jr RF, Reenstra WR, McNelly NA. Factors influencing fetal macrophage development: III. Immunocytochemical localization of cytokines and time-resolved expression of differentiation markers in organ-cultured rat lungs. Anat Rec. 1997;248:93–103.CrossRefPubMed Sorokin SP, Hoyt Jr RF, Reenstra WR, McNelly NA. Factors influencing fetal macrophage development: III. Immunocytochemical localization of cytokines and time-resolved expression of differentiation markers in organ-cultured rat lungs. Anat Rec. 1997;248:93–103.CrossRefPubMed
30.
31.
go back to reference Cutz E, Pan J, Yeger H, Domnik NJ, Fisher JT. Recent advances and contraversies on the role of pulmonary neuroepithelial bodies as airway sensors. Semin Cell Dev Biol. 2013;24:40–50.CrossRefPubMed Cutz E, Pan J, Yeger H, Domnik NJ, Fisher JT. Recent advances and contraversies on the role of pulmonary neuroepithelial bodies as airway sensors. Semin Cell Dev Biol. 2013;24:40–50.CrossRefPubMed
32.
go back to reference Youngson C, Nurse C, Yeger H, Cutz E. Oxygen sensing in airway chemoreceptors. Nature. 1993;365:153–5.CrossRefPubMed Youngson C, Nurse C, Yeger H, Cutz E. Oxygen sensing in airway chemoreceptors. Nature. 1993;365:153–5.CrossRefPubMed
33.
go back to reference Cutz E, Jackson A. Neuroepithelial bodies as airway oxygen sensors. Respir Physiol. 1999;115:201–14.CrossRefPubMed Cutz E, Jackson A. Neuroepithelial bodies as airway oxygen sensors. Respir Physiol. 1999;115:201–14.CrossRefPubMed
34.
go back to reference Lembrechts R, Brouns I, Schnorbusch K, Pintelon I, Timmermans JP, Adriaensen D. Neuroepithelial bodies as mechanotransducers in the intrapulmonary airway epithelium: involvement of TRPC5. Am J Respir Cell Mol Biol. 2012;47:315–23.CrossRefPubMed Lembrechts R, Brouns I, Schnorbusch K, Pintelon I, Timmermans JP, Adriaensen D. Neuroepithelial bodies as mechanotransducers in the intrapulmonary airway epithelium: involvement of TRPC5. Am J Respir Cell Mol Biol. 2012;47:315–23.CrossRefPubMed
35.
go back to reference Lembrechts R, Pintelon I, Schnorbusch K, Timmermans JP, Adriaensen D, Brouns I. Expression of mechanogated two-pore domain potassium channels in mouse lungs: special reference to mechanosensory airway receptors. Histochem Cell Biol. 2011;136:371–85.CrossRefPubMed Lembrechts R, Pintelon I, Schnorbusch K, Timmermans JP, Adriaensen D, Brouns I. Expression of mechanogated two-pore domain potassium channels in mouse lungs: special reference to mechanosensory airway receptors. Histochem Cell Biol. 2011;136:371–85.CrossRefPubMed
36.
go back to reference Pan J, Copland I, Post M, Yeger H, Cutz E. Mechanical stretch-induced serotonin release from pulmonary neuroendocrine cells: implications for lung development. Am J Physiol Lung Cell Mol Physiol. 2006;290:L185–93.CrossRefPubMed Pan J, Copland I, Post M, Yeger H, Cutz E. Mechanical stretch-induced serotonin release from pulmonary neuroendocrine cells: implications for lung development. Am J Physiol Lung Cell Mol Physiol. 2006;290:L185–93.CrossRefPubMed
37.
go back to reference Livermore S, Zhou Y, Pan J, Yeger H, Nurse CA, Cutz E. Pulmonary neuroepithelial bodies are polymodal airway sensors: evidence for CO2/H+ sensing. Am J Physiol Lung Cell Mol Physiol. 2015;308:L807–15.CrossRefPubMed Livermore S, Zhou Y, Pan J, Yeger H, Nurse CA, Cutz E. Pulmonary neuroepithelial bodies are polymodal airway sensors: evidence for CO2/H+ sensing. Am J Physiol Lung Cell Mol Physiol. 2015;308:L807–15.CrossRefPubMed
38.
go back to reference Li F, He J, Wei J, Cho WC, Liu X. Diversity of epithelial stem cell types in adult lung. Stem Cells Int. 2015;2015:728307.PubMedPubMedCentral Li F, He J, Wei J, Cho WC, Liu X. Diversity of epithelial stem cell types in adult lung. Stem Cells Int. 2015;2015:728307.PubMedPubMedCentral
40.
go back to reference Wansleeben C, Barkauskas CE, Rock JR, Hogan BL. Stem cells of the adult lung: their development and role in homeostasis, regeneration, and disease. Wiley Interdiscip Rev Dev Biol. 2013;2:131–48.CrossRefPubMed Wansleeben C, Barkauskas CE, Rock JR, Hogan BL. Stem cells of the adult lung: their development and role in homeostasis, regeneration, and disease. Wiley Interdiscip Rev Dev Biol. 2013;2:131–48.CrossRefPubMed
41.
go back to reference Rock JR, Hogan BL. Epithelial progenitor cells in lung development, maintenance, repair, and disease. Annu Rev Cell Dev Biol. 2011;27:493–512.CrossRefPubMed Rock JR, Hogan BL. Epithelial progenitor cells in lung development, maintenance, repair, and disease. Annu Rev Cell Dev Biol. 2011;27:493–512.CrossRefPubMed
42.
go back to reference Giangreco A, Arwert EN, Rosewell IR, Snyder J, Watt FM, Stripp BR. Stem cells are dispensable for lung homeostasis but restore airways after injury. Proc Natl Acad Sci U S A. 2009;106:9286–91.CrossRefPubMedPubMedCentral Giangreco A, Arwert EN, Rosewell IR, Snyder J, Watt FM, Stripp BR. Stem cells are dispensable for lung homeostasis but restore airways after injury. Proc Natl Acad Sci U S A. 2009;106:9286–91.CrossRefPubMedPubMedCentral
43.
go back to reference Sobonya RE. Pathology of chronic obstructive pulmonary disease. Curr Opin Pulm Med. 1995;1:125–8.PubMed Sobonya RE. Pathology of chronic obstructive pulmonary disease. Curr Opin Pulm Med. 1995;1:125–8.PubMed
44.
go back to reference Aguayo SM. Determinants of susceptibility to cigarette smoke. Potential roles for neuroendocrine cells and neuropeptides in airway inflammation, airway wall remodeling, and chronic airflow obstruction. Am J Respir Crit Care Med. 1994;149:1692–8.CrossRefPubMed Aguayo SM. Determinants of susceptibility to cigarette smoke. Potential roles for neuroendocrine cells and neuropeptides in airway inflammation, airway wall remodeling, and chronic airflow obstruction. Am J Respir Crit Care Med. 1994;149:1692–8.CrossRefPubMed
45.
go back to reference Vaughan AE, Chapman HA. Regenerative activity of the lung after epithelial injury. Biochim Biophys Acta. 1832;2013:922–30. Vaughan AE, Chapman HA. Regenerative activity of the lung after epithelial injury. Biochim Biophys Acta. 1832;2013:922–30.
46.
go back to reference Stovold R, Blackhall F, Meredith S, Hou J, Dive C, White A. Biomarkers for small cell lung cancer: neuroendocrine, epithelial and circulating tumour cells. Lung Cancer. 2012;76:263–8.CrossRefPubMed Stovold R, Blackhall F, Meredith S, Hou J, Dive C, White A. Biomarkers for small cell lung cancer: neuroendocrine, epithelial and circulating tumour cells. Lung Cancer. 2012;76:263–8.CrossRefPubMed
47.
go back to reference Sutherland KD, Proost N, Brouns I, Adriaensen D, Song JY, Berns A. Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung. Cancer Cell. 2011;19:754–64.CrossRefPubMed Sutherland KD, Proost N, Brouns I, Adriaensen D, Song JY, Berns A. Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung. Cancer Cell. 2011;19:754–64.CrossRefPubMed
48.
go back to reference Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.CrossRefPubMed Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.CrossRefPubMed
49.
50.
go back to reference Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034.CrossRefPubMedPubMedCentral Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034.CrossRefPubMedPubMedCentral
51.
go back to reference Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8:R19.CrossRefPubMedPubMedCentral Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8:R19.CrossRefPubMedPubMedCentral
52.
go back to reference Hong KU, Reynolds SD, Giangreco A, Hurley CM, Stripp BR. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am J Respir Cell Mol Biol. 2001;24:671–81.CrossRefPubMed Hong KU, Reynolds SD, Giangreco A, Hurley CM, Stripp BR. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am J Respir Cell Mol Biol. 2001;24:671–81.CrossRefPubMed
53.
go back to reference Perez-Novo CA, Claeys C, Speleman F, Van Cauwenberge P, Bachert C, Vandesompele J. Impact of RNA quality on reference gene expression stability. Biotechniques. 2005;39:52–54,56.CrossRefPubMed Perez-Novo CA, Claeys C, Speleman F, Van Cauwenberge P, Bachert C, Vandesompele J. Impact of RNA quality on reference gene expression stability. Biotechniques. 2005;39:52–54,56.CrossRefPubMed
54.
go back to reference Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006;27:126–39.CrossRefPubMed Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006;27:126–39.CrossRefPubMed
55.
go back to reference De Spiegelaere W, Cornillie P, Van Poucke M, Peelman L, Burvenich C, Van den Broeck W. Quantitative mRNA expression analysis in kidney glomeruli using microdissection techniques. Histol Histopathol. 2011;26:267–75.PubMed De Spiegelaere W, Cornillie P, Van Poucke M, Peelman L, Burvenich C, Van den Broeck W. Quantitative mRNA expression analysis in kidney glomeruli using microdissection techniques. Histol Histopathol. 2011;26:267–75.PubMed
56.
go back to reference Fend F, Emmert-Buck MR, Chuaqui R, Cole K, Lee J, Liotta LA, Raffeld M. Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol. 1999;154:61–6.CrossRefPubMedPubMedCentral Fend F, Emmert-Buck MR, Chuaqui R, Cole K, Lee J, Liotta LA, Raffeld M. Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol. 1999;154:61–6.CrossRefPubMedPubMedCentral
57.
go back to reference Cox ML, Eddy SM, Stewart ZS, Kennel MR, Man MZ, Paulauskis JD, Dunstan RW. Investigating fixative-induced changes in RNA quality and utility by microarray analysis. Exp Mol Pathol. 2008;84:156–72.CrossRefPubMed Cox ML, Eddy SM, Stewart ZS, Kennel MR, Man MZ, Paulauskis JD, Dunstan RW. Investigating fixative-induced changes in RNA quality and utility by microarray analysis. Exp Mol Pathol. 2008;84:156–72.CrossRefPubMed
58.
go back to reference Kerman IA, Buck BJ, Evans SJ, Akil H, Watson SJ. Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression. J Neurosci Methods. 2006;153:71–85.CrossRefPubMed Kerman IA, Buck BJ, Evans SJ, Akil H, Watson SJ. Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression. J Neurosci Methods. 2006;153:71–85.CrossRefPubMed
59.
go back to reference McGinley JN, Zhu Z, Jiang W, Thompson HJ. Collection of epithelial cells from rodent mammary gland via laser capture microdissection yielding high-quality RNA suitable for microarray analysis. Biol Proced Online. 2010;12:9026.CrossRefPubMed McGinley JN, Zhu Z, Jiang W, Thompson HJ. Collection of epithelial cells from rodent mammary gland via laser capture microdissection yielding high-quality RNA suitable for microarray analysis. Biol Proced Online. 2010;12:9026.CrossRefPubMed
60.
go back to reference Gurok U, Loebbert RW, Meyer AH, Mueller R, Schoemaker H, Gross G, Behl B. Laser capture microdissection and microarray analysis of dividing neural progenitor cells from the adult rat hippocampus. Eur J Neurosci. 2007;26:1079–90.CrossRefPubMed Gurok U, Loebbert RW, Meyer AH, Mueller R, Schoemaker H, Gross G, Behl B. Laser capture microdissection and microarray analysis of dividing neural progenitor cells from the adult rat hippocampus. Eur J Neurosci. 2007;26:1079–90.CrossRefPubMed
61.
go back to reference Wang HY, Owens JD, Shih JH, Li MC, Bonner RF, Mushinski JF. Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA. BMC Genomics. 2006;7:97.CrossRefPubMedPubMedCentral Wang HY, Owens JD, Shih JH, Li MC, Bonner RF, Mushinski JF. Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA. BMC Genomics. 2006;7:97.CrossRefPubMedPubMedCentral
62.
go back to reference Tamamaki N, Yanagawa Y, Tomioka R, Miyazaki JI, Obata K, Kaneko T. Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J Comp Neurol. 2003;467:60–79.CrossRefPubMed Tamamaki N, Yanagawa Y, Tomioka R, Miyazaki JI, Obata K, Kaneko T. Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J Comp Neurol. 2003;467:60–79.CrossRefPubMed
63.
go back to reference Bhattacherjee V, Mukhopadhyay P, Singh S, Roberts EA, Hackmiller RC, Greene RM, Pisano MM. Laser capture microdissection of fluorescently labeled embryonic cranial neural crest cells. Genesis. 2004;39:58–64.CrossRefPubMed Bhattacherjee V, Mukhopadhyay P, Singh S, Roberts EA, Hackmiller RC, Greene RM, Pisano MM. Laser capture microdissection of fluorescently labeled embryonic cranial neural crest cells. Genesis. 2004;39:58–64.CrossRefPubMed
64.
go back to reference Medeiros F, Rigl CT, Anderson GG, Becker SH, Halling KC. Tissue handling for genome-wide expression analysis - A review of the issues, evidence, and opportunities. Arch Pathol Lab Med. 2007;131:1805–16.PubMed Medeiros F, Rigl CT, Anderson GG, Becker SH, Halling KC. Tissue handling for genome-wide expression analysis - A review of the issues, evidence, and opportunities. Arch Pathol Lab Med. 2007;131:1805–16.PubMed
65.
go back to reference Vermeulen J, Derveaux S, Lefever S, De Smet E, De Preter K, Yigit N, De Paepe A, Pattyn F, Speleman F, Vandesompele J. RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts. BMC Res Notes. 2009;2:235.CrossRefPubMedPubMedCentral Vermeulen J, Derveaux S, Lefever S, De Smet E, De Preter K, Yigit N, De Paepe A, Pattyn F, Speleman F, Vandesompele J. RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts. BMC Res Notes. 2009;2:235.CrossRefPubMedPubMedCentral
66.
go back to reference Singh R, Maganti RJ, Jabba SV, Wang M, Deng G, Heath JD, Kurn N, Wangemann P. Microarray-based comparison of three amplification methods for nanogram amounts of total RNA. Am J Physiol Cell Physiol. 2005;288:C1179–89.CrossRefPubMed Singh R, Maganti RJ, Jabba SV, Wang M, Deng G, Heath JD, Kurn N, Wangemann P. Microarray-based comparison of three amplification methods for nanogram amounts of total RNA. Am J Physiol Cell Physiol. 2005;288:C1179–89.CrossRefPubMed
67.
go back to reference Nygaard V, Holden M, Loland A, Langaas M, Myklebost O, Hovig E. Limitations of mRNA amplification from small-size cell samples. BMC Genomics. 2005;6:147.CrossRefPubMedPubMedCentral Nygaard V, Holden M, Loland A, Langaas M, Myklebost O, Hovig E. Limitations of mRNA amplification from small-size cell samples. BMC Genomics. 2005;6:147.CrossRefPubMedPubMedCentral
68.
go back to reference King C, Guo N, Frampton GM, Gerry NP, Lenburg ME, Rosenberg CL. Reliability and reproducibility of gene expression measurements using amplified RNA from laser-microdissected primary breast tissue with oligonucleotide arrays. J Mol Diagn. 2005;7:57–64.CrossRefPubMedPubMedCentral King C, Guo N, Frampton GM, Gerry NP, Lenburg ME, Rosenberg CL. Reliability and reproducibility of gene expression measurements using amplified RNA from laser-microdissected primary breast tissue with oligonucleotide arrays. J Mol Diagn. 2005;7:57–64.CrossRefPubMedPubMedCentral
69.
go back to reference Li Y, Ali S, Philip PA, Sarkar FH. Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues. Cancer Detect Prev. 2003;27:405–11.CrossRefPubMed Li Y, Ali S, Philip PA, Sarkar FH. Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues. Cancer Detect Prev. 2003;27:405–11.CrossRefPubMed
70.
go back to reference Song H, Yao E, Lin C, Gacayan R, Chen MH, Chuang PT. Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis. Proc Natl Acad Sci U S A. 2012;109:17531–6.CrossRefPubMedPubMedCentral Song H, Yao E, Lin C, Gacayan R, Chen MH, Chuang PT. Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis. Proc Natl Acad Sci U S A. 2012;109:17531–6.CrossRefPubMedPubMedCentral
71.
go back to reference Guha A, Vasconcelos M, Cai Y, Yoneda M, Hinds A, Qian J, Li G, Dickel L, Johnson JE, Kimura S, et al. Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways. Proc Natl Acad Sci U S A. 2012;109:12592–7.CrossRefPubMedPubMedCentral Guha A, Vasconcelos M, Cai Y, Yoneda M, Hinds A, Qian J, Li G, Dickel L, Johnson JE, Kimura S, et al. Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways. Proc Natl Acad Sci U S A. 2012;109:12592–7.CrossRefPubMedPubMedCentral
72.
go back to reference Rawlins EL, Okubo T, Xue Y, Brass DM, Auten RL, Hasegawa H, Wang F, Hogan BL. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell. 2009;4:525–34.CrossRefPubMedPubMedCentral Rawlins EL, Okubo T, Xue Y, Brass DM, Auten RL, Hasegawa H, Wang F, Hogan BL. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell. 2009;4:525–34.CrossRefPubMedPubMedCentral
74.
go back to reference Stripp BR, Maxson K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol. 1995;269:L791–9.PubMed Stripp BR, Maxson K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol. 1995;269:L791–9.PubMed
75.
go back to reference Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K, Desai R, Escarpe PA, Hampl J, Laysang A, et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med. 2015;7:302ra136.CrossRefPubMedPubMedCentral Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K, Desai R, Escarpe PA, Hampl J, Laysang A, et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med. 2015;7:302ra136.CrossRefPubMedPubMedCentral
76.
go back to reference Park KS, Martelotto LG, Peifer M, Sos ML, Karnezis AN, Mahjoub MR, Bernard K, Conklin JF, Szczepny A, Yuan J, et al. A crucial requirement for Hedgehog signaling in small cell lung cancer. Nat Med. 2011;17:1504–8.CrossRefPubMedPubMedCentral Park KS, Martelotto LG, Peifer M, Sos ML, Karnezis AN, Mahjoub MR, Bernard K, Conklin JF, Szczepny A, Yuan J, et al. A crucial requirement for Hedgehog signaling in small cell lung cancer. Nat Med. 2011;17:1504–8.CrossRefPubMedPubMedCentral
77.
go back to reference Park K-S, Liang M-C, Raiser DM, Zamponi R, Roach RR, Curtis SJ, Walton Z, Schaffer BE, Roake CM, Zmoos A-F, et al. Characterization of the cell of origin for small cell lung cancer. Cell Cycle. 2014;10:2806–15.CrossRef Park K-S, Liang M-C, Raiser DM, Zamponi R, Roach RR, Curtis SJ, Walton Z, Schaffer BE, Roake CM, Zmoos A-F, et al. Characterization of the cell of origin for small cell lung cancer. Cell Cycle. 2014;10:2806–15.CrossRef
78.
go back to reference Collins BJ, Kleeberger W, Ball DW. Notch in lung development and lung cancer. Semin Cancer Biol. 2004;14:357–64.CrossRefPubMed Collins BJ, Kleeberger W, Ball DW. Notch in lung development and lung cancer. Semin Cancer Biol. 2004;14:357–64.CrossRefPubMed
79.
go back to reference Morimoto M, Liu Z, Cheng HT, Winters N, Bader D, Kopan R. Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate. J Cell Sci. 2010;123:213–24.CrossRefPubMedPubMedCentral Morimoto M, Liu Z, Cheng HT, Winters N, Bader D, Kopan R. Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate. J Cell Sci. 2010;123:213–24.CrossRefPubMedPubMedCentral
80.
go back to reference Ball D. Achaete-scute homolog-1 and Notch in lung neuroendocrine development and cancer. Cancer Lett. 2004;204:159–69.CrossRefPubMed Ball D. Achaete-scute homolog-1 and Notch in lung neuroendocrine development and cancer. Cancer Lett. 2004;204:159–69.CrossRefPubMed
81.
go back to reference Meder L, Konig K, Ozretic L, Schultheis AM, Ueckeroth F, Ade CP, Albus K, Boehm D, Rommerscheidt-Fuss U, Florin A, et al. NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas. Int J Cancer. 2016;138:927–38.CrossRefPubMed Meder L, Konig K, Ozretic L, Schultheis AM, Ueckeroth F, Ade CP, Albus K, Boehm D, Rommerscheidt-Fuss U, Florin A, et al. NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas. Int J Cancer. 2016;138:927–38.CrossRefPubMed
82.
go back to reference Henke RM, Meredith DM, Borromeo MD, Savage TK, Johnson JE. Ascl1 and Neurog2 form novel complexes and regulate Delta-like3 (Dll3) expression in the neural tube. Dev Biol. 2009;328:529–40.CrossRefPubMedPubMedCentral Henke RM, Meredith DM, Borromeo MD, Savage TK, Johnson JE. Ascl1 and Neurog2 form novel complexes and regulate Delta-like3 (Dll3) expression in the neural tube. Dev Biol. 2009;328:529–40.CrossRefPubMedPubMedCentral
83.
go back to reference Augustyn A, Borromeo M, Wang T, Fujimoto J, Shao C, Dospoy PD, Lee V, Tan C, Sullivan JP, Larsen JE, et al. ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers. Proc Natl Acad Sci U S A. 2014;111:14788–93.CrossRefPubMedPubMedCentral Augustyn A, Borromeo M, Wang T, Fujimoto J, Shao C, Dospoy PD, Lee V, Tan C, Sullivan JP, Larsen JE, et al. ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers. Proc Natl Acad Sci U S A. 2014;111:14788–93.CrossRefPubMedPubMedCentral
84.
go back to reference Perdigoto CN, Bardin AJ. Sending the right signal: Notch and stem cells. Biochim Biophys Acta. 1830;2013:2307–22. Perdigoto CN, Bardin AJ. Sending the right signal: Notch and stem cells. Biochim Biophys Acta. 1830;2013:2307–22.
85.
go back to reference Meuwissen R, Linn SC, Linnoila RI, Zevenhoven J, Mooi WJ, Berns A. Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model. Cancer Cell. 2003;4:181–9.CrossRefPubMed Meuwissen R, Linn SC, Linnoila RI, Zevenhoven J, Mooi WJ, Berns A. Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model. Cancer Cell. 2003;4:181–9.CrossRefPubMed
Metadata
Title
Selective gene expression analysis of the neuroepithelial body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
Authors
Line Verckist
Robrecht Lembrechts
Sofie Thys
Isabel Pintelon
Jean-Pierre Timmermans
Inge Brouns
Dirk Adriaensen
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Respiratory Research / Issue 1/2017
Electronic ISSN: 1465-993X
DOI
https://doi.org/10.1186/s12931-017-0571-4

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