Project description:Transcriptional profiling of Control and RbpjkCNULL lungs from E18.5. For microarray profiling, total RNA from E18.5 lungs was submitted for labeling and hybridization (Mouse Gene 1.0 ST Whole Genome Array, Affymetrix) to the Boston University Microarray Core facility. Clara cells (CCs) are a morphologically and operationally heterogeneous population of secretoglobin Scgb1a1-expressing secretory cells crucial for airway homeostasis and post-injury repair. Insights into the extent and origin of CC diversity have been hindered by the limited knowledge of markers of these cells and their precursors. To identify novel putative markers of CCs we characterized global changes in gene expression in embryonic lungs in which CCs were suppressed by conditional disruption of Notch signaling (Rbpjkcnull). Microarray profiling and Real Time PCR (qRT-PCR) identified eleven genes differentially downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. Remarkably, in situ hybridization (ISH) revealed overlapping but also distinct domains of expression of these genes in E18.5 controls. Notably, Reg3g, Chad, Gabrp and Lrrc26 were selectively expressed in CCs of proximal airways and Upk3a expression was highly enriched in a CC subpopulation surrounding Neuroepithelial Bodies (NEBs). All genes expressed in the adult airways were found to be expressed in adult CCs and downregulated by selective CC ablation in a Naphthalene model of injury. Flow cytometry-based isolation of CCs from different airway regions of adult B1-EGFP reporter mice and qRT-PCR corroborated the spatial enrichment in gene expression observed by ISH. Remarkably, although, Scgb3a2, Upk3a, Cyp2f2, Cbr2, Krt15 could be detected unambiguously in airway progenitors as early as E14.5, only Scgb3a2 and Upk3a had their expression suppressed by disruption of Notch signaling, implicating both genes as the earliest markers for CC differentiation. Our study supports the idea that the diversification of the CC phenotype already arises during embryonic development and identifies candidate markers that can be used to investigate this process. Control (n=3) versus RbpjkCNULL(n=3)

Project description:Transcriptional profiling of Control and RbpjkCNULL lungs from E18.5. For microarray profiling, total RNA from E18.5 lungs was submitted for labeling and hybridization (Mouse Gene 1.0 ST Whole Genome Array, Affymetrix) to the Boston University Microarray Core facility. Clara cells (CCs) are a morphologically and operationally heterogeneous population of secretoglobin Scgb1a1-expressing secretory cells crucial for airway homeostasis and post-injury repair. Insights into the extent and origin of CC diversity have been hindered by the limited knowledge of markers of these cells and their precursors. To identify novel putative markers of CCs we characterized global changes in gene expression in embryonic lungs in which CCs were suppressed by conditional disruption of Notch signaling (Rbpjkcnull). Microarray profiling and Real Time PCR (qRT-PCR) identified eleven genes differentially downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. Remarkably, in situ hybridization (ISH) revealed overlapping but also distinct domains of expression of these genes in E18.5 controls. Notably, Reg3g, Chad, Gabrp and Lrrc26 were selectively expressed in CCs of proximal airways and Upk3a expression was highly enriched in a CC subpopulation surrounding Neuroepithelial Bodies (NEBs). All genes expressed in the adult airways were found to be expressed in adult CCs and downregulated by selective CC ablation in a Naphthalene model of injury. Flow cytometry-based isolation of CCs from different airway regions of adult B1-EGFP reporter mice and qRT-PCR corroborated the spatial enrichment in gene expression observed by ISH. Remarkably, although, Scgb3a2, Upk3a, Cyp2f2, Cbr2, Krt15 could be detected unambiguously in airway progenitors as early as E14.5, only Scgb3a2 and Upk3a had their expression suppressed by disruption of Notch signaling, implicating both genes as the earliest markers for CC differentiation. Our study supports the idea that the diversification of the CC phenotype already arises during embryonic development and identifies candidate markers that can be used to investigate this process.

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Important role for secretoglobin(SCGB)3A2 in lung development

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