Analysis of Notch signaling-dependent gene expression in developing airways reveals diversity of Clara cells - PubMed (original) (raw)

Analysis of Notch signaling-dependent gene expression in developing airways reveals diversity of Clara cells

Arjun Guha et al. PLoS One. 2014.

Abstract

Clara cells (CCs) are a morphologically and operationally heterogeneous population of Secretoglobin Scgb1a1-expressing secretory cells that are crucial for airway homeostasis and post-injury repair. Analysis of the extent and origin of CC diversity are limited by knowledge of genes expressed in these cells and their precursors. To identify novel putative markers of CCs and explore the origins of CC diversity, we characterized global changes in gene expression in embryonic lungs in which CCs do not form due to conditional disruption of Notch signaling (Rbpjk(CNULL)). Microarray profiling, Real Time PCR (qRT-PCR), and RNA in situ hybridization (ISH) identified eleven genes downregulated in the E18.5 airways of Rbpjk(cnull) compared to controls, nearly half not previously known to mark CCs. ISH revealed that several genes had overlapping but distinct domains of expression of in the normal developing lung (E18.5). Notably, Reg3g, Chad, Gabrp and Lrrc26 were enriched in proximal airways, Hp in the distal airways and Upk3a in clusters of cells surrounding Neuroepithelial Bodies (NEBs). Seven of the eleven genes, including Reg3g, Hp, and Upk3a, were expressed in the adult lung in CCs in a pattern similar to that observed in the developing airways. qRT-PCR-based analysis of gene expression of CCs isolated from different airway regions of B1-EGFP reporter mice corroborated the spatial enrichment in gene expression observed by ISH. Our study identifies candidate markers for CC-precursors and CCs and supports the idea that the diversification of the CC phenotype occurs already during embryonic development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Microarray-based identification of mRNAs downregulated in RbpjkCNULL lungs at E18.5 and validation using qRT-PCR.

Genes downregulated greater than two-fold (p-value <0.05 and FDR-q value <0.28) are shown here. FC = fold change; FDR-q = Benjamini-Hochberg False Discovery Rate. A total of n = 3 lungs per condition from control (CTRL) and Shh cre/+; Rbp Flox/Flox (RbpjkCNULL) were used for the respective analyses.

Figure 2

Figure 2. Expression patterns of the genes identified by transcriptional profiling.

RNA in situ hybridization (ISH) analysis of the expression of candidate genes in in control and RbpjkCNULL lungs at E18.5. All genes were expressed in the airways in widespread (A–K) and localized (M–U) patterns and downregulated in the RbpjkCNULL lungs (B–L, N–V). Chad expression was detected in precursors of the cartilage (O, asterisk) and this expression was unaffected in RbpjkCNULL (P, asterisk). Arrows in the panels indicate comparable regions of the airways. Red arrows indicate that the panels have been vertically ordered with respect to Fold Change (FC) in gene expression reported by microarray profiling. ISH on CTRL and RbpjkCNULL sections was performed simultaneously and the staining was developed for the same period for time. Tr = Trachea, Lu = Lung.

Figure 3

Figure 3. Several genes downregulated in RbpjkCNULL at E18.5 are expressed in adult CCs.

(A–H) Transcripts that were broadly expressed from trachea to terminal bronchiole (arrowhead) in cells that expressed Scgb1a1 (B, D, F, H, arrows, brown = Scgb1a1 protein). Cbr2 was expressed along the entire airway both, in cells that express Scgb1a1 (H, arrows) and others (H, asterisks). (I–N) Transcripts that were expressed in restricted patterns in the airways in cells that expressed Scgb1a1. Hp expression was negligible in the trachea (I, left inset) and mainstem bronchi (bracketed region shown at higher resolution in right inset) but high in the terminal bronchiole (I, arrowhead). Hp expressing cells in the intrapulmonary airways co-expressed Scgb1a1 (J, arrows). Reg3g expression detected in the trachea (K, left inset) and mainstem bronchi (bracketed region shown at higher resolution in right inset) but not in the terminal bronchiole (K, arrowhead). Many Reg3g expressing cells in the proximal airways co-localized with Scgb1a1 and a few cells did not (L, asterisk). Upk3a expressing cells were rare (M, arrow) and only detected in the vicinity of NEBs (Cgrp, red). (N) _Upk3a_-creER expressing cells were distributed throughout the intrapulmonary airways (arrowheads mark the bronchioalveolar duct junction). _Upk3a_-creER expressing cells in the adult lung (red, arrows) were clustered around NEBS (green) and expressed Scgb1a1 (white).

Figure 4

Figure 4. Impact of Naphthalene injury on the expression of genes identified by transcriptional profiling.

(A) qRT-PCR analysis of gene expression in mock-treated (corn oil, CTRL) and Naphthalene-treated (Nap) lungs 2 day post-injection (n = 3 lungs per condition). (B–G) ISH for Reg3g (B–C) and Hp (D–G in CTRL (B, D–E and Nap-treated lungs (F–G respectively. E, G are higher magnification images of airways shown in D, F respectively.

Figure 5

Figure 5. Comparison of gene expression in CCs derived from proximal (trachea) and distal (intrapulmonary) airways of B1-EGFP mice.

(A) Isolation and characterization of B1-EGFP expressing (GFPHigh) epithelial (Epcam+) cells from the trachea and intrapulmonary airways. Dot plots (central panels) and IHC of cytospins (right panels) showing that the isolated cells from the respective pools expressed Scbg1a1. (B) qRT-PCR-based analysis of the relative expression of eleven genes in tracheal and intrapulmonary GFPHigh Epcam+ cells.

Figure 6

Figure 6. Expression patterns of the genes identified by transcriptional profiling at E14.5.

(A) qRT-PCR analysis of gene expression in CTRL and RbpjkCNULL lungs from E14.5 (B) ISH showing expression of the eleven genes in lungs from control and RbpjkCNULL at E14.5. Genes expressed at E14.5 shown in left panel.

Figure 7

Figure 7. Summary of gene expression patterns in embryonic and adult lung.

Cartoon summarizing the domains of expressions of the genes identified in Fig. 1 in the embryonic (E18.5) and adult lung based on ISH (blue, green) and qRT-PCR (orange) analysis.

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