Transcriptomic analysis comparing mouse strains with extreme total lung capacities identifies novel candidate genes for pulmonary function

Abstract

Background: Failure to attain peak lung function by early adulthood is a risk factor for chronic lung diseases. Previously, we reported that C3H/HeJ mice have about twice total lung capacity (TLC) compared to JF1/MsJ mice. We identified seven lung function quantitative trait loci (QTL: Lfnq1-Lfnq7) in backcross/intercross mice derived from these inbred strains. We further demonstrated, superoxide dismutase 3, extracellular (Sod3), Kit oncogene (Kit) and secreted phosphoprotein 1 (Spp1) located on these Lfnqs as lung function determinants. Emanating from the concept of early origin of lung disease, we sought to identify novel candidate genes for pulmonary function by investigating lung transcriptome in C3H/HeJ and JF1/MsJ mice at the completion of embryonic development, bulk alveolar formation and maturity.

Methods: Design-based stereological analysis was performed to study lung structure in C3H/HeJ and JF1/MsJ mice. Microarray was used for lung transcriptomic analysis [embryonic day 18, postnatal days 28, 70]. Quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical analysis were used to confirm selected differences.

Results: Stereological analysis revealed decreased alveolar number density, elastin to collagen ratio and increased mean alveolar volume in C3H/HeJ mice compared to JF1/MsJ. Gene ontology term "extracellular region" was enriched among the decreased JF1/MsJ transcripts. Candidate genes identified using the expression-QTL strategy include: ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1), formyl peptide receptor 1 (Fpr1), gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1); histocompatibility 2 genes: class II antigen E beta (H2-Eb1), D region locus 1 (H2-D1), and Q region locus 4 (H2-Q4); leucine rich repeat containing 6 (testis) (Lrrc6), radial spoke head 1 homolog (Rsph1), and surfactant associated 2 (Sfta2). Noteworthy genes selected as candidates for their consistent expression include: Wnt inhibitor factor 1 (Wif1), follistatin (Fst), chitinase-like 1 (Chil1), and Chil3.

Conclusions: Comparison of late embryonic, adolescent and adult lung transcript profiles between mouse strains with extreme TLCs lead to the identification of candidate genes for pulmonary function that has not been reported earlier. Further mechanistic investigations are warranted to elucidate their mode of action in determining lung function.

Keywords: Asthma; Chronic obstructive pulmonary disease; Lung development; Transcriptomics; WNT Signaling.

Fig. 1

Comparative expression of lung Wnt inhibitor factor 1 (Wif1) and frizzled homolog 6 (Fzd6) and chitinase 3 like 3 (CHIL3) in JF1/MsJ and C3H/HeJ mice. a Expression of lung Wif1 mRNA in JF1/Msf was increased compared with C3H/HeJ mice at postnatal (P) days P28 and P70. b Expression of lung Fzd6 mRNA in JF1/Msf was decreased compared to C3H/HeJ mice at P28 and P70. Quantitative real-time polymerase chain reaction was performed, and the comparative cycle number threshold (C_T) method (ΔΔC_T) was used [ΔC_T = C_T (gene)-C_T(Actb)]. Data are presented as expression relative to time-matched C3H/HeJ level (means ± SE; n = 5 mice/strain/stage). *Significantly different from C3H/HeJ (ANOVA followed by all pairwise multiple-comparison procedures with Holm-Sidak method, P < 0.05). c Western blot analysis of CHIL3 from total lung homogenate of P28 C3H/HeJ and JF1/MsJ mice (n = 3 mice/strain; female). Lung transcript expression showed > 15 fold decreased Chil3 expression in Jf1/MsJ compared to C3H/HeJ. β-actin (ACTB) was used as the control. Photograph is representative of at least three observations

Fig. 2

Localization of Wnt inhibitor factor 1 (WIF1) protein in the lungs of four weeks old female C3H/HeJ and JF1/MsJ mice (n = 5). Increased WIF1 immunostaining was detected in the b, e airway epithelium and h alveolar type I and type II cells of JF1/MsJ lungs compared with those of c, f, i C3H/HeJ mice. a, d, g Sections incubated phosphate-buffered saline (PBS control) without primary antisera

Fig. 3

Localization of follistatin (FST) protein in the lungs of four weeks old female C3H/HeJ and JF1/MsJ mice (n = 5). Increased of FST immunostaining was detected in the b, e airways and h alveolar type I and type II cells of JF1/MsJ lungs compared with those of c, f, i C3H/HeJ mice. a, d, g Sections incubated phosphate-buffered saline (PBS control) without primary antisera

Fig. 4

Localization of frizzled homolog 6 (FZD6) protein in the lungs of four weeks old female C3H/HeJ and JF1/MsJ mice (n = 5). Decreased FZD6 immunostaining was detected in the b, e airways and h alveolar type I and type II cells of JF1/MsJ lungs compared with those of c, f, i C3H/HeJ mice. a, d, g Sections incubated phosphate-buffered saline (PBS control) without primary antisera