Temporal dynamics of the developing lung transcriptome in three common inbred strains of laboratory mice reveals multiple stages of postnatal alveolar development

Abstract

To characterize temporal patterns of transcriptional activity during normal lung development, we generated genome wide gene expression data for 26 pre- and post-natal time points in three common inbred strains of laboratory mice (C57BL/6J, A/J, and C3H/HeJ). Using Principal Component Analysis and least squares regression modeling, we identified both strain-independent and strain-dependent patterns of gene expression. The 4,683 genes contributing to the strain-independent expression patterns were used to define a murine Developing Lung Characteristic Subtranscriptome (mDLCS). Regression modeling of the Principal Components supported the four canonical stages of mammalian embryonic lung development (embryonic, pseudoglandular, canalicular, saccular) defined previously by morphology and histology. For postnatal alveolar development, the regression model was consistent with four stages of alveolarization characterized by episodic transcriptional activity of genes related to pulmonary vascularization. Genes expressed in a strain-dependent manner were enriched for annotations related to neurogenesis, extracellular matrix organization, and Wnt signaling. Finally, a comparison of mouse and human transcriptomics from pre-natal stages of lung development revealed conservation of pathways associated with cell cycle, axon guidance, immune function, and metabolism as well as organism-specific expression of genes associated with extracellular matrix organization and protein modification. The mouse lung development transcriptome data generated for this study serves as a unique reference set to identify genes and pathways essential for normal mammalian lung development and for investigations into the developmental origins of respiratory disease and cancer. The gene expression data are available from the Gene Expression Omnibus (GEO) archive (GSE74243). Temporal expression patterns of mouse genes can be investigated using a study specific web resource (http://lungdevelopment.jax.org).

Keywords: Gene expression; Genomics; Laboratory mice; Lung development; Time series.

Figure 1. Comparison of mouse strains, time points, and platforms for genome wide expression profiling of murine lung development.

Shaded cells indicate time points included in published transcriptional profiles of mouse lung development. Images reflect the major anatomical changes at each embryonic stage of development.

Figure 2. Global patterns of sample variation across lung development.

Plots of PCA scores (y-axis) for strain-independent principal components 1–3 along developmental time points and stages (x-axis). Time points: embryonic (E); postnatal (P). Stages: whole embryo (WE); embryonic (EMB); pseudoglandular (PSG); canalicular (CAN); saccular (SAC); alveolar (ALV1-4); mature lung (MAT). (A) PCA scores for principal components 1–3 (averaged across all three strains) across all developmental time points. (B) PCA scores for principal components 1–3 plotted for each mouse strain.

Figure 3. Regression modeling of gene expression as a function of strain and developmental stage.

Results of the linear regression analysis performed on PCA scores from strain-dependent principal components (PC 4–10). (A) Plots of least square means (y-axis) showing stage effects. (B) Plots of least square means (y-axis) illustrating strain effects. (C) Annotation enrichment results for characteristic gene sets with positive or negative loadings on PCs 4–10.

Figure 4. Regression analysis supports nine molecular stages of lung development.

Each set of colored bars represents the least square means of the stage effects for principal components 1–3. A summary of annotation enrichment categories are shown for the positive and negative characteristic gene sets from each component (complete results provided in Data S6).

Figure 5. Comparison of mouse and human embryonic Developing Lung Characteristic Subtranscriptome (DLCS) gene sets.

Schematic of workflow for comparing embryonic DLCS gene sets from mouse and human. Mouse genes were converted to human homolog gene symbols to facilitate comparison. Results of annotation enrichment analysis for the unique and overlapping genes are listed. Complete results available in Data S9.

Figure 6. Post-natal expression patterns of genes associated with alveolarization and angiogenesis.

Schematic of anti-correlated patterns of gene expression levels for genes associated with alveolarization and angiogenesis (solid line) versus negative regulation of angiogenesis (dotted line). The patterns illustrate functional genomics basis of the four stages of alveolar development proposed in this study.