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

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Introduction

Materials and Methods

Tissue and RNA

Data processing

Principal Component Analysis (PCA)

Regression analysis

Differential gene expression analysis: Significance of Microarrays (SAM)

Quantitative Real-Time PCR (qPCR) validation of strain-specific gene expression

Annotation term enrichment analysis

Mouse and human gene homology

Results

Principal component and regression analysis reveals nine stages of murine lung development

Strain-independent principal components 1–3 define a murine developing lung characteristic subtranscriptome (mDLCS)

The murine developing lung characteristic subtranscriptome

Strain-specific patterns of gene expression during lung development

  • C3H different from indistinguishable pair AJ/B6 (PC4)

  • B6 different from indistinguishable pair AJ/C3H (PC6, PC7)

  • AJ different from indistinguishable pair B6/C3H (PC8)

  • All strains different (PC5, PC9, PC10)

C3H different from AJ and/or B6

B6 different from AJ and/or C3H

AJ different from B6 and C3H

Comparative analysis of the mouse and human embryonic developing lung characteristic subtranscriptomes

Discussion and Conclusion

Principal components 1–3 suggest that lung development utilizes superimposed periodic patterns of transcriptional control

Alveolarization in mouse has four distinct transcriptional stages suggesting waves of vascularization and innervation

PCs 1–3 define a “Developing Lung Characteristic Subtranscriptome” in mouse

Strain specific gene expression during lung development

Comparison of mouse and human embryonic lung development

Supplemental Information

Genotyping protocol for determining the sex of mouse embryos

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Summarized gene expression values for lung development in three inbred mouse strains

The mouse lung development gene expression data were filtered to remove genes that had low levels of temporal expression variation.

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Results from principal component analysis of lung development gene expression: gene loading values

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Results from principal component analysis of lung development gene expression: principal component sample scores

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Results of differential gene expression analysis across stages of lung development using SAM

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Results of annotation enrichment analysis for the mouse Developing Lung Characteristic Subtranscriptome (mDLCS)

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Explanation of annotation term enrichment analysis

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Genes identified in previous transcriptional profiling studies of mouse lung development

The genes included in these lists were compared to the genes included in the mouse Developing Lung Characteristic Subtranscriptome (mDLCS) defined in this study.

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Mouse genes annotated with ontology terms for respiratory phenotype and lung development

The genes included in these lists were compared to the genes included in the mouse Developing Lung Characteristic Subtranscriptome (mDLCS) defined in this study.

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Relationships between gene loading values and PCA score

(A) Plots of gene loading values for PC 1–10. (B) Plot of PCA scores for PC1 across lung development time points sampled in this study highlighting a dramatic shift in gene loading values just before birth. (C) Mean expression levels of two representative genes with inverse loading values from PC1. Black bars, expression of Nasp (positive loading value); light grey bars, expression of Gpr116 (negative loading value). Error bars reflect one standard error from the mean. Complete PCA results of gene loading values and PCA scores found in Data S3 and S4, respectively.

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Principal component analysis reveals strain-dependent transcriptional patterns throughout lung development Plots of PCA scores by strain and stage for principal components showing strain-dependent correlations

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Genes associated with pulmonary vascularization or angiogenic rest are differentially expressed during postnatal lung development Plot of Z-scores (y-axis) for gene expression levels of selected genes associated with pulmonary vascularization across stages of lung development (x-axis)

Each error bar is constructed using 1 standard error from the mean. The patterns of expression illustrate the concept of periods of “angiogenic rest” during alveolarization. Potent angiogenic factors (Vegfa, Tgfb1, Ang) and known regulators of pulmonary vascularization (Adora2b, Adrb2, Lgals3) have elevated expression levels at alveolar stages (ALV1 and/or ALV4). Genes associated with the negative regulation of angiogenesis (Thbs2, Agt) and vascular stabilization/maturation factors (Angpt1, Angpt2, Serpine1, Igf1) show an inverse relationship of expression levels.

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Genes associated with alveolarization are differentially expressed across multiple postnatal alveolar stages

Plot of Z-scores (y-axis) for gene expression levels of selected genes associated with lung alveolus development. Each error bar is constructed using 1 standard error from the mean. Transcription factors (Hopx, Nkx2-1, Errfi1), growth factors (Fgfr2, Vegfa) and genes involved in pulmonary surfactant production (Sftpd, Abca3, Gpr116, Napsa) are differentially expressed between ALV1-ALV2 and/or ALV3-ALV4.

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Genes associated with axon guidance are differentially expressed across multiple stages of postnatal alveolarization

Plot of Z-scores (y-axis) for gene expression levels of selected genes associated with axon guidance and neurogenesis. Each error bar is constructed using 1 standard error from the mean.

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Expression profiles of known and novel cell differentiation markers throughout lung development

Heatmap showing the relative expression of 35 genes previously associated with cellular differentiation in the lung. Each expression profile is relative to the average expression of that gene across all time points. Solid blue indicates 1.5-fold decrease relative to average; yellow indicates 1.5-fold increase. Gene loading values (PC1-3) are shown to right of heatmap; dark red shading indicates that a gene is within the top 5% of contributors to that respective PC; light red or dark red squares indicate genes that were captured by the mDLCS.

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Genes associated with chemotaxis are differentially expressed between strains during postnatal alveolarization

Plot of Z-scores (y-axis) for gene expression levels of genes associated with chemotaxis. Each error bar is constructed using 1 standard error from the mean. (A) Genes associated with chemotaxis with higher expression levels in AJ or C3H relative to B6. (B) Genes associated with immune-related chemotaxis with higher expression levels in C3H relative to AJ or B6. Alveolar stages (ALV1-4) highlighted in red.

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Genes associated with pulmonary innervation are differentially expressed in B6 relative to AJ or C3H during the EMB stage of lung development Plot of Z-scores (y-axis) for gene expression levels of genes associated with pulmonary innervation expressed lower in B6 relative to AJ or C3H

Each error bar is constructed using 1 standard error from the mean. Embryonic stage (EMB) highlighted in orange.

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Genes that are differentially expressed in AJ relative to C3H or B6

Plot of Z-scores (y-axis) for gene expression levels of genes differentially expressed in AJ relative to C3H or B6. Each error bar is constructed using 1 standard error from the mean.

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Expression profiles of genes associated with Wnt signaling and fibrosis across lung development with comparisons between strains Summary of strain effects and significant stage by strain effects for genes involved in Wnt signaling (A) and fibrosis (B)

Light red highlighting indicates genes that were included in the mDLCS; dark red highlighting indicates genes in the top 5% of contributors to a given PC. Heatmaps contrast expression levels in A/J (left) or C3H/HeJ (right) with C57BL/6J. Solid blue indicates a 1-fold increase in expression between strains; solid yellow indicates a 1-fold decrease in expression between strains.

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qPCR validation of strain-specific differences in gene expression during lung development

ΔCt = cycle threshold, normalized to the geometric mean of three control genes (Actb, Rpl10, Rpl13a). Variation between biological replicates (detected by microarray) did not significantly impact trends of strain-variation when quantified by qPCR. Each error bar constructed using one standard error from the mean. Significant differences detected by Tukey multiple comparisons ANOVA between strains. P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

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Postnatally induced toll-like receptor, lymphocyte antigen, chemokine receptor/ligand and interleukin genes with high loading values on PC1 The shift in gene expression patterns from prenatal development (EMB-CAN) to postnatal development (SAC-MAT) captured by PC1 (see Fig. S1) is characterized by expression changes in genes associated with immune system function

PCA-derived loading values (for PC1-3) also shown for each of these genes.

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Differences in strain-dependent gene expression at postnatal day 13 of murine lung development

Mean differences (Mean diff.) in relative expression units (A.U.) between strains analyzed by Tukey multiple testing corrected ANOVA. Results predominately conform to the trends of strain-dependent expression detected by microarray (shown next to each gene name on left) as seen by comparing qPCR significance to regression significance.

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Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Kyle J. Beauchemin conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Julie M. Wells conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.

Alvin T. Kho contributed reagents/materials/analysis tools, reviewed drafts of the paper, secured funding.

Vivek M. Philip contributed reagents/materials/analysis tools, reviewed drafts of the paper.

Daniela Kamir analyzed the data, reviewed drafts of the paper.

Isaac S. Kohane reviewed drafts of the paper, secured funding.

Joel H. Graber conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.

Carol J. Bult conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper, secured funding.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

All animal work was performed in accordance with Jackson Laboratory Animal Care and Use Committee (ACUC) protocol 101011.

Microarray Data Deposition

The following information was supplied regarding the deposition of microarray data: GEO GSE74243.

Data Availability

The following information was supplied regarding data availability:

The raw data has been supplied as Supplemental files.

Funding

The research reported in this publication was funded in part by The Jackson Laboratory (JAX) Faculty Development Fund, the Maine Cancer Foundation, NIH HD068250, P30CA034196, and HL91124. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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