Transcriptional programs controlling perinatal lung maturation

Abstract

The timing of lung maturation is controlled precisely by complex genetic and cellular programs. Lung immaturity following preterm birth frequently results in Respiratory Distress Syndrome (RDS) and Broncho-Pulmonary Dysplasia (BPD), which are leading causes of mortality and morbidity in preterm infants. Mechanisms synchronizing gestational length and lung maturation remain to be elucidated. In this study, we designed a genome-wide mRNA expression time-course study from E15.5 to Postnatal Day 0 (PN0) using lung RNAs from C57BL/6J (B6) and A/J mice that differ in gestational length by ∼30 hr (B6<A/J). Comprehensive bioinformatics and functional genomics analyses were used to identify key regulators, bioprocesses and transcriptional networks controlling lung maturation. We identified both temporal and strain dependent gene expression patterns during lung maturation. For time dependent changes, cell adhesion, vasculature development, and lipid metabolism/transport were major bioprocesses induced during the saccular stage of lung development at E16.5-E17.5. CEBPA, PPARG, VEGFA, CAV1 and CDH1 were found to be key signaling and transcriptional regulators of these processes. Innate defense/immune responses were induced at later gestational ages (E18.5-20.5), STAT1, AP1, and EGFR being important regulators of these responses. Expression of RNAs associated with the cell cycle and chromatin assembly was repressed during prenatal lung maturation and was regulated by FOXM1, PLK1, chromobox, and high mobility group families of transcription factors. Strain dependent lung mRNA expression differences peaked at E18.5. At this time, mRNAs regulating surfactant and innate immunity were more abundantly expressed in lungs of B6 (short gestation) than in A/J (long gestation) mice, while expression of genes involved in chromatin assembly and histone modification were expressed at lower levels in B6 than in A/J mice. The present study systemically mapped key regulators, bioprocesses, and transcriptional networks controlling lung maturation, providing the basis for new therapeutic strategies to enhance lung function in preterm infants.

Figure 1. Dynamic changes in lung mRNAs with advancing gestational age.

(A) Heatmap of the 1938 mRNAs that were similarly altered in lung from both C57BL/6J (B6) and A/J mouse strains (E15.5 – PN0). Samples were normalized using the mean value of three E15.5 mice from each strain as the baseline. The intensity in red-to-green color indicates the up and down regulation of mRNAs, respectively. Each row represents a single gene, and each column represents a developmental time point for A/J or B6 mice. For each time point, samples from three biological replicates were averaged. (B) and (C) represent the top 20 induced and repressed genes during lung maturation in A/J and B6 mice respectively. The full descriptions for gene symbols can be found under the section of “Abbreviations”.

Figure 2. Most changed transcription factors (TF) and signaling molecules (SM) and their dynamic expression profiles at different gestational stages.

TF/SMs changed during lung maturation were identified by two-way ANOVA with a cut-off of p-value<0.001 and fold change >1.25. Altered TF/SMs were clustered on the basis of their initial change occurring at E16.5, E17.5, and E18.5 or later, labeled as A) E16 up, B) E17 up, C) E18–19 up, D) E16 down, E) E17 down and F) E18–19 down respectively. Most of the TF/SMs show similar dynamic expression profiles in A/J vs. B6 mouse strains. The x-axis represents the gestational ages and the y-axis (in log₂ scale) represents relative mRNA using E15.5 as the baseline. |Fold| represents the absolute value of fold change between the peak expression of a given gene (E15.5 – PN0) and its baseline expression at E15.5. Profiles are expressed as the means ± SEM of 3 animals per time point. The full descriptions for gene symbols can be found under the section of “Abbreviations”.

Figure 3. Dynamic changes in TF/SMs during lung maturation.

(A) mRNAs encoding TF/SMs that increased from E17.5 were functionally enriched in regulation of angiogenesis. Vegfa was identified as an important regulatory hub. (B) mRNAs encoding TF/SMs regulating angiogenesis were highly correlated with Vegfa expression (Pearson's correlation coefficients r>0.85). (C) TFSMs induced from E18.5 or later was functionally enriched in genes regulating immune/defense response, cell differentiation and phosphorylation. (D) Biological association networks of TF/SMs indicate that the JAK/STAT1 and AP-1 complexes form important hubs. Cytokine-cytokine receptor interaction was the most enriched signaling pathway at this gestational age. Genes/proteins are represented as nodes, and the biological relationships between two nodes are represented as an edge (line). Solid lines denote direct interactions and dotted lines indirect interactions between nodes. TFs are outlined in red. Cytokines and cytokine receptors are outlined in blue. TF/SMs decreasing from E17.5 to PN0 were functionally enriched in (E) cell cycle and (F) chromatin assembly/organization. In the proposed cell cycle related sub-network (E), Foxm1, Myc, Nfyb and Tfdp1 formed important TF-hubs (red nodes). The Plk1 signaling pathway was a highly enriched pathway (blue nodes). In the chromatin assembly and histone modification related sub-network (F), the high mobility group family, chromobox family, SWI/SNF family and mini-chromosome maintenance proteins formed important hubs (red nodes).

Figure 4. Identification of temporal dependent lung mRNA expression patterns and predicted functions.

mRNAs that changed in both strains during lung maturation were clustered into 6 temporally dependent expression patterns using STEM . The x-axis represents the gestational ages and the y-axis represents relative mRNA using E15.5 as the baseline. Profiles are expressed as the means ± SEM of 3 animals per time point. Most highly enriched functional categories for each cluster were identified using DAVID (http://david.abcc.ncifcrf.gov/summary.jsp).

Figure 5. Transcriptional regulatory networks (TRN) of major gene clusters that changed during lung maturation.

(A) Proposed TRN for C-79 genes and its matched TF/SMs. (B) Proposed TRN for C-71 genes and its matched TF/SMs. Nodes in bold were TF/SMs that were predicted to serve as important regulatory hubs in the network.

Figure 6. Identification of strain dependent changes in mRNAs during lung maturation.

(A) Heatmap of mRNAs expressed differentially in two mouse strains at E18.5 but were not changed comparing A/J at E19.5 to B6 at E18.5. Two major expression patterns were revealed by hierarchical clustering, one is induced with advancing gestational age, and the other is repressed with advancing gestational age. (B) and (C) Analysis of enrichment of functional terms associated with genes in the two clusters. Top functional terms (ranked by p -value) are listed.

Figure 7. Functional classes of mRNAs associate with strain dependent changes.

Genes involved in (A) surfactant homeostasis and (B) innate defense were expressed at significantly higher levels in B6 than in A/J mice at E18.5. (C) Histone associated genes and (D) genes involved in chromatin assembly/disassembly were expressed at significantly lower levels in B6 than in A/J mice at E18.5. (E) RT-PCR validation of selected mRNAs encoding chromatin regulators in lungs from B6 and A/J mice at E18.5 is shown. Results are expressed as the means ± SEM of 3 animals per time point.

Figure 8. Changes of lung mRNAs related to the Notch Signaling Pathway were influenced by strain.

(A) Representation of mRNAs changed during lung maturation in KEGG Notch Signaling Pathway (map04330). Red nodes indicate mRNAs that were significantly increased, while dark green indicates decreased expression during lung maturation. Light green indicates no significant change in gene expression or data not available. (B) Genes encoding a number of key components in the Notch Signaling Pathway were expressed at significantly higher levels in A/J than in B6 mice at E18.5 (*: P<0.05). Results are expressed as the means ± SEM of 3 animals per time point.

Figure 9. Schematized depiction of temporal and strain dependent effects on lung maturation.

(A) Representation of bioprocesses and predicted key regulators changed dynamically with advancing gestation. (B) Strain effects underlying lung maturation are shown. Innate immune responses and surfactant production are critical and connected processes that play positive roles to promote lung maturation, while epigenetic regulators are likely to play a repressive role by altering chromatin structure and controlling the cell cycle. The precise balance of these positive and negative gene networks is likely a critical determinant that coordinates the timing of lung maturation.