Retinal transcriptome of neonatal mice after optic nerve injury

Abstract

Background: The axonal growth capacity of retinal ganglion cells decreases dramatically within the first day of birth, and the axonal regeneration after injury in mature mammals is very limited. Here, this study aimed to delineate the transcriptomic changes associated with altered axonal growth capacity and to identify the key genes associated with axonal regeneration by the RNA sequencing (RNA-Seq) analysis.

Methods: The whole retinas from the mice of embryonic day (E) 20, postnatal day (P) 1 and P3 were collected at 6 hours after optic nerve crush (ONC). Differentially expressed genes (DEGs) for ONC or ages were identified by the RNA-Seq analysis. K-means analysis was conducted for the clustering of DEGs based on expression patterns. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and Gene Set Enrichment analysis (GSEA). Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the DEGs selected from the RNA-Seq analysis.

Results: In total, 5,408 DEGs were identified for ages, and 2,639 DEGs in neonatal mouse retina after ONC. K-means analysis revealed 7 clusters in age-DEGs and 11 clusters in ONC-DEGs. The GO, KEGG and GSEA pathway analyses identified significantly enrichment of DEGs in the visual perception and phototransduction for the age effect, and the break repair, neuron projection guidance, and immune system pathway for the ONC. PPI analysis identified hub genes in the axon-related gene cluster. The expressions of Mlc1, Zfp296, Atoh7, Ecel1, Creb5, Fosb, and Lcn2, thought to be involved in RGC death and axonal growth were validated by qRT-PCR.

Conclusions: This study, for the first time, delineated the gene expression changes following ON injury in embryonic and neonatal mice, providing a new resource of age- and injury-driven data on axonal growth capacity.

Fig 1. Quantitative analysis of retinal gene expression.

(A) Boxplot shows the overall range and distribution of FPKM value of gene expression of all the samples. (B) PCA analysis shows the differentiation among all the samples. (C) Pearson correlation between all the samples. The more correlation coefficient closer to 1, the more similar the expression pattern between the samples. (D) Cluster analysis of genes among samples. The color of the heat map indicated the relative gene expression. Differentially expressed genes analysis. (E-G, I-K) The volcano map shows the differentially expressed genes in different comparison groups. (H) The Venn diagram showing the common regulated DEGs during the develop of E20-P1-P3. (L) The Venn diagram showing the overlapping regulated DEGs after ONC.

Fig 2. K-means and enrichment analyses (GO, KEGG) of age-DEGs.

(A) K‐means clustering of the differential expression gene with age (K = 7). Each box is a DEG cluster. Each line indicates one differential expression gene. The blue line is the average trend of all DEGs in the cluster. (B) Gene ontology biological process term enrichment of the seven K‐means clusters. The top results are plotted.

Fig 3. K-means and enrichment analyses (GO, KEGG) of ONC-DEGs.

(A) K‐means clustering of the differential expression gene with age (K = 11). Each box is a DEG cluster. Each line indicates one differential expression gene. The blue line is the average trend of all DEGs in the cluster. (B) Gene ontology biological process term enrichment of the seven K‐means clusters. The top results are plotted.

Fig 4

(A-B) Construction of PPI (protein-protein interaction) networks of age-DEGs and ONC-DEGs. Line color indicates the type of interaction evidence. (C-H) GSEA analyses of age-DEGs (C-E) and ONC-DEGs (F-H). The y-axis represents enrichment score, and the x-axis denotes genes (vertical black lines) represented in gene sets. The colored band at the bottom represents the degree of correlation of genes with the INR phenotype (red for positive and blue for negative correlation).

Fig 5. Validation of selected DEGs with qRT-PCR.

(A–D) Statistical graph shows mRNA expression in the control groups with age, normalized to an average expression of 1.0 in the E20 group. (E–L) Statistical graph shows mRNA expression in the ONC and control groups, normalized to an average expression of 1.0 in the control group. Data are represented as Means ± SD; n = 3; *p < 0.05; **p < 0.01; ***p < 0.001.