GSE113719
Transcriptional respose to Wnt activation regulates the regenerative capacity of the murine postnatal cochlea
Purpose: Wnt activation can induce regeneration in the early postnatal cochlea, but the regenerative capacity of the postnatal cochlea diminishes between P0 to P5. The goal of this study was to utilize RNA-Seq for the overall evaluation of expression patterns, comparing P1 and P4 newborn cochlea with and without Wnt activation. Methods: Sox2-CreER; ß-cateninflox(Exon3)/+ mice were used to stabilize ß-catenin in Sox2+ cochlear supporting cells. Tamoxifen was injected to the mother at either P1 or P4 to induce Cre activation in the pups. mRNA libraries were generated from treated and untreated pups 3 days after injection in triplicates (overall 12 samples) using ThruPLEX DNA-seq kit (Rubicon Genomics) and then sequenced using Illumina NextSeq500 Single-End 75bp. Reads were aligned to mm10 and quality control was assessed using FastQC and Qualimap followed by FeatureCounts, Sailfish v0.10.1 and tximport. Differential expression was evaluated using DESeq2 and subsequent pathway analysis was performed using Ingenuity Pathway Analysis Software (Qiagen). Results: 40-50 million reads were detected per sample. PCA and Pearson correlation demonstrated a clear segragation of the samples by age that was supported by the identification of 2914 differentially expressed genes between P1 and P4 samples. Additional affect of treatment resulted is 530 differentially expressed genes between treated and untreated samples at P1 and 304 genes were differentially expressed between treated and untreated samples at P4. Clear activation of the Wnt pathway was apparent at both time-points after treatment. However, activation of downstream pathways that may be involved in regeneration was detected at P1 but not at P4, mainly the involvement of cell-cycle and proliferation as well as Erbb2 associated genes. Conclusions: Our results suggest that the Wnt pathway is still active and can be further induced at early and late postnatal stages in a similar manner. However, at early stages, exogenous Wnt activation potentially promotes an arrest of maturation progression or even partial dedifferentiation of supporting cells into a less mature state that enables regeneration, while the downstream affect is not as prominenet later on. Proliferative capacity in the early postnatal cochlea is correlated with a unique transcriptional response that diminishes in the older cochlea, including a robust increase in the expression of cell cycle genes and cellular growth and development genes. Our results suggest that the downstream transcriptional response to Wnt activation is the key factor mediating a regenerative response, or a lack-thereof, in the mammalian cochlea and that efforts to stimulate regeneration in the adult auditory organ may require recapitulating the transcriptional response of the early neonatal cochlea to exogenous Wnt activation and subsequent alterations of silenced state of Wnt target genes. . Overall design: Samples were generated in triplicates from treated (tamoxifen injection to Sox2-CreER; ß-cateninflox(Exon3)/+ mice leading to ß-catenin stabilization) and untreated (tamoxifen injection to mice without CreER) pups at P4 (injected at P1) and P7 (injected at P4). During the analysis process, treated samples were compared to untreated at every time-point as well as between time-points.