Next Generation Sequencing Reveals Gene Expression Patterns in the Zebrafish Inner Ear Following Growth Hormone Injection

Loss of hair cells due to acoustic trauma results in the loss of hearing. In humans, unlike other vertebrates, the mechanism of hair cell regeneration is not possible. The molecular mechanisms that underlie this regeneration in nonmammalian vertebrates remain elusive. To understand the gene regulation during hair cell regeneration, our previous microarray study on zebrafish inner ears found that growth hormone (GH) was significantly upregulated after noise exposure. In this current study, we utilized Next Generation Sequencing (NGS) to examine the genes and pathways that are significantly regulated in the zebrafish inner ear following sound exposure and GH injection. Four groups of 20 zebrafish each were exposed to a 150 Hz tone at 179 dB re 1μPa RMS for 40 h. Zebrafish were injected with either salmon GH, phosphate buffer or zebrafish GH antagonist following acoustic exposure, and one baseline group received no acoustic stimulus or injection. RNA was extracted from ear tissues at 1 and 2 days post-trauma, and cDNA was synthesized for NGS. The reads from Illumina Pipeline version SCS 2.8.0 were aligned using TopHat and annotated using Cufflinks. The statistically significant differentially expressed transcripts were identified using Cuffdiff for six different pairwise comparisons and were analyzed using Ingenuity Pathway Analysis. I found significant regulation of growth factors such as GH, prolactin and fibroblast growth factor receptor 2, different families of solute carrier molecules, cell adhesion molecules such as CDH17 and CDH23, and other transcription factors such as Fos, FosB, Jun that regulate apoptosis. Analysis of the cell proliferation network in the GH-injected condition compared to buffer-injected day 1 showed significant up-regulation of GH while downregulation of apoptotic transcription factors was found. In contrast, the antagonist-injected condition compared to the GH-injected condition showed an opposite pattern in which up-regulation of apoptotic transcription factors were found while GH was down-regulated. A number of other transcripts (e.g., POMC, SLC6A12, TMEM27, HNF4A, CDH17 and FGFR2) that showed up-regulation in GH-injected condition showed down-regulation in antagonist-injected condition. These results strongly suggest that injection of exogenous GH potentially has a protective role in the zebrafish inner ear following acoustic trauma