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

The Transcriptomics to Proteomics of Hair Cell Regeneration: Looking for a Hair Cell in a Haystack

Mature mammals exhibit very limited capacity for regeneration of auditory hair cells, while all non-mammalian vertebrates examined can regenerate them. In an effort to find therapeutic targets for deafness and balance disorders, scientists have examined gene expression patterns in auditory tissues under different developmental and experimental conditions. Microarray technology has allowed the large-scale study of gene expression profiles (transcriptomics) at whole-genome levels, but since mRNA expression does not necessarily correlate with protein expression, other methods, such as microRNA analysis and proteomics, are needed to better understand the process of hair cell regeneration. These technologies and some of the results of them are discussed in this review. Although there is a considerable amount of variability found between studies owing to different species, tissues and treatments, there is some concordance between cellular pathways important for hair cell regeneration. Since gene expression and proteomics data is now commonly submitted to centralized online databases, meta-analyses of these data may provide a better picture of pathways that are common to the process of hair cell regeneration and lead to potential therapeutics. Indeed, some of the proteins found to be regulated in the inner ear of animal models (e.g., IGF-1) have now gone through human clinical trials

Oxford Nanopore Technology: A Promising Long-Read Sequencing Platform To Study Exon Connectivity and Characterize Isoforms of Complex Genes

The central dogma states that the genetic information contained in DNA flows to RNA through the process of transcription which, in turn, can result in protein synthesis through translation. Alternative splicing is a mechanism by which multiple mRNA isoforms are generated from a single gene. Ultracomplex genes, characterized by their ability to encode hundreds to thousands of isoforms, arise from a combination of multiple splicing events. Our understanding of alternative splicing improved vastly in the past decade due to the advent of next-generation sequencing (NGS) technologies. The NGS technologies are powerful and have enabled scientists to measure the expression of genes and isoforms digitally, assemble genomes, reconstruct transcriptomes and clinicians to cater treatments that are specific to an individual’s genetic makeup. While NGS technologies have many strengths, the shorter read lengths generated from these platforms limit their ability to study exon connectivity over long distances and this information is often inferred through statistical means rather than direct measurement. Additionally, the repetitive regions in the genome represents a special case where the short reads have inherent difficulty in joining two adjacent different contigs into a scaffold. The third-generation sequencing technologies, characterized by their ability to generate ultra-long reads can be used to address these limitations. Here, I have used the Oxford Nanopore (ONT) MinION device to first demonstrate the utility of nanopore technology to sequence long reads to identify exon connectivity using the Drosophila Rdl, MRP, Mhc and Dscam1 genes. I extended this approach to sequence full-length cDNAs generated from SIRV spike-in RNA to determine the quantitative ability of the platform. These experiments demonstrate the ability of ONT platform to deconvolute isoforms and by sequencing Drosophila ultracomplex genes, I also show that ONT can identify previously unannotated exons and RNA editing sites over long distances. By using direct RNA sequencing, I demonstrate the ability to sequence full-length Eno2 RNA molecules and that a majority of the reads were sequenced full-length

Oxford Nanopore Technology: A Promising Long-Read Sequencing Platform To Study Exon Connectivity and Characterize Isoforms of Complex Genes

The central dogma states that the genetic information contained in DNA flows to RNA through the process of transcription which, in turn, can result in protein synthesis through translation. Alternative splicing is a mechanism by which multiple mRNA isoforms are generated from a single gene. Ultracomplex genes, characterized by their ability to encode hundreds to thousands of isoforms, arise from a combination of multiple splicing events. Our understanding of alternative splicing improved vastly in the past decade due to the advent of next-generation sequencing (NGS) technologies. The NGS technologies are powerful and have enabled scientists to measure the expression of genes and isoforms digitally, assemble genomes, reconstruct transcriptomes and clinicians to cater treatments that are specific to an individual’s genetic makeup. While NGS technologies have many strengths, the shorter read lengths generated from these platforms limit their ability to study exon connectivity over long distances and this information is often inferred through statistical means rather than direct measurement. Additionally, the repetitive regions in the genome represents a special case where the short reads have inherent difficulty in joining two adjacent different contigs into a scaffold. The third-generation sequencing technologies, characterized by their ability to generate ultra-long reads can be used to address these limitations. Here, I have used the Oxford Nanopore (ONT) MinION device to first demonstrate the utility of nanopore technology to sequence long reads to identify exon connectivity using the Drosophila Rdl, MRP, Mhc and Dscam1 genes. I extended this approach to sequence full-length cDNAs generated from SIRV spike-in RNA to determine the quantitative ability of the platform. These experiments demonstrate the ability of ONT platform to deconvolute isoforms and by sequencing Drosophila ultracomplex genes, I also show that ONT can identify previously unannotated exons and RNA editing sites over long distances. By using direct RNA sequencing, I demonstrate the ability to sequence full-length Eno2 RNA molecules and that a majority of the reads were sequenced full-length

The Transcriptomics to Proteomics of Hair Cell Regeneration: Looking for a Hair Cell in a Haystack

microarray