BioProject

Unlike adult mammals, adult frogs regrow and regenerate their optic nerve following a crush injury. More...

Unlike adult mammals, adult frogs regrow and regenerate their optic nerve following a crush injury. Using Translational Ribosome Affinity Purification (TRAP), a method to isolate mRNAs actively undergoing translation in a target cell population, we have generated a transcriptional profile by RNA-Seq for retinal ganglion cells (RGC) during the period of recovery following an optic nerve injury. Based on bioinformatics analysis using the JGI 9.1 Xenopus laevis gene models, our results reveal a profound shift in the composition of actively translating mRNAs during the early stages of RGC regeneration: as factors involved in cell signaling are rapidly downregulated, and those involved in core metabolism are upregulated. We identified one highly upregulated gene in response to injury, uchl1, which coupled to downregulation of the synucleins (snca, scng), was previously implicated in neurodegenerative diseases. Our injury-screen in Xenopus identified a previously unknown gene, gng8, as being associated with the regenerative process. Our generated online database provides the Xenopus community a valuable resource for the identification of genes involved in the regeneration process to target for future functional studies. Overall design: To investigate the changes in gene expression that occur as retinal ganglion cells (RGCs) recover and regrow following injury, we have used the TRAP method. With TRAP we are able to isolate the actively translating pool of mRNAs from a specific cell type, in this case RGCs. To do this, we created lines of transgenic frogs which express an eGFP-tagged variant of the large ribosomal protein rpl10a under the control of an RGC-specific promoter from the islet2b locus. In our experimental framework, we quantify gene expression changes in RGCs recovering from optic nerve crush by comparing mRNA levels in samples collected from the eye undergoing a surgical crush (right) to the contralateral eye (left). At discrete time points following optic nerve crush in the left eye, both eyes are rapidly dissected and the ribosome-associated RNAs purified from tissue extracts using eGFP antibodies conjugated to magnetic beads. To control for the effects of surgery on RGCs, gene expression was also quantified in animals that underwent sham surgeries with no optic nerve crush ("sham" samples). To control for the systemic effects of the surgical procedure per se, gene expression was also quantified in animals that did not undergo any surgery ("naive" sample). These mRNA pools were used to construct libraries for RNA-Seq using poly(A) selection and 2x multiplexing. Less...