January 1, 2017;
Unlike adult mammals, adult frogs regrow their optic nerve
following a crush injury, making Xenopus laevis a compelling model for studying the molecular mechanisms that underlie neuronal regeneration. Using Translational Ribosome Affinity Purification (TRAP), a method to isolate ribosome-associated mRNAs from a target cell population, we have generated a transcriptional profile by RNA-Seq for retinal ganglion
) during the period of recovery following an optic nerve
injury. Based on bioinformatic analysis using the Xenopus laevis 9.1 genome assembly, our results reveal a profound shift in the composition of ribosome-associated mRNAs during the early stages of RGC
regeneration. As factors involved in cell signaling are rapidly down-regulated, those involved in protein biosynthesis are up-regulated alongside key initiators of axon
development. Using the new genome assembly, we were also able to analyze gene expression profiles of homeologous gene pairs arising from a whole-genome duplication in the Xenopus lineage. Here we see evidence of divergence in regulatory control among a significant proportion of pairs. Our data should provide a valuable resource for identifying genes involved in the regeneration process to target for future functional studies, in both naturally regenerative and non-regenerative vertebrates.
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Fig. 1. Profiling retinal ganglion cell regeneration. (A) The effects of axonal injury on retinal ganglion cells (RGCs) in the optic tectum can be visualized using frog lines expressing GFP under the control of an RGC-specific promoter (islet2b). An example time series shows the key transition point falls between 3 and 7 days post-injury, with full recovery occurring by 210 days (210x) post-injury. (B) Quantification of mean GFP fluorescence intensity in the tectum, as seen in panel (A). Data were averaged from at least 5 biological replicates per day and error bars represent the standard deviation from the mean. (C) In this study, gene expression in RGCs is directly compared between a right eye in which the optic nerve has undergone a surgical crush (Crush) to the untreated left eye of the same animal (Control) for various days after surgery (1, 3, 7, 11). Additional controls include a sham surgery (Sham), non-surgical animals (Naïve), and RNA from whole retina (Total RNA). (D) To allow for tissue specific isolation of ribosome-associated mRNAs from RGCs, a transgenic line of Xenopus laevis is used that expresses an eGFP tagged variant of rpl10a under the control of an RGC-specific promoter (islet2b). (E) Following retina dissection, ribosome-associated RNAs in RGCs are purified using eGFP coated magnetic beads; subsequent poly(A) selection enriches for mRNA species. This mRNA fraction is then used for RT-qPCR validation and RNA-Seq library construction.
Fig. 2. RNA-Seq read alignment and analysis. (A) Proportion of RNA-Seq reads that were mapped to the Xenopus laevis 9.1 genome assembly across experimental naïve (−), contralateral control (c) and crush (x) samples demonstrates good mapping rates across the various days post-crush (Dpc). (B) Read counts, expressed as FPKM, for each gene sequenced in the crushed (FPKMC; x-axis) versus control eye (FPKMX; y-axis) showing both up- and down-regulation in the days following injury. (C) Comparison of changes in gene expression following optic nerve crush between homeolog-pairs (L x-axis, S y-axis) across the post-injury time course, shows overall correlation between pairs with clear outliers. (D) The magnitude of difference between changes in gene expression among homeolog-pairs, quantified by Euclidean distance, increases over time after optic nerve injury.
Fig. 3. Independent biological validation of RNA-Seq. The up- and down-regulation of select factors in response to optic nerve injury described in the TRAP RNA-Seq analysis was independently verified by in situ hybridization, RT-qPCR and immunofluorescence. (A) In situ hybridization demonstrates that uchl1 transcript is up-regulated unilaterally in RGCs by 11 days post-injury (Day 11x). Autofluorescence of photoreceptor cells (PR) shows no injury-dependent variability. (B) Semi-quantitative analysis of fluorescence intensity from uchl1 in situ, as shown in panel A, shows a significant up-regulation of uchl1 mRNA in the crushed eye relative to control eye in the RGC layer by day 11 (p <0.01). Results were averaged for three retinal sections from each of three different individuals and data represent arbitrary fluorescence units (au). (C) Gene expression levels assayed using RT-qPCR for uchl1 and snca (bars) are highly correlated with changes in fold gene expression quantified by RNA-Seq (line graph). (D) As predicted by RNA-Seq, protein levels for sncg decrease over the experimental time course in injured RGCs, as seen by immunofluorescence. (E) Semi-quantitative analysis of fluorescence intensity in the RGC layer immunostained for sncg protein, as in panel D, shows strong down-regulation of sncg in crush relative to control eyes by day 11. This boxplot shows averaged results for two retinal sections from each of four different individuals and values represent arbitrary fluorescence units (au). In A and D, location of the RGCs observed in the ganglion cell layer (GCL) and highlighted by brackets, photoreceptor cell (PR) outer (ONL) and inner (INL) nuclear and inner plexiform (IPL) layers was visualized using DAPI. The scale bar is 50 µm.
Fig. 4. Global changes in RGC gene expression following optic nerve injury. (A) The differences in transcript abundance in each sample was compared pair-wise using the Euclidean distance between FPKM values. In this heat map, the brightest blue represents the most closely related FPKM profiles. Control samples are highly similar, while samples from the optic-nerve crush show large shifts in gene expression pattern in the days following injury. (B) Hierarchical clustering of genes sequenced in RGCs across the experimental time course. Clusters of up- and down-regulated genes shows strong correspondence to groups identified by k-means clustering (groups 1 & 2 yellow, up-regulated; groups 3 & 4 blue, down-regulated; group 5 grey, unchanged).
Fig. 5. Functional specificity in the set of transcripts that are up- and down-regulated following retinal ganglion crush. (A) Heat maps showing hierarchical clustering of transcripts that are up- (left) and down-regulated (right), identified by k-means clustering, following retinal ganglion crush. (B) The set of Gene Ontology biological process terms that are significantly over represented in each group of transcripts. The evidence for enrichment of each term (log10(p-value)) is plotted for the group of up- (left) and down-regulated genes (right); there is little overlap in significantly enriched terms.
Fig. 6. RGC-enriched factors. (A) Gene families that are highly enriched in naïve TRAP samples compared to total retina include key RGC-specific factors along-side novel groups. (B) When we compare gene expression in crush versus control samples, we see that optic nerve injury leads to strong down-regulation of many of these RGC-enriched genes.
Fig. 7. Gene groups differentially regulated following optic nerve injury. (A) Many axonal regeneration-associated genes (RAGs) and development associated factors are up-regulated following crush (tubulin, gap43, klf6), while some factors shown to inhibit axonal growth are down-regulated (klf4). (B & C) Stress response pathways shown to be up-regulated by optic nerve injury in other vertebrate species are up-regulated in Xenopus laevis RGCs as well. (D) Some members of the Jak/Stat signaling pathway are dramatically up-regulated. (A–D) In all panels, we compare the log2 ratio of expression levels for genes in the crush versus control samples. Genes with names not appended “.L” or “.S” (hmox1, IL-10), represent symbols not yet assigned in the Xenopus laevis 9.1 genome assembly. In these cases, data are shown from read alignments to a transcript reference (detailed in Material and methods).
Continued neurogenesis is not a pre-requisite for regeneration of a topographic retino-tectal projection.