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Fig. 1 | BMC Genomics

Fig. 1

From: Comparative gene expression profiling between optic nerve and spinal cord injury in Xenopus laevis reveals a core set of genes inherent in successful regeneration of vertebrate central nervous system axons

Fig. 1

Using Xenopus laevis to discover prospective core genetic programs for functional recovery from central nervous system (CNS) injury. aXenopus laevis occupies a transition point in the phylogenetic decline (green to red) of functional recovery after CNS injury in vertebrates. Like other anurans (yellow), X. laevis regenerates optic axons to restore vision throughout life, but only successfully regenerates spinal cord axons as tadpoles. b A three-tissue comparison was designed to parse out core sets of genes most closely associated with successful CNS axon regeneration. Injury-induced gene expression profiles (RNA-seq) were compared between two regenerative tissues [stage 53 tadpole hindbrain after spinal cord transection (SCI) and 1–3 month, post-metamorphic, juvenile frog eye after optic nerve crush (ONC)] and a non-regenerative tissue [1–3 month, post-metamorphic, juvenile frog hindbrain after SCI] to find injury-induced genes that were uniquely shared between the regenerative CNS tissues but not with the non-regenerative one. c Previous histological, electrophysiological, and behavioral studies in X. laevis were consulted to select three time points after optic nerve crush (ONC) and spinal cord transection (SCI) for making suitable comparisons among the tissues - an early trauma phase, when damaged axons first begin to cross the lesion site (3 days), a peak period of maximal regenerative axon outgrowth (7 days for SCI & 11 days for ONC), and a late period, after regenerative axon regrowth is largely completed, but synaptic refinement and behavioral recovery continues (3 weeks) [11, 33, 40, 42, 43, 98, 126, 128, 152, 160]. d Scale drawing of the CNS superimposed on the outline of a juvenile frog, to illustrate the injury sites and harvested tissues (ONC and SCI surgeries were done in separate animals; the tadpole is not illustrated, but its hindbrain and spinal cord transection site were similar in location to those of juvenile frog). For tadpole SCI, hindbrains were harvested from operated animals and age-matched unoperated controls (5 pooled hindbrains per biological replicate, with 3 paired injury and control replicates per time point). For juvenile frog SCI, the same unoperated controls were used for all three SCI time points (5 animals pooled into each of 3 biological replicates). For ONC, both eyes of juvenile frogs receiving an orbital nerve crush on the right side were harvested; the right eye provided the ONC samples and the left, contralateral, unoperated eye provided the control (6 pooled eyes for each of three biological replicates per time point). Three biological replicates of surgically naive eyes were also collected (see text). Red bars indicate the anatomical locations of the optic nerve crush and spinal cord lesions. Dotted red arrows indicate the trajectories of the axons injured by the surgeries whose cell bodies are located in the tissues sampled for RNA-seq. e Diagram summarizing the workflow of the study (see text for details). f13 Summary characteristics of the RNA-seq data. f1, a histogram of the number of successfully aligned reads in each of the 51 samples (17 conditions, 3 biological replicates each). f2, an example of histograms of expression values [log10(FPKM)] per gene, averaged across the biological replicates, normalized for the total number of genes assayed (Gene Density). Data for the 1 week SCI tadpole hindbrain (gray) is superimposed upon that of its age-matched control (blue). The inflection point (dotted vertical line) was used to set a threshold for the fpkm of actively expressed genes. Values below this were categorized as representing no expression. f3, Whisker plot summarizing the data dispersion for all 17 conditions (3 biological replicates per condition). The median log10(FPKM) is represented as a horizontal line through the box, which in turn delimits the 2nd (lower) and 3rd (upper) quartiles of the data. Whiskers illustrate the 1st and 4th quartiles, with their minimum and maximum values, respectively. Abbreviations: CNS, central nervous system; Cntrl, control; FPKM, fragments per kilobase of exon mapped; Juv., juvenile frog; ONC, optic nerve crush; SCI, spinal cord injury; Tad., tadpole; Tx, transection; Unop, unoperated

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