XB-ART-57252BMC Genomics August 5, 2020; 21 (1): 540.
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.
BACKGROUND: The South African claw-toed frog, Xenopus laevis, is uniquely suited for studying differences between regenerative and non-regenerative responses to CNS injury within the same organism, because some CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs. Tissues from these CNS regions (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) were used in a three-way RNA-seq study of axotomized CNS axons to identify potential core gene expression programs for successful CNS axon regeneration. RESULTS: Despite tissue-specific changes in expression dominating the injury responses of each tissue, injury-induced changes in gene expression were nonetheless shared between the two axon-regenerative CNS regions that were not shared with the non-regenerative region. These included similar temporal patterns of gene expression and over 300 injury-responsive genes. Many of these genes and their associated cellular functions had previously been associated with injury responses of multiple tissues, both neural and non-neural, from different species, thereby demonstrating deep phylogenetically conserved commonalities between successful CNS axon regeneration and tissue regeneration in general. Further analyses implicated the KEGG adipocytokine signaling pathway, which links leptin with metabolic and gene regulatory pathways, and a novel gene regulatory network with genes regulating chromatin accessibility at its core, as important hubs in the larger network of injury response genes involved in successful CNS axon regeneration. CONCLUSIONS: This study identifies deep, phylogenetically conserved commonalities between CNS axon regeneration and other examples of successful tissue regeneration and provides new targets for studying the molecular underpinnings of successful CNS axon regeneration, as well as a guide for distinguishing pro-regenerative injury-induced changes in gene expression from detrimental ones in mammals.
PubMed ID: 32758133
PMC ID: PMC7430912
Article link: BMC Genomics
Species referenced: Xenopus laevis
Genes referenced: abcb1 acsbg2 aldoa apoe cntrl coq8a cyb5r2 ebf3 enpp2 ezh2 fabp3 fabp7 fads1 fxyd1 hbe1 hes5.1 idh1 irf8 jarid2 kcnn3 kdm7a krt78.5 lep mapk8 mcm6 mex3a ocm1 otop3 pdf pkd2 plp1 prmt1 prph rplp1 slc38a4 slc9a3r2 snrpd3 socs3 sox11 suz12 tf ttl tuba1a tubb2b ugt8 znf395
GO keywords: neural tissue regeneration
GEO Series: GSE137844: NCBI
Article Images: [+] show captions
|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). f1–3 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|
|Fig. 2 Temporal patterns of gene expression and shared injury-response genes between regenerative vs. non-regenerative tissues. a Regenerative tissues [i.e., SCI tadpole hindbrain (SCI Tadpole) and ONC juvenile frog eye (ONC Juvenile)] shared similar temporal patterns of numbers of significant (FDR < 0.05) differentially expressed genes, which differed markedly from that of the non-regenerative tissue [SCI juvenile frog hindbrain (SCI Juvenile)]. Whereas the expression response of the two regenerative tissues peaked during the mid recovery phase (1 week/11 days), that of the non-regenerative tissue peaked at the early, post trauma phase (3 days). Up- and down-regulated genes are shown in green and red, respectively; S & L gene homeologs were tallied separately. b Plot illustrating the percentage of annotated genes that were significantly (FDR < 0.05) differentially expressed with injury (100% = 24,382 genes). Additional_File1_Differential_Expression_Analysis_by_Cuffdif.xlsm contains the CuffDiff2 output files from which A and B were derived. c - e UpSet plots illustrating the number of genes overlapping between the samples indicated by the circles below each bar at 3 days (c), 7/11 days (d), and 3 weeks (e) after injury. Numbers of shared up- and down-regulated genes are indicated above and below each bar, respectively. The maximum number of overlapping genes between the two successfully regenerative tissues (DESR: Differentially Expressed in Successful Regeneration) occurred during the peak phase of regenerative CNS axon outgrowth. Additional_File4_DESR_Data.xlsm contains the DESR data|
|Fig. 3 Eigenvector representation of the Principal Component Analyses (PCA) of gene expression profiles. Eigenvectors depict relative degrees of similarity among data sets, as indicated. Black points represent individual genes plotted against the principal axes of similarity (PC1, PC2). a, PCA of all 17 experimental conditions and controls. b, PCA of SCI hindbrain samples and their unoperated controls. c, PCA of ONC operated eye expression profiles, as well as those of their paired, contralateral unoperated control eyes and eyes of uninjured animals (surgically naive). Abbreviations: Cntrl, control hindbrain; Juv, juvenile (1–3 month post-metamorphic) frog; ONC, optic nerve crush; PC1, principal component axis 1; PC2, Principal Component axis 2; SCI, spinal cord injured; Tx, spinal cord transection; Unop, unoperated eye, contralateral to the ONC; Wk, week. Additional_File5_PCA_Scatterplot.pdf shows PCA scatterplots|
|Fig. 4 Grouping DESR genes by known functions provided insights into processes underlying successful CNS axon regeneration. a Curating DESR genes based on functions documented in the scientific literature (see text for details) parsed them into eleven categories. Vertical boxes outline functional categories present at all three time points. Boxes with arrows list prominent functional sub-categories at the different time points (see text for details). Green shades, upregulated genes; red shades, down-regulated genes (S & L homeologs tallied separately).b Pie charts display data from (a) according to each category’s relative contribution (%) to the total number of DESR genes and are scaled in size to reflect the total number of DESR genes at each time point (N). Additional_File6_DESR_Functional_Categories.pdf contains a list of the DESR genes, separated by time point and category, along with relevant literature citations supporting the functional categorization. Because functions are mostly based on mammalian studies, and Xenopus generally has more than one homeolog for each human gene, they are listed without regard to which homeolog is differentially expressed. Detailed data for individual homeologs are in Additional_File4_DESR_Data.xlsm|
|Fig. 5 KEGG pathway analyses of DESR genes expressed across multiple time points implicated the Adipocytokine signaling pathway as playing a prominent role in successful CNS axon regeneration. Black pentagons identify DESR’s. Ellipses indicate differential expression (DE; FDR < 0.05) in response to injury (up or down) in at least one tissue, during at least one time point. The Adipocytokine signaling pathway image was obtained from KEGG (Kyoto Encyclopedia of Genes and Genomes). Additional_File7_Adipocytokine_Signaling_Pathway_Gene_Expression_Data.xlsm contains an Excel spreadsheet with all 115 genes (S&L homeologs are separate entries) belonging to this pathway, together with their δFPKM values [log2(fold change, injury/control)], and their p and q (FDR-adjusted p) values for differential expression, at each time point, for all three tissues|
|Fig. 7. Cellular localization of select DESR genes by in situ hybridization of retina at the peak phase of regenerative axon outgrowth after optic nerve injury. Genes are as indicated in their respective panels and represent a range of fold-change values (0.03 < |log2(fold change)| < 3), and FDRs (0.002–0.05), as well as relatively low (FPKM < 50) and high (FPKM > 100) levels of expression. Examples of up-regulated (a – e) and down-regulated (f, g) genes are included. Column 1 (left), operated eye; column 2 (right), contralateral unoperated eye from the same animal and processed on the same slide as that of its adjacent column. Arrows indicate cells of the retinal ganglion cell layer, which comprises the neurons that regenerate an axon. Abbreviations: RGC, retinal ganglion cells; INL, inner nuclear layer; PR, photoreceptors. Scale bar in G2 applies to all panels|
|Additional File 5 Scatterplot representation of the Principal Component Analyses (PCA) of gene expression profiles. Ellipses group biological replicates for each indicated condition (experimentals, solid squares; controls, empty squares), indicating variation among samples. AC, PCA of tadpole and juvenile hindbrain after spinal cord injury (SCI), and of juvenile frog after optic nerve crush (ONC), respectively. In C, expression profiles from the operated eye were compared with those of the contralateral, unoperated eyes within the same animals. D, PCA of all 17 conditions combined, supporting the tissue-specific nature of gene expression profiles. Conditions were the same as in AC, except that data from eyes of animals receiving no injury was included (open triangles, Frog Eye, Unop). E, PCA of tadpole and juvenile frog hindbrain samples after spinal cord injury, supporting the conclusion that the differences in gene expression profiles between the time points at which numbers of differentially expressed genes reached their peaks (3 days in juvenile frog hindbrain and 1 week in tadpole hindbrain) were more than just a kinetic difference in the timing of expression of the same differentially expressed genes. Abbreviations: ONC, optic nerve crush; PC1, principal component axis 1; PC2, Principal Component axis 2; SCI, spinal cord injured; TX, spinal cord transected; Unop ONC unoperated eye, contralateral to the operated eye; Frog Eye, Unop eyes from unoperated animals; wk, week.|
References [+] :
Abe, Mammalian target of rapamycin (mTOR) activation increases axonal growth capacity of injured peripheral nerves. 2010, Pubmed