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. 2022 Jul 27;11(15):2320.
doi: 10.3390/cells11152320.

Transcriptomes of Injured Lamprey Axon Tips: Single-Cell RNA-Seq Suggests Differential Involvement of MAPK Signaling Pathways in Axon Retraction and Regeneration after Spinal Cord Injury

Li-Qing Jin  1   2 , Yan Zhou  3 , Yue-Sheng Li  4 , Guixin Zhang  1   2 , Jianli Hu  1   2 , Michael E Selzer  1   2   5
Affiliations

Transcriptomes of Injured Lamprey Axon Tips: Single-Cell RNA-Seq Suggests Differential Involvement of MAPK Signaling Pathways in Axon Retraction and Regeneration after Spinal Cord Injury

Li-Qing Jin et al. Cells. .

Abstract

Axotomy in the CNS activates retrograde signals that can trigger regeneration or cell death. Whether these outcomes use different injury signals is not known. Local protein synthesis in axon tips plays an important role in axon retraction and regeneration. Microarray and RNA-seq studies on cultured mammalian embryonic or early postnatal peripheral neurons showed that axon growth cones contain hundreds to thousands of mRNAs. In the lamprey, identified reticulospinal neurons vary in the probability that their axons will regenerate after axotomy. The bad regenerators undergo early severe axon retraction and very delayed apoptosis. We micro-aspirated axoplasms from 10 growing, 9 static and 5 retracting axon tips of spinal cord transected lampreys and performed single-cell RNA-seq, analyzing the results bioinformatically. Genes were identified that were upregulated selectively in growing (n = 38), static (20) or retracting tips (18). Among them, map3k2, csnk1e and gtf2h were expressed in growing tips, mapk8(1) was expressed in static tips and prkcq was expressed in retracting tips. Venn diagrams revealed more than 40 components of MAPK signaling pathways, including jnk and p38 isoforms, which were differentially distributed in growing, static and/or retracting tips. Real-time q-PCR and immunohistochemistry verified the colocalization of map3k2 and csnk1e in growing axon tips. Thus, differentially regulated MAPK and circadian rhythm signaling pathways may be involved in activating either programs for axon regeneration or axon retraction and apoptosis.

Keywords: MAPK pathway; axon regeneration; circadian rhythm; local protein synthesis; single cell RNA-seq; spinal cord injury.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sampling axoplasm by micro-aspiration from growing, static and retracting axon tips. (A/B), (C/D), (E/F), three SCs containing a growing (+21.5 µm), a static and a retracting (−13.6 µm, 1) tip, respectively. Another tip (2) disappeared after 3.8 h of observation (F). Insets in (B,D,F): fluorescent images of glass micropipettes containing axoplasm following successful micro-aspiration. White lines: Alignment of fiduciary landmarks (local neuronal cell bodies). Green lines connect white dots labeling the leading edge of axon tips in the initial fluorescent images. Six green numbers indicate the times when the 1st and 2nd images were captured. The six arrowheads point to fiduciary landmarks. In the four small square regions of (A–D), brightness and contrast have been adjusted to show fiduciary landmarks.
Figure 2
Figure 2
Flowchart for the construction of NGS libraries and sequencing. Axoplasms from 10 growing, 9 static and 5 retracting tips were collected, reverse transcribed and pre-amplified with a SMART-Seq Single Cell Kit (Takara Bio, San Jose, CA, USA). To construct the NGS libraries, cDNAs were tagmented and amplified with a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA). DNA qualities from two reactions were checked with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), as shown in the two right panels. Deep sequencing was performed on an Illumina Novaseq 6000 System with paired-end reads (2 × 100 bp).
Figure 3
Figure 3
Hierarchical cluster analysis of gene expression in growing, static and retracting axon tips. Heat maps were constructed for those genes that showed significant differential expression patterns between growing (G), static (S) and retracting (R) axon tips: (A) A representative heat map cluster showing genes that were expressed significantly more in G than in S tips. (B) A similar heat map for genes that were expressed more in G tips than in R tips. (C) A similar heat map for genes that were expressed more in R tips than in G tips. The blue-to-red gradation represents levels in gene expression, from low to high. (D) Three gene lists identified that were consistently expressed more in G tips than in S and R tips (G > S and R, 38 genes), more in S tips than in G and R tips (S > G and R, 20 genes) or more in R tips than in G and S tips (R > G and S, 18 genes). Among them, map3k2 and csnk1e (red font) were significantly upregulated in G tips more than in S tips or R tips and are involved either in “MAPK-signaling-cascades” or in “circadian rhythms”, respectively. The gene mapk8(1) (green font) in the “S > G and R” list participates in “MAPK-signaling-cascades”, and the gene prkcq (purple font) in the “R > G and S” list participates in the T cell receptor signaling pathway.
Figure 4
Figure 4
Volcano maps showing the consistently greater expressions of map3k2 and csnk1e in G tips than in R tips or S tips: (A) Volcano plot of genes that were differentially expressed in G vs. R tips. Blue dots: 513 genes that were comparatively upregulated (right side) or downregulated (left side) in G tips compared with R tips (p values < 0.01). Red and Green dots represent the genes map3k2 and csnk1e, respectively. (B) Volcano plot of 151 genes that were differentially expressed in G tips vs. S tips (p < 0.01).
Figure 5
Figure 5
GO enrichment analysis of DEGs in G vs. R tips. A list of genes that were differentially expressed between growing tips and retracting tips (1488 genes) were analyzed by DAVID enrichment analysis (v. 6.8): (A) GO terms for genes were expressed more highly in G tips than R tips (FC > 1.5, FDR < 0.1). The horizontal axis represents the −log10(FDR) of the significant GO terms. The x-intercept of the vertical red line indicates the standard cutoff value, where −log10FDR = 1.301 (FDR = 0.05). Numbers beside the bars indicate the list hit numbers vs. list total numbers. Red terms are genes directly involved in protein synthesis; Green terms are for mitochondrial function; Blue terms are for the modification of DNA or chromosomes; the Pink term is for the ubiquitin conjugation pathway; and the Black terms are for other cell functions. (B) Scatter plot for genes expressed more highly in G tips than R tips (p < 0.05, FC > 1.5). Color dots represent categories of GO terms similar to those in (A).
Figure 6
Figure 6
GO enrichment analysis of DEGs in S vs. R tips. A list of genes that were differentially expressed between static tips and retracting tips (1067 genes) and that were identified and analyzed by DAVID enrichment analysis: (A) GO terms for genes expressed more highly in S than in R tips (FC > 1.5, FDR < 0.2). The x-intercept of the vertical red line indicates the standard cutoff value, in which −log10FDR = 1.301 (FDR = 0.05). Red terms are genes directly involved in protein synthesis; Green terms are mitochondrial function genes; Blue terms are genes involved in the modification of DNA or chromosomes; and Black terms are genes involved in other cell functions. (B) Scatter plot for genes expressed more highly in S tips than R tips (p < 0.05, FC > 1.5). Color dots represent categories of GO terms, similar to those in (A).
Figure 7
Figure 7
Multiple transcripts for proteins of MAPK signaling pathways were identified in growing, static and retracting axon tips. Genes obtained by RNA-seq from axoplasms of growing (G; 5097 genes), static (S; 4105 genes) and retracting (R; 2318 genes) tips are mapped in a Venn diagram. Genes participating in MAPK pathways in each sub-group were identified (DAVID) and listed inside the gray frames. Red text is used for the conventional MAPK genes: mapk1 = erk, mapk14 = p38 and mapk8 = jnk. The Venn analysis suggests that the Erk/, Jnk/ and p38/MAPK pathways are heavily involved in axon regeneration dynamics.
Figure 8
Figure 8
q-PCR validation of mRNA expression for key genes found in growing, static and retracting tips, including conventional MAPK genes. Blue, orange and gray bars represent gene expression levels in growing (G), static (S) and retracting (0R) tips, respectively (means ± SEM, n = 4). R tips contain higher levels of erk, jnk2, p38β and usp22 genes than do G tips, whereas G tips have more p38α, map3k2 and csnk1e than do R tips. * p < 0.05 by one-way ANOVA, followed by Tukey’s test.
Figure 9
Figure 9
Cellular localization of map3k2 and csnk1e in frozen sections of naïve lamprey brains and spinal cords. (A) Western blot of proteins prepared from lamprey brains (Br) or spinal cords (SC), probed with antibodies against map3k2 and csnk1e. A single band at approximately 34 and 44 KDa was labeled with antibodies to map3k2 and csnk1e, respectively, in either brains or spinal cords. (B,C) Chromogenic IHC of coronal brain sections stained for map3k2 and csnk1e, respectively. Rostral is up. The two insets at the bottom right of (B,C) are enlarged images of the I1 neurons framed by the respective white boxes. Positive immunostaining is in brown; counterstaining by hematoxylin is in light blue. (D) Spinal cord coronal sections immuno-fluorescently triple stained for map3k2 (red), csnk1e (green) and DAPI (blue). The three asterisks (*) are inside faintly stained uninjured axons. CC: central canal. (E1–E3) are monochromatic enlarged micrographs from the framed region in (D). (E4) is (E1–E3) superimposed, showing local neurons (1–3) and glial cells (4).
Figure 10
Figure 10
Colocalization of map3k2 and csnk1e in growing axon tips. (A/B), (C/D) and (E/F): Three spinal cords containing either a growing tip (G-tip; A/B), a static tip (S-tip; C/D) or a retracting tip (R-tip; E/F), respectively (labeled with white asterisks). Their growth statuses were determined by fluorescence photomicrography, with 3–4 h intervals between captures, as indicated by the times listed in green at the top right of each frame. Spinal cords were frozen immediately and sectioned. (G/H), (J/K), and (M/N): Immunofluorescence for map3k2 (red) and csnk1e (green). Axon tips from Growing or Static axons were stained by both map3k2 (G,J, red arrows) and csnk1e (H,K, green arrows). Retracting tips showed only background staining (M,N). (I,L,O) are the superimposed images of (G/H), (J/K) and (M/N), respectively. The blue color in (I,L,O) is DAPI stain. Thus, growing tips, and to a lesser degree static tips, expressed both map3k2 and csnk1e, whereas retracting tips showed only faint, diffuse map3k2 staining. Six white arrowheads point to fiduciary landmarks. Brightness and contrast in six small square regions in (A–D) are adjusted to show fiduciary landmarks.
Figure 11
Figure 11
Network analysis of gene expression related to “map3k2”. Blue to red graduation represents gene expressions from low to high. Diamond, round or triangle nodes represent genes that are predominant in growing, static or retracting tips, respectively. Rectangular gray nodes represent genes that are important but not present in the sequencing lists. Six nodes (bottom left) are isolated because of their indirect linkage to the network (ptprga, hhipl1, acox, stk38b), or possibly different connections from their isoforms [mapk8(2), mapk14(β)]. Node mapk1 (erk) is referenced for its particularly high expression level (2.9) in retracting tips. Enrichment analysis indicated that nodes in this network are related to the “MAPK signaling pathway” or to the “JNK cascade”.
Figure 12
Figure 12
Three conventional MAPK functions in the regulation of axon regeneration coordinately and differentially. The central triangle with the red gradient indicates the platform formed by the levels of erk expression in the three types of axon tips: highest in retracting tips (100%), followed by growing tips (56%) and static tips (16%). Five isoforms (jnk1, jnk2, p38α, p38β and p38γ) of two other conventional MAPKs (jnk/p38) are differentially expressed in G, S or R tips. The highest expression levels were for jnk2 in R tips (100%) and for p38α in G tips (97%). This was followed by jnk1 in S tips (80%), p38γ in R tips (14%) and p38β in R tips (8%). In R tips, expression levels for erk were 8.7-fold higher than for jnk2. MAPKKK and MAPKK are listed on the left and right sides of the three circles, respectively, based on the type of tip in which they were most highly expressed (G, S or R), listed in order of expression levels, from highest (top) to lowest (bottom).
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Grants and funding

This research was funded by the Shriners Hospitals for Children Grants, grant number: SHC-85101, SHC-85400 and SHC-85220 to M.E.S., and funded by the NIH, grant number: NS097846 and NS092876 to M.E.S.
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