Neural Regeneration Research

RESEARCH ARTICLE
Year
: 2022  |  Volume : 17  |  Issue : 6  |  Page : 1334--1342

Identification of key genes involved in recovery from spinal cord injury in adult zebrafish


Wen-Yuan Shen1, Xuan-Hao Fu1, Jun Cai2, Wen-Chang Li2, Bao-You Fan1, Yi-Lin Pang1, Chen-Xi Zhao1, Muhtidir Abula1, Xiao-Hong Kong3, Xue Yao1, Shi-Qing Feng1,  
1 International Science and Technology Cooperation Base of Spinal Cord Injury, Tianjin Key Laboratory of Spine and Spinal Cord, Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, China
2 Tianjin Medicine and Health Research Center, Tianjin Institute of Medical & Pharmaceutical Sciences, Tianjin, China
3 School of Medicine, Nankai University, Tianjin, China

Correspondence Address:
Shi-Qing Feng
International Science and Technology Cooperation Base of Spinal Cord Injury, Tianjin Key Laboratory of Spine and Spinal Cord, Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin
China
Xue Yao
International Science and Technology Cooperation Base of Spinal Cord Injury, Tianjin Key Laboratory of Spine and Spinal Cord, Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin
China

Abstract

Zebrafish are an effective vertebrate model to study the mechanisms underlying recovery after spinal cord injury. The subacute phase after spinal cord injury is critical to the recovery of neurological function, which involves tissue bridging and axon regeneration. In this study, we found that zebrafish spontaneously recovered 44% of their swimming ability within the subacute phase (2 weeks) after spinal cord injury. During this period, we identified 7762 differentially expressed genes in spinal cord tissue: 2950 were up-regulated and 4812 were down-regulated. These differentially expressed genes were primarily concentrated in the biological processes of the respiratory chain, axon regeneration, and cell-component morphogenesis. The genes were also mostly involved in the regulation of metabolic pathways, the cell cycle, and gene-regulation pathways. We verified the gene expression of two differentially expressed genes, clasp2 up-regulation and h1m down-regulation, in zebrafish spinal cord tissue in vitro. Pathway enrichment analysis revealed that up-regulated clasp2 functions similarly to microtubule-associated protein, which is responsible for axon extension regulated by microtubules. Down-regulated h1m controls endogenous stem cell differentiation after spinal cord injury. This study provides new candidate genes, clasp2 and h1m, as potential therapeutic intervention targets for spinal cord injury repair by neuroregeneration. All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical & Pharmaceutical Sciences (approval No. IMPS-EAEP-Q-2019-02) on September 24, 2019.



How to cite this article:
Shen WY, Fu XH, Cai J, Li WC, Fan BY, Pang YL, Zhao CX, Abula M, Kong XH, Yao X, Feng SQ. Identification of key genes involved in recovery from spinal cord injury in adult zebrafish.Neural Regen Res 2022;17:1334-1342


How to cite this URL:
Shen WY, Fu XH, Cai J, Li WC, Fan BY, Pang YL, Zhao CX, Abula M, Kong XH, Yao X, Feng SQ. Identification of key genes involved in recovery from spinal cord injury in adult zebrafish. Neural Regen Res [serial online] 2022 [cited 2022 Jan 21 ];17:1334-1342
Available from: http://www.nrronline.org/text.asp?2022/17/6/1334/327360


Full Text

 Introduction



Spinal cord injury (SCI) causes serious complications and permanent disability, thus placing a heavy burden on society and families (Bai et al., 2014; Kang et al., 2018; Kim et al., 2018; Huo et al., 2021). Effective repair of nerve injury has always posed difficulty for neuroscience researchers (Hutson and Di Giovanni, 2019; Wang et al., 2019; Yang et al., 2019). In stark contrast to mammals, adult zebrafish can recover to pre-injury levels after complete spinal cord transection, showing their superior spinal cord regeneration capacity (Pan et al., 2013; El-Daher and Becker, 2020). Therefore, zebrafish are an ideal model to investigate the mechanisms underlying spinal cord regeneration (Cigliola et al., 2020).

For the past 20 years, zebrafish spinal cord regeneration studies have shed light on mammalian SCI regeneration. For example, semaphorin 4D contributed to the successful regeneration of adult zebrafish axons and the recovery of swimming ability after SCI (Peng et al., 2017; Li et al., 2020). Similar successes involve L1 (Becker et al., 2004), L1.2 (Chen et al., 2016), major vault protein (Pan et al., 2013), E2f4 (a member of the DREAM complex) (Sasagawa et al., 2016), recombinant tenascin c (Yu et al., 2011a), syntenin-a (Yu and Schachner, 2013), activating transcription factor 6 (Ji et al., 2020), and miR-133b (Yu et al., 2011b; Danilov et al., 2020). In addition, Liu et al. (2016) suggested that Mcam promotes neurogenesis and angiogenesis to repair SCI. In another study, Fang et al. (2014) reported that Hmgb1 promotes recovery from SCI by stimulating neural regeneration and angiogenesis. Guo et al. (2011) and Ogai et al. (2012) showed that Sox11b affects neural regeneration and the differentiation fate of endogenous neural stem cells after SCI. Mokalled et al. (2016) showed that Ctgfa is involved in the formation of bridge-like structures in spinal cord regeneration. Glial bridges are also required for natural spinal cord regeneration in rats (Tran et al., 2018). These results indicate that zebrafish and mammals have both common and unique mechanisms related to spinal cord regeneration. Thus, studying the mechanisms behind zebrafish SCI regeneration provides new research targets for mammalian SCI regeneration.

The subacute phase, which is approximately 2 weeks post-injury, is a key time point for adult zebrafish spinal cord regeneration, in which the key processes, such as tissue bridging and proliferating, are involved (Cigliola et al., 2020). According to Reimer et al. (2008), newly proliferated motor neurons migrate to the damaged area to replace lost neurons. The peak for motor neuron proliferation, induced by injury, occurs on the 14th day after SCI (Reimer et al., 2008). Mokalled et al. (2016) conducted RNA sequencing of spinal cord cells 2 weeks after SCI in zebrafish. Therefore, we used this dataset to analyze differences in gene expression between the control and injury groups. We also performed real-time quantitative polymerase chain reaction (RT-qPCR) verification on the key genes that were screened out. The purpose of this study was to find key genes and pathways for axon regeneration after SCI in zebrafish and to provide new ideas and targets for the treatment of SCI in mammals, using bioinformatics analysis.

 Materials and Methods



Animals

We obtained one hundred 6-month-old adult female zebrafish from Shanghai FishBio Co., Ltd, Shanghai, China. Zebrafish were kept in 3.5-liter tanks on a 14-hour light and 10-hour dark cycle at 28°C and fed twice a day. All zebrafish experiments complied with the standards of the Animal Ethics Committee of Tianjin Institute of Medical & Pharmaceutical Sciences (approval No. IMPS-EAEP-Q-2019-02) on September 24, 2019.

SCI modeling

One hundred zebrafish with body lengths of 2.5–3.0 cm were divided into two equal groups using a random-number table and a double-blind procedure (n = 50 for each group). The zebrafish were subjected to spinal cord transection or sham surgery, as previously described (Fang et al., 2012; Peng et al., 2017). Briefly, zebrafish were anesthetized by immersion in 0.033% ethyl 3-aminobenzoate methanesulfonate (MS-222, MilliporeSigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 3–5 minutes. A longitudinal incision was made on the zebrafish’s side with fine scissors (15000-00, Fine Science Tools GmbH, Heidelberg, Germany) to expose the spine. For the SCI group, the spinal cord was cut with scissors (15000-03, Fine Science Tools, Inc.) between two vertebrae about 4 mm caudal to the brainstem/spinal cord transitional junction. The SCI fish had a straight body posture and were not able to swim freely after surgery. For the sham group, an incision was made to expose the spinal cord, but no SCI was performed.

Swim tracking

Swim tracking analysis was performed on zebrafish at 3, 7, and 14 days after SCI in a 22 cm × 10 cm × 15 cm tank containing 5 cm-deep water. Each fish’s swim path was recorded with a digital camera (Ordro HDV-V7, Shenzhen Boya Times Technology Co., Ltd., Shenzhen, China) for 5 minutes, and the total swimming pattern was quantified using the Animal Tracker program of ImageJ software (Schneider et al., 2012).

RNA sequencing dataset

We used an online dataset of RNA sequencing results from spinal cord tissue of zebrafish 2 weeks after SCI to analyze changes in subacute-phase locomotion and to perform RT-qPCR on genes of interest. The original GSE77025 dataset was downloaded from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). The dataset was submitted by Mokalled et al. (2016) and is based on the GPL14875 platform (Illumina HiSeq 2000 [Danio rerio]). The GSE77025 dataset includes two sham-injured spinal cord samples and two transected SCI samples (Mokalled et al., 2016). According to a study by Mokalled et al. (2016), adult zebrafish underwent complete spinal cord transection, and the spinal cord tissue was collected 2 weeks post-injury. Total RNA from the spinal cord tissue of the zebrafish was extracted 2 weeks later for transcriptome sequencing. The sham-operated group was used as the control.

Quality assessment of RNA sequencing data

We used STAR (Dobin et al., 2013) version 2.2.1 to align reads and quantify transcripts. We then used quantile normalization of transcripts to reduce batch effects and systematic biases from sequencing. Differentially expressed gene (DEG) analysis was performed by DESeq2 (Love et al., 2014) version 1.26.0, followed by principal component analysis (PCA) and Pearson correlation analysis.

Identification of differentially expressed genes

DESeq2 version 1.26.0 was used to identify DEGs (|log2FoldChange| > 0.5 and false discovery rate [FDR] < 0.001). The volcano plot of the DEGs was drawn using the volcano plot package (Chan, 2018). Then, the DEGs were sorted from high-to-low expression, and the top 410 higher expressed genes (DHEGs) were obtained that play the most important role after SCI. Finally, we determined the top 50 genes in the DHEGs and drawn the heat map (Chan, 2018).

Function and pathway enrichment analysis of differentially expressed genes

Gene ontology (GO) analysis is a common method of gene-function annotation that can be used to analyze transcriptome data (Ashburner et al., 2000). Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for studying advanced functions and biological systems at the molecular level, and it is especially useful for genomic sequencing data (Ogata et al., 1999). DEGs were characterized by GO and KEGG enrichment using Metascape (http://metascape.org) (Zhou et al., 2019).

Protein-protein interaction network and transcription regulation

To further understand the specific molecular mechanisms by which genes regulate neural-repair-related pathways after SCI, we constructed a protein-protein interaction (PPI) network using the STRING database (Szklarczyk et al., 2017). A combined score of > 400 was set. The darker the color of the edge connecting two nodes, the greater the combined score. Cytoscape software (Shannon et al., 2003) version 3.7.1 was employed to analyze the network. The NetworkAnalyzer tool in Cytoscape was used to compute node degree value, which was mapped to node height and width. Therefore, larger nodes resulted in greater node degree values. The fill color and border paint of the nodes represent log2FoldChange and gene expression after SCI, respectively.

RT-qPCR analysis

At 2 weeks after SCI or sham surgery, total RNA was extracted from the 2-mm segment of spinal cord directly caudal to the lesion site using an Eastep Total RNA extraction kit (Promega, Madison, WI, USA) in accordance with the manufacturer’s protocol. Then, 500 ng of total RNA was reverse transcribed using a TransScript First-Strand cDNA synthesis kit (TransGen Biotech, Beijing, China). RT-qPCR was subsequently performed with TransStart Green qPCR SuperMix (TransGen Biotech), and products were detected with a LightCycler 96 (Roche, Basel, Switzerland). The primers used are shown in [Table 1]. Relative expression of gene transcripts was assessed using the 2–ΔΔCt method, for which ΔΔCt = (Cttarget gene – CtGAPDH) injury – (Cttarget gene – CtGAPDH) sham. Three independent experiments were conducted for each condition, with 10 zebrafish in each group.{Table 1}

Statistical analysis

Swim tracking and RT-qPCR data were expressed as the mean ± SEM. GraphPad Prism version 8.02 software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. The Student’s t-test was used to compare two independent groups. P < 0.05 was considered statistically significant.

 Results



Zebrafish partially recover locomotor ability 2 weeks post-spinal cord injury

The subacute phase is critical to zebrafish spinal cord recovery, when glial bridge formation and spinal cord regeneration occur (Mokalled et al., 2016). Therefore, the 2-week time point was used to investigate the mechanisms controlling locomotor recovery. The study design is presented in [Figure 1]A. First, a zebrafish SCI model was produced, and sham surgery was used as the control [Figure 1]B. Zebrafish with “C” and “S” shapes were considered to have damaged spines and were excluded on day 3 after SCI to ensure the consistency of SCI surgery results. Motor function of zebrafish was observed on days 3, 7, and 14 after SCI [Figure 1]C and [Figure 1]D. The results show that zebrafish did not recover at 3 days or 7 days after SCI. They recovered 44% of their swimming ability 14 days after SCI (P < 0.001). {Figure 1}

RNA sequencing data information and quality

The RNA sequencing dataset GSE77025 (Mokalled et al., 2016) was used to investigate gene-expression changes in the adult zebrafish spinal cord 2 weeks after complete transection. The dataset included two sham-injured spinal cord samples and two SCI samples. PCA analysis showed that the two groups are distributed in different regions, indicating an obvious difference in transcriptomes between the control and injury groups (data not shown). Pearson correlation analysis was used for data quality control. The transcriptomes of the two groups (SCI and sham) are similar when the correlation coefficient is close to 1. Pearson correlation analysis showed that there are significant differences in the transcriptomes of the control and injury groups (Additional Figure 1). Our results show that the data have good repeatability, are reliable, and can be used for subsequent bioinformatics analysis.

Identification of differentially expressed genes

To target the genes that play the most important role after SCI, we first obtained the DEGs after SCI by using DESeq2 (|log2FoldChange| > 0.5 and FDR < 0.001). In total, 7762 DEGs were identified after SCI, including 2950 up-regulated and 4812 down-regulated DEGs. We sorted the DEGs from highest-to-lowest-expressed genes in the injury group and obtained the top 410 higher expressed genes (DHEGs). Finally, we obtained the top 50 up-regulated and down-regulated DHEGs [Figure 2], which are also strongly expressed. We predicted these genes would play important roles in SCI repair. {Figure 2}

GO and KEGG pathway enrichment of differentially expressed genes

DEGs were characterized by GO and KEGG enrichment using Metascape. The top 10 pathways that reached a statistically significant value (P < 0.05) are shown in [Figure 3] and [Table 2] and [Table 3]. For the GO analysis, the up-regulated genes are mainly enriched in respiratory chain, synapse, neuron part, cellular component morphogenesis, and Ca2+-binding pathways. The up-regulated DEGs relate to neuronal repair and regeneration. The down-regulated genes are mainly enriched in RNA processing, DNA metabolic process, chromosome organization, and cell cycle. For the KEGG pathway analysis, the signaling pathways of up-regulated genes are mainly enriched in oxidative phosphorylation, NADH dehydrogenase (ubiquinone) pathway, and endocytosis. The signaling pathways of down-regulated differential genes are mainly enriched in RNA transport, protein processing in endoplasmic reticulum, and cell cycle. The GO and KEGG analyses of down-regulated DEGs show that these genes are mainly involved in cellular replication.{Figure 3}{Table 2}{Table 3}

Distribution of the top 50 genes into the top 10 GO pathways

Through GO analysis, we found that the up-regulated genes are enriched in synapses and neurons. Hence, we assumed that up-regulated genes are beneficial for SCI repair. We distributed the top 50 genes into the top 10 GO pathways to determine how the top 50 up-regulated genes perform certain functions and to identify the most important genes after SCI [Figure 4]A. Most of the top 30 of the top 50 genes fall into at least one pathway. Furthermore, calb2b, sptan1, map4l, stmn2a, clasp2, and ache fall into at least three pathways, which implies that these genes may play key roles in their respective pathways. These genes are related to the components and repair of neurons, such as respiratory chain, synapse, neuron part, and Ca2+ binding. We also distributed the top 50 down-regulated genes into the top 10 GO pathways. The results showed that h1m and ddx3xb fall into four pathways [Figure 4]B, and all the genes are involved in cellular replication.{Figure 4}

Key genes were verified by RT-qPCR

Key genes in [Figure 4]A and [Figure 4]B are involved in multiple pathways, suggesting that they play an important role in spinal cord repair. Therefore, we performed RT-qPCR to verify the expression changes of calb2b, sptan1, map41, stmn2a, clasp2, ache, h1m, and ddx3xb after SCI. The mRNAs encoding calb2b, sptan1, map41, stmn2a, clasp2, and ache are up-regulated, and the mRNAs encoding h1m and ddx3xb are down-regulated in the RNA sequencing dataset for SCI. According to Mokalled et al. (2016), ctgfa is up-regulated 2 weeks after zebrafish spinal cord crush injury (P < 0.05). Ctgfa was used as a positive control for this dataset, and it was also up-regulated consistently, as previously reported (Mokalled et al., 2016) [Figure 5]A. RT-qPCR results verified that clasp2 is up-regulated (1.46 times, P < 0.05) and h1m is down-regulated (0.46 times, P < 0.001) after SCI [Figure 5]A. The expression changes are consistent with the RNA sequencing (RNAseq) transcriptome data. The RT-qPCR results show that sptan1 and stmn2a were unchanged and calb2b, map4l, and ache were downregulated to 0.48, 0.33, and 0.62 times the sham group, respectively, and ddx3xb was up-regulated to 1.69 times the sham group (Additional Figure 2). {Figure 5}

Protein-protein interaction networks of Clasp2 and H1m

To further predict the specific molecular mechanisms of Clasp2 and H1m for regulating nerve injury repair-related pathways after SCI, protein-protein interaction networks were created for Clasp2 protein or H1m protein using the STRING database version 11.0 (https://string-db.org/). PPI network analysis showed the interactions among the DEGs. There were nine DEGs interacting with Clasp2 [Figure 5]B. Pafah1b1a (Drerup et al., 2010) is involved in the development of the nervous system, and Ckap5 is associated with microtubule dynamics (Cassimeris et al., 2009; Chanez et al., 2015). There were 18 DEGs interacting with H1m [Figure 5]C; most of their functions are related to cell cycle and embryonic development, such as Cdc6 (Yao et al., 2017), Ccna1 (Beaudoin et al., 2018), and Nanog (He et al., 2020; Palfy et al., 2020).

 Discussion



Adult zebrafish spinal cord regeneration is multi-faceted. We chose to identify key genes by using an established bioinformatics database. Mokalled et al. (2016) generated RNAseq data from zebrafish with spinal cord injury and found ctgfa to be the key to zebrafish spinal cord regeneration by promoting formation of glial bridge-like structures. By further analysis of the same dataset, we identified that clasp2 and h1m may also play a key role in spinal cord regeneration.

We screened 2950 up-regulated genes and 4812 down-regulated genes by genetic differential analysis. Similar to previous studies (Fu et al., 2019; Li et al., 2020), we found that some up-regulated genes were also highly expressed in regenerated neurons 2 weeks after SCI. In the GO analysis, the up-regulated genes were mainly enriched in respiratory chain, synapse, neuron part, cellular component morphogenesis, and Ca2+ binding. The respiratory chain is in the inner membrane of mitochondria and undergoes oxidative phosphorylation via the electron transport chain to supply ATP required for metabolism (Mazat et al., 2020). Mitochondria are damaged after SCI, resulting in electron transport chain dysfunction, mitochondrial-membrane-potential disappearance, and ATP synthesis obstruction (DiMauro and Schon, 2003; Li et al., 2019). There is evidence that axon regeneration requires the consumption of 50% of the intracellular ATP (Smith and Gallo, 2018), and hence it relies heavily on the complete respiratory chain to provide energy for axon regeneration (Weng et al., 2017). In this study, proteins expressed by the up-regulated genes participate in the respiratory chain pathway by contributing to the synthesis of ATP and providing energy for axon regeneration. Ca2+ plays a significant role in signal transduction, but its overload activates a variety of protein lysosomes and lipid lysosomes, leading to plasma membrane damage, decreased ATP production, and ultimately skeletal proteolysis (Hall and Springer, 2004; Bandura and Feng, 2019). Ca2+ is also involved in nerve conduction between synapses, and Ca2+ imbalance hinders neurological recovery after SCI (Pivovarova and Andrews, 2010). The proteins expressed by the up-regulated genes in this study participate in the Ca2+-binding pathway, which helps in eliminating the neuron damage caused by Ca2+ overload (Hall and Springer, 2004; Sullivan et al., 2004). Cell proliferation also plays an important role in secondary injury after SCI (Ren et al., 2018). During the acute stage of injury, the glial scar (caused by the proliferation of astrocytes) and the inflammatory response (caused by microglia cells) can limit the scope of injury and remove toxic metabolites over time (Sofroniew, 2018; Tran et al., 2018; Li et al., 2019). However, the inflammatory role maintained by microglia during late axonal growth is detrimental (Totoiu and Keirstead, 2005; Witcher et al., 2015; Tsarouchas et al., 2018). Chondroitin sulfate proteoglycans in the glial scar inhibit axon outgrowth through the protein tyrosine phosphatase sigma (PTPσ) (O’Shea et al., 2017; Tran et al., 2018). Therefore, it is important to regulate the proliferation of neural cells according to the spatiotemporal changes of the microenvironment after injury (Fouad et al., 2005; Duncan et al., 2018). The microenvironment of SCI regions can be regulated by target genes or pathways to promote SCI repair (Fan et al., 2018). In addition, the regulation of these target genes or pathways has been applied to transplanted exogenous stem cells, so that the cells have good nerve-regeneration ability (Lu et al., 2017; Duncan et al., 2018).

In the KEGG pathway analysis, the signaling pathways of DEGs were mainly enriched in oxidative phosphorylation, NADH dehydrogenase (ubiquinone) pathway, RNA transport, protein processing in endoplasmic reticulum, and cell cycle. These pathways focus on the cell cycle and oxidative stress. The results of GO enrichment analysis were also similar. Results from the GO and KEGG analyses suggest that these differentially expressed genes promote regeneration in two ways. On one hand, these genes promote cell metabolism, such as oxidative phosphorylation and NADH dehydrogenase (ubiquinone) pathway, while on the other hand, the genes influence the fate of cells, such as by controlling RNA transport, DNA degradation, and protein processing. Acute inflammation is intertwined with axon regeneration, as described previously in zebrafish (Bollaerts et al., 2017). The acute inflammatory phase is beneficial to central nervous system regeneration in zebrafish. The anti-inflammatory drug dexamethasone significantly inhibits the regeneration of motor neurons in zebrafish (Ohnmacht et al., 2016). However, the inflammatory response is suppressed in the first week post-injury without any chronic consequences in zebrafish (Ceci et al., 2018). Interestingly, in this RNAseq data, DEGs were not significantly enriched in a specific inflammatory pathway. This could be due to the time period used (14 days post-injury), which was beyond the acute phase of inflammation. Because RNAseq data were obtained in the whole spinal cord tissue homogenate, we could not figure out the distinctive expression pattern of inflammatory cells such as microglia or macrophages. Therefore, single cell sequencing may be used in the future to better understand the function of inflammation in specific neuronal or immune cell types.

We distributed the top 50 up-regulated genes into the top 10 GO pathways. The results showed that calb2b, sptan1, map4l, stmn2a, clasp2, and ache fall into at least three pathways. Moreover, the top 50 down-regulated genes, including h1m and ddx3xb, fall into at least four pathways. The RT-qPCR results show that the expression of clasp2 and h1m is consistent with the sequencing results. These genes may play key roles in their respective pathways.

Microtubules play an important role in the formation and maintenance of axons and dendrites in neurons. Clasp2 is a microtubule plus-end tracking protein that promotes the stabilization of dynamic microtubules (Maki et al., 2015). Because it is induced in zebrafish upon SCI, we hypothesized that it has a pro-regenerative role. Clasp2 mediates growth-cone microtubules and is in exquisite balance with the co-regulation of GSK3 (Hur et al., 2011). It has also been reported that Clasp2 regulates neuronal polarity and synaptic function, and it has been found that overexpression of Clasp2 can lead to an increase in presynaptic-terminal circumference and total synapse number (Beffert et al., 2012). Dillon et al. (2017) demonstrated that Clasp2 regulates neuronal production and controls neuronal migration in mice. Through PPI network analysis, we found that Clasp2 interacts with microtubule-related proteins. Ckap5 plays an important role in axon growth and regulates the growth cone by promoting interaction between microtubules and F-actin (Lowery et al., 2013; Slater et al., 2019). Pafah1b1a (also known as lissencephaly-1 or LIS1) activates dynein, which facilitates microtubule transportation that is critical for axon regeneration (Elshenawy et al., 2020; Htet et al., 2020; Marzo et al., 2020; McKenney, 2020). We hypothesized that Clasp2 may promote the growth of neuronal axons and growth cones [Figure 6]. Neurogenesis is a key process in zebrafish spinal cord regeneration (Ceci et al., 2018). Neurogenesis may come from transdifferentiation as well as from direct differentiation from neural stem cells or progenitor cells. Notwithstanding transcription factors, non-coding RNAs, such as microRNAs, play an emerging role in transdifferentiation (Sabater et al., 2020). Therefore, further mining of the RNAseq data for the microRNAome or investigating single-cell RNAseq techniques are needed to study neurogenesis.{Figure 6}

In this study, h1m is down-regulated after SCI in zebrafish. We hypothesized that H1m may block spinal cord regeneration. Hence after injury, reduced H1m expression may trigger the spontaneous regeneration programmed in adult zebrafish. The exact mechanism needs further elucidation. H1m is a linker histone that is in the nucleus of primordial germ cells and plays an important role in zebrafish embryonic growth. When the zebrafish embryo develops to the gastrulation stage, the expression of h1m in primordial germ cells and somatic cells declined rapidly (Muller et al., 2002). In Xenopus, Nap-1 was shown to be the main chaperone of H1m. It mediated chromosome aggregation during mitosis (Muller et al., 2002). These studies show that H1m may modulate regeneration at the chromatin level. PPI network analysis has also revealed that H1m interacts with ubiquitin b (Ubb) and several cell cycle-related proteins. The studies found that knocking down UBB in mice affected the differentiation of neural stem cells into neurons (Jung et al., 2018; Lim et al., 2019). Therefore, in this study, upregulation of Ubb may contribute to the differentiation of neural stem cells into neurons by promoting ubiquitin-related degradation of H1m. There are many down-regulated proteins that interact with H1m, such as Cdc6, Ccna1, and Nanog. Cdc6 is crucial for the initiation of DNA replication and regulation of the early stage of DNA replication (Yao et al., 2017). Ccna1 is expressed in the testis, brain, and leukemia cell lines, and is thought to play an important role in controlling the meiotic cell cycle in the germ line (Beaudoin et al., 2018). Nanog plays an important role in embryonic development by blocking embryonic stem cell differentiation (He et al., 2020; Palfy et al., 2020). Downregulation of Nanog after SCI may promote the differentiation of stem cells, such as the differentiation of neural stem cells into neurons. Therefore, down-regulation of H1m after SCI promotes differentiation of endogenous neural stem cells into neurons by altering the rate of interactions with these proteins [Figure 6].

There are limitations to our study. The dataset we used contains only two sequenced samples for each group, so the evidence may not be strong enough to draw meaningful conclusions. However, we verified the candidate genes, identified using the dataset, by RT-qPCR, which ensures the credibility of the experimental results. In the RT-qPCR experiment, we performed three independent replicates with 10 zebrafish in each group. The mechanism for Clasp2 promotion of axon regeneration after SCI has not been verified, and different neuronal cell types may have different regulation patterns (Hur et al., 2011). There is evidence that Clasp2 plays a role in promoting axon regeneration in vivo, but it remains unclear whether Clasp2 promotes motor-function recovery from SCI. We need to discover the temporal and spatial expression patterns of these genes as well as the specific cell-types that express these genes. With our established swim assay, the effects of key genes’ knockdown and overexpression on swim ability should be conducted to investigate the role of these genes in spinal cord regeneration. Furthermore, we need to clarify whether these genes are the key to boosting regeneration in mammalian species by first studying these genes in rodents or even non-human primates.

Zebrafish can recover 44% of their motor ability within 2 weeks after SCI. We analyzed RNA sequencing data from the GEO database for zebrafish 2 weeks after SCI. Through genetic differential analysis, GO, and KEGG, we identified the key genes for acute-phase SCI recovery. RT-qPCR confirmed that clasp2 was up-regulated after SCI and h1m was down-regulated after SCI, both of which are involved in multiple signaling pathways. Up-regulated Clasp2 is a microtubule-binding protein that is associated with Ckap5 and is responsible for axon elongation modulated by microtubules. Pafah1b1a is a dynein-associated protein responsible for microtubule transportation. H1m down-regulation may be attributed to modulation of endogenous stem cell differentiation upon SCI. Taken together, findings from this study reveal that up-regulation of Clasp2 promoted axonal regeneration and down-regulation of H1m promoted endogenous neural stem cell differentiation. These changes may exist as key contributors to zebrafish spinal recovery in the acute phase [Figure 6]. Clasp2 and H1m are potential therapeutic targets for spinal cord regeneration. Our study provides new candidate genes for the repair of SCI.

Author contributions: Study design: WYS, XHF, JC; data analysis: WYS, XHF; experimental proofreading: JC, WCL; paper writing: WYS, XHF; critical revision of the manuscript for intellectual content: BYF, YLP, CXZ, MA, XHK, XY, SQF; fundraising: WYS, XY, SQF. All authors approved the final version of the paper.

Conflicts of interest: The authors declare that there are no conflicts of interest associated with this manuscript.

Editor note: SQF is an Editorial Board member of Neural Regeneration Research. He was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal’s standard procedures, with peer review handled independently of this Editorial Board member and their research groups.

Financial support: This work was financially supported by the National Natural Science Foundation of China (No. 81972074; to XY), the National Key R&D Project (No. 2019YFA0112100; to SQF), Tianjin Key Research and Development Plan, Key Projects for Science and Technology Support (No. 19YFZCSY00660; to SQF), Tianjin Medical University General Hospital Youth Incubation Fund (No. ZYYFY2018003; to WYS). The funding sources had no role in study conception and design, data analysis or interpretation, paper writing or decision to submit this paper for publication.

Institutional review board statement: All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical & Pharmaceutical Sciences (approval No. IMPS-EAEP-Q-2019-02) on September 24, 2019. The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Additional files:

Additional Figure 1: Pearson correlation of samples[SUPPORTING:1].

Additional Figure 2: RT-qPCR verified partially differentially expressed genes (DEGs)[SUPPORTING:2].

Funding: This work was financially supported by the National Natural Science Foundation of China (No. 81972074; to XY), the National Key R&D Project (No. 2019YFA0112100; to SQF), Tianjin Key Research and Development Plan, Key Projects for Science and Technology Support (No. 19YFZCSY00660; to SQF), Tianjin Medical University General Hospital Youth Incubation Fund (No. ZYYFY2018003; to WYS).

References

1Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25:25-29.
2Bai M, Yin HP, Li SW, Du ZC (2014) Bone filling mesh container for osteoporotic vertebral fractures with posterior vertebral wall breakage: it is effective for prevention of bone cement leakage. Zhongguo Zuzhi Gongcheng Yanjiu 18:7545-7549.
3Bandura J, Feng ZP (2019) Current understanding of the role of neuronal calcium sensor 1 in neurological disorders. Mol Neurobiol 56:6080-6094.
4Beaudoin JD, Novoa EM, Vejnar CE, Yartseva V, Takacs CM, Kellis M, Giraldez AJ (2018) Analyses of mRNA structure dynamics identify embryonic gene regulatory programs. Nat Struct Mol Biol 25:677-686.
5Becker CG, Lieberoth BC, Morellini F, Feldner J, Becker T, Schachner M (2004) L1.1 is involved in spinal cord regeneration in adult zebrafish. J Neurosci 24:7837-7842.
6Beffert U, Dillon GM, Sullivan JM, Stuart CE, Gilbert JP, Kambouris JA, Ho A (2012) Microtubule plus-end tracking protein CLASP2 regulates neuronal polarity and synaptic function. J Neurosci 32:13906-13916.
7Bollaerts I, Van Houcke J, Andries L, De Groef L, Moons L (2017) Neuroinflammation as fuel for axonal regeneration in the injured vertebrate central nervous system. Mediators Inflamm 2017:9478542.
8Cassimeris L, Becker B, Carney B (2009) TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability. Cell Motil Cytoskeleton 66:535-545.
9Ceci M, Mariano V, Romano N (2018) Zebrafish as a translational regeneration model to study the activation of neural stem cells and role of their environment. Rev Neurosci 30:45-66.
10Chan BKC (2018) Data analysis using R programming. Adv Exp Med Biol 1082:47-122.
11Chanez B, Goncalves A, Badache A, Verdier-Pinard P (2015) Eribulin targets a ch-TOG-dependent directed migration of cancer cells. Oncotarget 6:41667-41678.
12Chen T, Yu Y, Hu C, Schachner M (2016) L1.2, the zebrafish paralog of L1.1 and ortholog of the mammalian cell adhesion molecule L1 contributes to spinal cord regeneration in adult zebrafish. Restor Neurol Neurosci 34:325-335.
13Cigliola V, Becker CJ, Poss KD (2020) Building bridges, not walls: spinal cord regeneration in zebrafish. Dis Model Mech doi: 10.1242/dmm.044131.
14Danilov CA, Gu Y, Punj V, Wu Z, Steward O, Schonthal AH, Tahara SM, Hofman FM, Chen TC (2020) Intravenous delivery of microRNA-133b along with Argonaute-2 enhances spinal cord recovery following cervical contusion in mice. Spine J 20:1138-1151.
15Dillon GM, Tyler WA, Omuro KC, Kambouris J, Tyminski C, Henry S, Haydar TF, Beffert U, Ho A (2017) CLASP2 links reelin to the cytoskeleton during neocortical development. Neuron 93:1344-1358.e5.
16DiMauro S, Schon EA (2003) Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656-2668.
17Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15-21.
18Drerup CM, Wiora HM, Morris JA (2010) Characterization of the overlapping expression patterns of the zebrafish LIS1 orthologs. Gene Expr Patterns 10:75-85.
19Duncan GJ, Manesh SB, Hilton BJ, Assinck P, Liu J, Moulson A, Plemel JR, Tetzlaff W (2018) Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination. Nat Commun 9:3066.
20El-Daher F, Becker CG (2020) Neural circuit reorganisation after spinal cord injury in zebrafish. Curr Opin Genet Dev 64:44-51.
21Elshenawy MM, Kusakci E, Volz S, Baumbach J, Bullock SL, Yildiz A (2020) Lis1 activates dynein motility by modulating its pairing with dynactin. Nat Cell Biol 22:570-578.
22Fan B, Wei Z, Yao X, Shi G, Cheng X, Zhou X, Zhou H, Ning G, Kong X, Feng S (2018) Microenvironment imbalance of spinal cord injury. Cell Transplant 27:853-866.
23Fang P, Lin JF, Pan HC, Shen YQ, Schachner M (2012) A surgery protocol for adult zebrafish spinal cord injury. J Genet Genomics 39:481-487.
24Fang P, Pan HC, Lin SL, Zhang WQ, Rauvala H, Schachner M, Shen YQ (2014) HMGB1 contributes to regeneration after spinal cord injury in adult zebrafish. Mol Neurobiol 49:472-483.
25Fouad K, Schnell L, Bunge MB, Schwab ME, Liebscher T, Pearse DD (2005) Combining Schwann cell bridges and olfactory-ensheathing glia grafts with chondroitinase promotes locomotor recovery after complete transection of the spinal cord. J Neurosci 25:1169-1178.
26Fu H, Han G, Li H, Liang X, Hu D, Zhang L, Tang P (2019) Identification of key genes and pathways involved in the heterogeneity of intrinsic growth ability between neurons after spinal cord injury in adult Zebrafish. Neurochem Res 44:2057-2067.
27Guo Y, Ma L, Cristofanilli M, Hart RP, Hao A, Schachner M (2011) Transcription factor Sox11b is involved in spinal cord regeneration in adult zebrafish. Neuroscience 172:329-341.
28Hall ED, Springer JE (2004) Neuroprotection and acute spinal cord injury: a reappraisal. NeuroRx 1:80-100.
29He M, Zhang R, Jiao S, Zhang F, Ye D, Wang H, Sun Y (2020) Nanog safeguards early embryogenesis against global activation of maternal beta-catenin activity by interfering with TCF factors. PLoS Biol 18:e3000561.
30Htet ZM, Gillies JP, Baker RW, Leschziner AE, DeSantis ME, Reck-Peterson SL (2020) LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes. Nat Cell Biol 22:518-525.
31Huo CC, Zheng Y, Lu WW, Zhang TY, Wang DF, Xu DS, Li ZY (2021) Prospects for intelligent rehabilitation techniques to treat motor dysfunction. Neural Regen Res 16:264-269.
32Hur EM, Saijilafu, Lee BD, Kim SJ, Xu WL, Zhou FQ (2011) GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules. Genes Dev 25:1968-1981.
33Hutson TH, Di Giovanni S (2019) The translational landscape in spinal cord injury: focus on neuroplasticity and regeneration. Nat Rev Neurol 15:732-745.
34Ji Z, Zhou ZL, Hao Q, Zhao L, Cui C, Huang SB, Yang YL, Shen YQ (2020) Activating transcription factor 6 contributes to functional recovery after spinal cord injury in adult Zebrafish. J Mol Neurosci doi:10.1007/s12031-020-01691-9.
35Jung BK, Park CW, Ryu KY (2018) Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro. Sci Rep 8:2629.
36Kang Y, Ding H, Zhou H, Wei Z, Liu L, Pan D, Feng S (2018) Epidemiology of worldwide spinal cord injury: a literature review. J Neurorestoratol 6:1-9.
37Kim HY, Kumar H, Jo MJ, Kim J, Yoon JK, Lee JR, Kang M, Choo YW, Song SY, Kwon SP, Hyeon T, Han IB, Kim BS (2018) Therapeutic efficacy-potentiated and diseased organ-targeting nanovesicles derived from mesenchymal stem cells for spinal cord injury treatment. Nano Lett 18:4965-4975.
38Li H, Wang C, He T, Zhao T, Chen YY, Shen YL, Zhang X, Wang LL (2019) Mitochondrial transfer from bone marrow mesenchymal stem cells to motor neurons in spinal cord injury rats via gap junction. Theranostics 9:2017-2035.
39Li JH, Shi ZJ, Li Y, Pan B, Yuan SY, Shi LL, Hao Y, Cao FJ, Feng SQ (2020) Bioinformatic identification of key candidate genes and pathways in axon regeneration after spinal cord injury in zebrafish. Neural Regen Res 15:103-111.
40Lim D, Park CW, Ryu KY, Chung H (2019) Disruption of the polyubiquitin gene Ubb causes retinal degeneration in mice. Biochem Biophys Res Commun 513:35-40.
41Liu CJ, Xie L, Cui C, Chu M, Zhao HD, Yao L, Li YH, Schachner M, Shen YQ (2016) Beneficial roles of melanoma cell adhesion molecule in spinal cord transection recovery in adult zebrafish. J Neurochem 139:187-196.
42Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550.
43Lowery LA, Stout A, Faris AE, Ding L, Baird MA, Davidson MW, Danuser G, Van Vactor D (2013) Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth. Neural Dev 8:22.
44Lu P, Ceto S, Wang Y, Graham L, Wu D, Kumamaru H, Staufenberg E, Tuszynski MH (2017) Prolonged human neural stem cell maturation supports recovery in injured rodent CNS. J Clin Invest 127:3287-3299.
45Maki T, Grimaldi AD, Fuchigami S, Kaverina I, Hayashi I (2015) CLASP2 has two distinct TOG domains that contribute differently to microtubule dynamics. J Mol Biol 427:2379-2395.
46Marzo MG, Griswold JM, Markus SM (2020) Pac1/LIS1 stabilizes an uninhibited conformation of dynein to coordinate its localization and activity. Nat Cell Biol 22:559-569.
47Mazat JP, Devin A, Ransac S (2020) Modelling mitochondrial ROS production by the respiratory chain. Cell Mol Life Sci 77:455-465.
48McKenney RJ (2020) LIS1 cracks open dynein. Nat Cell Biol 22:515-517.
49Mokalled MH, Patra C, Dickson AL, Endo T, Stainier DY, Poss KD (2016) Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish. Science 354:630-634.
50Muller K, Thisse C, Thisse B, Raz E (2002) Expression of a linker histone-like gene in the primordial germ cells in zebrafish. Mech Dev 117:253-257.
51Ogai K, Hisano S, Mawatari K, Sugitani K, Koriyama Y, Nakashima H, Kato S (2012) Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish. Neurochem Int 61:1202-1211.
52Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M (1999) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 27:29-34.
53Ohnmacht J, Yang Y, Maurer GW, Barreiro-Iglesias A, Tsarouchas TM, Wehner D, Sieger D, Becker CG, Becker T (2016) Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish. Development 143:1464-1474.
54O’Shea TM, Burda JE, Sofroniew MV (2017) Cell biology of spinal cord injury and repair. J Clin Invest 127:3259-3270.
55Palfy M, Schulze G, Valen E, Vastenhouw NL (2020) Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation. PLoS Genet 16:e1008546.
56Pan HC, Lin JF, Ma LP, Shen YQ, Schachner M (2013) Major vault protein promotes locomotor recovery and regeneration after spinal cord injury in adult zebrafish. Eur J Neurosci 37:203-211.
57Peng SX, Yao L, Cui C, Zhao HD, Liu CJ, Li YH, Wang LF, Huang SB, Shen YQ (2017) Semaphorin4D promotes axon regrowth and swimming ability during recovery following zebrafish spinal cord injury. Neuroscience 351:36-46.
58Pivovarova NB, Andrews SB (2010) Calcium-dependent mitochondrial function and dysfunction in neurons. FEBS J 277:3622-3636.
59Reimer MM, Sorensen I, Kuscha V, Frank RE, Liu C, Becker CG, Becker T (2008) Motor neuron regeneration in adult zebrafish. J Neurosci 28:8510-8516.
60Ren H, Chen X, Tian M, Zhou J, Ouyang H, Zhang Z (2018) Regulation of Inflammatory cytokines for spinal cord injury repair through local delivery of therapeutic agents. Adv Sci (Weinh) 5:1800529.
61Sabater L, Locatelli L, Oakley F, Hardy T, French J, Robinson SM, Sen G, Mann DA, Mann J (2020) RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat. Sci Rep 10:21708.
62Sasagawa S, Nishimura Y, Hayakawa Y, Murakami S, Ashikawa Y, Yuge M, Okabe S, Kawaguchi K, Kawase R, Tanaka T (2016) E2F4 promotes neuronal regeneration and functional recovery after spinal cord injury in Zebrafish. Front Pharmacol 7:119.
63Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671-675.
64Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498-2504.
65Slater PG, Cammarata GM, Samuelson AG, Magee A, Hu Y, Lowery LA (2019) XMAP215 promotes microtubule-F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopus laevis. J Cell Sci 132.
66Smith GM, Gallo G (2018) The role of mitochondria in axon development and regeneration. Dev Neurobiol 78:221-237.
67Sofroniew MV (2018) Dissecting spinal cord regeneration. Nature 557:343-350.
68Sullivan PG, Rabchevsky AG, Keller JN, Lovell M, Sodhi A, Hart RP, Scheff SW (2004) Intrinsic differences in brain and spinal cord mitochondria: implication for therapeutic interventions. J Comp Neurol 474:524-534.
69Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, Santos A, Doncheva NT, Roth A, Bork P, Jensen LJ, von Mering C (2017) The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 45:D362-368.
70Totoiu MO, Keirstead HS (2005) Spinal cord injury is accompanied by chronic progressive demyelination. J Comp Neurol 486:373-383.
71Tran AP, Warren PM, Silver J (2018) The biology of regeneration failure and success after spinal cord injury. Physiol Rev 98:881-917.
72Tsarouchas TM, Wehner D, Cavone L, Munir T, Keatinge M, Lambertus M, Underhill A, Barrett T, Kassapis E, Ogryzko N, Feng Y, van Ham TJ, Becker T, Becker CG (2018) Dynamic control of proinflammatory cytokines IL-1beta and TNF-alpha by macrophages in zebrafish spinal cord regeneration. Nat Commun 9:4670.
73Wang S, Huang Y, Yan Y, Zhou H, Wang M, Liao L, Wang Z, Chen D, Ji D, Xia X, Liu F, Huang J, Xiong K (2019) Calpain2 but not calpain1 mediated by calpastatin following glutamate-induced regulated necrosis in rat retinal neurons. Ann Anat 221:57-67.
74Weng YL, An R, Cassin J, Joseph J, Mi R, Wang C, Zhong C, Jin SG, Pfeifer GP, Bellacosa A, Dong X, Hoke A, He Z, Song H, Ming GL (2017) An intrinsic epigenetic barrier for functional axon regeneration. Neuron 94:337-346.
75Witcher KG, Eiferman DS, Godbout JP (2015) Priming the inflammatory pump of the CNS after traumatic brain injury. Trends Neurosci 38:609-620.
76Yang LX, Yang LK, Zhu J, Chen JH, Wang YH, Xiong K (2019) Expression signatures of long non-coding RNA and mRNA in human traumatic brain injury. Neural Regen Res 14:632-641.
77Yao L, Chen J, Wu X, Jia S, Meng A (2017) Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype. Hum Mol Genet 26:4168-4180.
78Yu Y, Schachner M (2013) Syntenin-a promotes spinal cord regeneration following injury in adult zebrafish. Eur J Neurosci 38:2280-2289.
79Yu YM, Cristofanilli M, Valiveti A, Ma L, Yoo M, Morellini F, Schachner M (2011a) The extracellular matrix glycoprotein tenascin-C promotes locomotor recovery after spinal cord injury in adult zebrafish. Neuroscience 183:238-250.
80Yu YM, Gibbs KM, Davila J, Campbell N, Sung S, Todorova TI, Otsuka S, Sabaawy HE, Hart RP, Schachner M (2011b) MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish. Eur J Neurosci 33:1587-1597.
81Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C, Chanda SK (2019) Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 10:1523.