Bioinformatic identification of key candidate genes and pathways in axon regeneration after spinal cord injury in zebrafish

Abstract

Zebrafish and human genomes are highly homologous; however, despite this genomic similarity, adult zebrafish can achieve neuronal proliferation, regeneration and functional restoration within 6–8 weeks after spinal cord injury, whereas humans cannot. To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury, and to explore the key genes and pathways of axonal regeneration after spinal cord injury, microarray GSE56842 was analyzed using the online tool, GEO2R, in the Gene Expression Omnibus database. Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes. Finally, we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals. A total of 636 differentially expressed genes were obtained, including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained. A protein-protein interaction network contained 480 node genes and 1976 node connections. We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score. The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish. Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish. Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells, such as Schwann cells or neural progenitor cells, after spinal cord injury in zebrafish. Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish, providing targets for treatment of spinal cord injury in mammals.

Chinese Library Classification No. R446; R364; R741

Introduction

In mammals, spinal cord injury (SCI) is a destructive neurological disorder that often results in the loss of sensory and motor functions (Yu et al., 2016; Dyck and Karimi-Abdolrezaee, 2018; Sharma et al., 2019). After SCI, the non-regenerative characteristics of the central nervous system lead to neuronal death in the spinal cord (Pinto and Gotz, 2007; Wei et al., 2019). In contrast to mammals, adult zebrafish can achieve neuronal proliferation, regeneration and functional restoration within 6–8 weeks after SCI via several regenerative processes that evade cell death (Becker et al., 1998; Briona et al., 2015). Zebrafish and human genomes are highly homologous; therefore, zebrafish is used as a regeneration model (Howe et al., 2013). However, not all axons regenerate after SCI in adult zebrafish (Vajn et al., 2013) and the mechanisms of recovery after SCI in zebrafish and the significant differences between zebrafish axons are not fully understood.

In the past decade, there have been many studies on SCI in zebrafish that have identified SCI regeneration mechanisms that are similar between zebrafish and mammals. For example, fibroblast growth factor (FGF) signaling in zebrafish can promote regeneration after SCI by means of multiple mechanisms (Goldshmit et al., 2012). Similarly, many studies have demonstrated that FGF1 and FGF2 can protect neurons and promote axonal regeneration and recovery of movement function in mammalian SCI models (Kuo et al., 2011; Zhang et al., 2013). Other similar mechanisms involve bcl-2, phospho-Akt and miR-133b (Seki et al., 2003; Yu et al., 2011; Ogai et al., 2012; Zhang et al., 2016; Theis et al., 2017). Study of zebrafish regeneration mechanisms after SCI leads to understanding the zebrafish regeneration process and provides new research targets for SCI regeneration in mammals.

In recent years, microarray technology has been extensively used to study various biological mechanisms and a growing number of studies have used microarray technology to study the pathophysiology of diseases (Vogelstein et al., 2013; Hao et al., 2018). The purpose of this study was to identify the key genes and pathways involved in zebrafish axonal regeneration after SCI using bioinformatic methods and to provide new ideas and therapeutic targets for the treatment of mammalian SCI.

Materials and Methods

Identification of differentially expressed genes (DEGs)

The original GSE56842 datasets were downloaded from NCBI GEO (available online: http://www.ncbi.nlm.nih.gov/geo/). These datasets were submitted by Vajn et al. (2013) and are based on the GPL1319 Platform (Affymetrix Zebrafish Genome Array, Affymetrix Technologies, Santa Clara, CA, USA). The data were derived from a complete spinal cord transection with fluoro-ruby retrograde tracing performed at the 8^th vertebral level in adult zebrafish. The fluoro-ruby dye labeled all the neurons in the brain that projected their axons to the 8^th vertebra. Three weeks later, fluoro-emerald was injected 4 mm distal to the spinal cord transection site, thereby labeling all neurons that regenerated their axons to this level. The zebrafish were sacrificed 1 week after fluoro-emerald tracing and the brains were enzymatically dissociated. Cells were sorted using fluorescence-activated cell sorting. The GSE56842 dataset contains nine samples, including three non-regenerated neuron samples, three regenerated neuron samples, and three non-lesion samples. In the present study, the regenerated neuron samples and non-regenerated neuron samples were selected for analysis.

The original gene expression profile data were analyzed on the Morpheus website (available online: https://software.broadinstitu te.org/morpheus/) to obtain a heat map of the most significant DEGs. GEO2R was used to obtain the DEGs (available online: https://www.ncb i.nlm.nih.gov/geo/geo2r/). We considered P < 0.05 to show statistical significance and [logFC] > 1 as the cut-off value.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

GO analysis is a common method of gene function annotation that can be used for the analysis of transcriptome data (Ashburner et al., 2000, 2006). KEGG is a database resource for studying advanced functions and biological systems from the molecular level, and is especially useful for genomic sequencing data (Ogata et al., 1999). DAVID (available online: https://david.ncifcrf.gov/) was used to analyze candidate DEG functions and pathway enrichment. P < 0.05 was considered statistically significant.

Module screening from integration of protein-protein interaction (PPI) networks

STRING (available online: http://string-db.org) (Franceschini et al., 2013) was employed to evaluate the interaction among DEGs. Cytoscape software (Shannon et al., 2003) was used to analyze the PPI networks. To calculate the number of interconnections, the network analyzer plug-in was used to filter PPI hub genes. The modules of PPI networks were obtained by using Molecular Complex Detection in Cytoscape, and Molecular Complex Detection scores > 5 were used. Finally, DAVID was used to analyze module pathway enrichment. P < 0.05 was considered statistically significant.

Results

Identification of DEGs

Taking P < 0.05 and [logFC] > 1 as inclusion conditions, we identified 636 DEGs, including 255 upregulated and 381 downregulated DEGs, in the regenerated neuron samples compared with the non-regenerated neuron samples from the GSE56842 expression profile dataset. Using the Morpheus website, we obtained a heat map of the top 50 upregulated and downregulated DEGs, revealing the top 100 significantly DEGs (Figure 1).

Figure 1.

Heat map of the top 50 up-regulated and down-regulated differentially expressed genes.

The three columns on the left are the axon-non-regenerated neuron samples. The three columns on the right are the axon-regenerated neuron samples. Blue indicates down-regulation and red indicates up-regulation.

GO analysis of DEGs

All DEGs were uploaded to DAVID and the overrepresented GO categories and KEGG pathways were identified. The top 30 enriched GO terms are shown in Figure 2; they were mainly in the biological process group, such as multicellular organism development, cell differentiation, and negative regulation of neurogenesis. In the molecular function group, DEGs were mainly enriched in structural constituent of cytoskeleton, cytochrome-c oxidase activity, and protein dimerization activity. There were only two GO terms in the cellular component group, mitochondrial respiratory chain complex IV, and spectrin. The upregulated DEGs were mainly enriched in cytochrome-c oxidase activity, structural constituent of cytoskeleton, mitochondrial respiratory chain complex IV, PDZ domain binding, and spectrin (Table 1). The downregulated DEGs were mainly enriched in multicellular organism development, negative regulation of neurogenesis, regulation of transcription, positive regulation of sequence-specific DNA binding transcription, and regulation of transcription DNA-templated (Table 1).

Figure 2.

Top 30 significantly enriched gene ontology (GO) terms of differentially expressed genes in regenerated neurons.

The green bars represent cellular component. The red bars represent molecular function. The blue bars represent biological process. The length of the bar represents the –log (P-value); credibility increases with bar length. The ordinate is the GO terms, and the abscissa is the –log (P-value) of GO terms.

Table 1.

Top 10 significant enriched gene Gene Ontology (GO) terms of up-regulated and down-regulated differentially expressed genes (DEGs) in regenerated neurons

Term	Description	Count	P-value
Up-regulated
GO: 0004129	Cytochrome-c oxidase activity	6	1.68E-05
GO: 0005200	Structural constituent of cytoskeleton	6	2.98E-04
GO: 0005751	Mitochondrial respiratory chain complex IV	4	5.75E-04
GO: 0030165	PDZ domain binding	4	5.89E-04
GO: 0008091	Spectrin	3	7.43E-04
GO: 0006814	Sodium ion transport	6	1.54E-03
GO: 0006811	Ion transport	15	2.44E-03
GO: 0005890	Sodium:potassium-exchanging ATPase complex	3	2.54E-03
GO: 0005543	Phospholipid binding	4	5.09E-03
GO: 0005388	Calcium-transporting ATPase activity	3	9.17E-03
Down-regulated
GO: 0007275	Multicellular organism development	31	1.76E-07
GO: 0050768	Negative regulation of neurogenesis	5	3.42E-05
GO: 0006355	Regulation of transcription, DNA-templated	46	3.60E-05
GO: 0051091	Positive regulation of sequence-specific DNA binding transcription Factor activity	5	7.20E-05
GO: 0006351	Transcription, DNA-templated	30	1.34E-04
GO: 0045666	Positive regulation of neuron differentiation	5	2.26E-04
GO: 0007219	Notch signaling pathway	7	2.73E-04
GO: 0046983	Protein dimerization activity	12	3.39E-04
GO: 0030154	Cell differentiation	14	3.63E-04
GO: 0042803	Protein homodimerization activity	10	4.14E-04

PDZ: Post-synaptic density protein 95, Drosophila disc large tumor suppressor and zona occludens 1.

Signaling pathway enrichment analysis

As shown in Table 2, the upregulated genes were mainly enriched in cardiac muscle contraction, oxidative phosphorylation, adrenergic signaling in cardiomyocytes, Wnt signaling pathway, and oocyte meiosis. The downregulated genes were mainly enriched in biosynthesis of antibiotics, glycine, serine and threonine metabolism, transforming growth factor beta (TGF)-β signaling pathway, and biosynthesis of amino acids. As shown in Figure 3, the top 30 enriched pathways were obtained. The most significantly enriched pathways of all DEGs were biosynthesis of antibiotics, cardiac muscle contraction, fatty acid degradation, carbon metabolism, and Wnt signaling pathway.

Table 2.

Enrichment KEGG pathways of up-regulated and down-regulated DEGs in regenerated neurons

Pathway	Name	Gene count	P value	Genes
Up-regulated DEGs
dre04260	Cardiac muscle contraction	9	1.54E-04	COX6B1, ATP1B1B, ATP1B2A, COX6A1, COX5AB, ATP1A3B, ATP1B2B, COX4I1L, COX7A2A
dre00190	Oxidative phosphorylation	10	4.27E-04	NDUFA4, NDUFB5, ATPV0E2, SDHC, COX6B1, COX6A1, COX5AB, COX4I1L, COX7A2A, ATP6V1F
dre04261	Adrenergic signaling in cardiomyocytes	10	4.66E-03	CAMK2D1, ATP2B2, PLCB4, CAMK2D2, ATP1B1B, ATP1B2A, ATP1A3B, ATP1B2B, PPP1CB, ATP2B1A
dre04310	Wnt signaling pathway	8	1.96E-02	CAMK2D1, PRICKLE1A, PLCB4, CAMK2D2, PPP3R1B, PPP3CB, RHOCB, RHOAB
dre04114	Oocyte meiosis	7	2.27E-02	CAMK2D1, YWHAZ, ANAPC13, CAMK2D2, PPP3R1B, PPP3CB, PPP1CB
dre00071	Fatty acid degradation	4	3.27E-02	GCDHB, CPT1AB, ACAT1, ACSL6
dre04145	Phagosome	7	4.08E-02	MARCO, ATPV0E2, TUBA2, MRC1A, TUBB2, TUBA8L2, ATP6V1F
Down-regulated DEGs
dre01130	Biosynthesis of antibiotics	13	1.28E-03	HADHAA, CYP51, SHMT2, AK3, ADH5, HADHB, FDFT1, ENO1B, NME5, PAPSS2B, IDH1, PSAT1, PAICS
dre00260	Glycine, serine and threonine metabolism	5	9.67E-03	SHMT2, TDH, BHMT, MAO, PSAT1
dre04350	TGF-beta signaling pathway	7	1.04E-02	INHBB, MYCB, BMPR1AA, SMURF2, DCN, BMPR1AB, ID2B
dre01230	Biosynthesis of amino acids	6	2.12E-02	GLULB, SHMT2, GPT2L, IDH1, PSAT1, ENO1B
dre04330	Notch signaling pathway	5	2.29E-02	NOTCH3, JAG1B, NOTCH1A, HER6, LFNG
dre00062	Fatty acid elongation	4	2.72E-02	HADHAA, HACD2, ELOVL7A, HADHB
dre01200	Carbon metabolism	7	2.94E-02	HADHAA, SHMT2, GPT2L, ADH5, IDH1, PSAT1, ENO1B

DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; TGF: transforming growth factor. The full names of the genes are shown in Additional Table 1.

Figure 3.

Top 30 significantly enriched pathways of DEGs in regenerated neurons.

The ordinate is KEGG signaling pathways, and the abscissa is the –log (P-value) of pathways. DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes.

PPI network and module analysis

The PPI network contained 480 nodes and 1976 edges. The top 10 hub nodes were obtained and included ras homolog gene family, member Ab (RHOAB); catenin (cadherin-associated protein), beta 1 (CTNNB1); YES proto-oncogene 1 (YES1); phosphoribosylaminoimidazole carboxylase (PAICS); mitogen-activated protein kinase 4 (MAPK4); Rho family GTPase 3a (RND3A); hydroxyacyl-CoA dehydrogenase, alpha subunit a (HADHAA); Rho family GTPase 3b (RND3B); fibroblast growth factor receptor 2 (FGFR2); and v-myc avian myelocytomatosis viral oncogene homolog b (MYCB). The top 10 hub genes and their corresponding degrees are shown in Table 3. Two significant modules with high corresponding degrees were obtained using Molecular Complex Detection in Cytoscape. As shown in Figure 4, module 1 consisted of nine nodes and 28 edges, and module 2 consisted of 56 nodes and 173 edges. Through pathway enrichment analysis, we conclude that nodes of module 1 were mainly enriched in focal adhesion, tight junctions, and regulation of actin cytoskeleton (Table 4), and nodes of module 2 were mainly enriched in cardiac muscle contraction, cytokine-cytokine receptor interaction, and TGF-β signaling pathway (Table 5).

Table 3.

Top 10 core genes and their corresponding degree (score), and expression in neurons with axonal regeneration

Gene	Full name of gene	Degree	Up or Down
RHOAB	Ras homolog gene family, member Ab	71	Up
CTNNB1	Catenin (cadherin-associated protein), beta 1	69	Down
YES1	YES proto-oncogene 1	60	Down
PAICS	Phosphoribosylaminoimidazole carboxylase	54	Down
MAPK4	Mitogen-activated protein kinase 4	49	Down
RND3A	Rho family GTPase 3a	43	Down
HADHAA	Hydroxyacyl-CoA dehydrogenase, alpha subunit a	42	Down
RND3B	Rho family GTPase 3b	39	Up
FGFR2	Fibroblast growth factor receptor 2	35	Down
MYCB	Myelocytomatosis viral oncogene homolog b	35	Down

CoA: Coenzyme A; GTP: guanosine triphosphate.

Figure 4.

The top two modules with high corresponding degrees from the protein-protein interaction network.

(A) The first module and nodes. (B) The second module and nodes. The full names of the proteins are shown in Additional Table 2.

Table 4.

The enriched pathways in module 1

Term	P-value	Count	%	Genes
Focal adhesion	8.77E-04	4	50.0	CRKL, PTENB, BCAR1, RHOAB
Tight junction	9.89E-03	3	37.5	PTENB, RHOAB, YES1
Regulation of actin cytoskeleton	2.24E-02	3	37.5	CRKL, BCAR1, RHOAB

The full names of the genes are shown in Additional Table 3.

Table 5.

The enriched pathways in module 2

Term	P-value	Count	%	Genes
Cardiac muscle contraction	2.00E-03	5	9.80	COX6B1, COX6A1, COX5AB, TPM1, ATP1A3A
Cytokine-cytokine receptor interaction	2.12E-03	6	11.76	KITA, FLT4, VEGFAA, BMPR1AA, BMPR1AB, CXCL12A
TGF-beta signaling pathway	2.24E-03	5	9.80	INHBB, MYCB, BMPR1AA, SMURF2, BMPR1AB
Melanogenesis	7.17E-03	5	9.80	KITA, FZD7B, CALM1A, FZD7A, CTNNB1
Oxidative phosphorylation	7.74E-03	5	9.80	NDUFA4, NDUFB5, COX6B1, COX6A1, COX5AB
Biosynthesis of antibiotics	4.18E-02	5	9.80	GOT1, PAPSS2B, OGDHL, IDH1, LDHBB

TGF: Transforming growth factor. The full names of the genes are shown in Additional Table 4.

Discussion

In this study, 636 DEGs were obtained from analysis of cohort profile datasets of GSE56842. GO and KEGG pathway enrichment analyses were then performed. From the GO analysis results, the up-regulated DEGs were involved in cytochrome-c oxidase activity, spectrin, and phospholipid binding, and down-regulated DEGs were involved in negative regulation of neurogenesis, positive regulation of neuron differentiation, Notch signaling and cell differentiation. Spectrins consist of two α and two β subunits and are widely expressed. They have the ability to assemble neuronal excitable regions (Berghs et al., 2000; Yang et al., 2007). Many studies indicate that breakdown of spectrins is an important mechanism in neurodegenerative diseases and injuries. For example, the lack of αII spectrin in mice and zebrafish leads to the death of embryos and larvae because of a stunted nervous system (Stankewich et al., 2011). In fact, spectrins are potent substrates for calpain, and their degradation has been related to brain injury (Schafer et al., 2009). A recent study showed that αII spectrin is critical for the development and synaptogenesis of dendrites and axons (Wang et al., 2018). In the peripheral nervous system of zebrafish, spectrins in Schwann cells can promote the myelination of axons (Susuki et al., 2011). Moreover, Schwann cells are extensively distributed after SCI in zebrafish (Hui et al., 2010). We suggest that spectrins may synergize with Schwann cells to promote nerve regeneration after SCI in zebrafish, and may also promote repair after SCI in mammals.

In most multicellular organisms, the Notch signaling pathway is a highly conserved cellular signaling system that contains four receptors, Notch1–4 (Artavanis-Tsakonas et al., 1999; Kumar et al., 2016). In mammals, Notch1 expression is enhanced in response to injury, and activation of Notch signaling prevents spinal cord progenitor cells from producing neurons. This inhibitory effect can be inhibited in vitro by attenuating the Notch signaling pathway (Yamamoto et al., 2001). In addition, Notch signaling inhibits motor neuron generation and progenitor proliferation in the injured ventromedial spinal cord. In zebrafish at 14 days post SCI, Notch1a and Notch1b are significantly upregulated in the spinal cord. Altogether, Notch signaling is a negative signal for nerve regrowth, and inhibiting Notch signaling can enhance the regeneration of spinal motor neurons in adult vertebrates (Dias et al., 2012).

From the KEGG analysis results, the upregulated DEGs were mainly enriched in cardiac muscle contraction, oxidative phosphorylation, adrenergic signaling in cardiomyocytes and Wnt signaling, while downregulated DEGs were mainly enriched in the biosynthesis of antibiotics, glycine, serine and threonine metabolism, TGF-β, biosynthesis of amino acids, Notch signaling pathways and fatty acid elongation. Upregulated DEGs were enriched in cardiac muscle contraction. Adrenergic signaling in cardiomyocytes is mainly because upregulated DEGs contain some genes for cytochrome oxidase, sodium-potassium ATPase and calcium channel. These illustrate that the basal metabolism of neurons is active after axonal regeneration. The Wnt signaling pathway is crucial in development and growth and is associated with cell differentiation, polarization, and migration during development. Wnt signaling is based on one canonical β-catenin-dependent and two β-catenin-independent non-canonical Wnt pathways: the planar cell polarity signaling pathway and the Ca²⁺ Wnt pathway (Taciak et al., 2018). After damage in animals with the ability to regenerate, the activation of Wnt/ß-catenin signaling is enhanced, and attenuating activation of the signaling can inhibit regeneration (Yokoyama et al., 2007). A previous study in zebrafish showed that Wnt/ß-catenin signaling is activated in glia after SCI, and when the signaling pathway is suppressed, neurogenesis is reduced and axon regrowth fails (Briona et al., 2015).

The TGF-β family, which works mainly through the SMAD pathway is composed of evolutionarily conserved polypeptides that have essential functions in homeostasis, growth and development of cells and tissues. However, they also function in the pathogenesis of diseases, including neuronal degeneration and tumor formation (Yamagata et al., 2005; Ueberham et al., 2006; Chalmers and Love, 2007; Jaskova et al., 2014). TGF-β is also an anti-inflammatory cytokine in the injured spinal cord after SCI (Semple-Rowland et al., 1995; Nakamura et al., 2003). A previous study suggested that SCI can lead to elevation of TGF-β, which might lead to the degeneration of neurons and the formation of glial scar after SCI (Joko et al., 2013). Both neuronal degeneration and glial scar are factors that hinder repair after SCI (Yuan and He, 2013; Sofroniew, 2018). Another study showed that inhibition of TGF-β1 in rats can promote axonal regeneration and preservation, and also reduce the formation of glial scar after SCI (Kohta et al., 2009). In mammals, TGF-β hinders nerve regeneration after SCI, and the down-regulated DEGs in the present study are enriched in the TGF-β pathway, indicating that TGF-β may also play a role in inhibiting nerve regeneration in zebrafish SCI.

We identified the top 10 degree hub genes: RHOAB, CTNNB1, YES1, PAICS, MAPK4, RND3A, HADHAA, RND3B, FGFR2, and MYCB. Catenin β-1 is encoded by the CTNNB1 gene. Catenin β-1 participates in the formation of a cadherin protein complex and is also a signal converter of the Wnt signaling pathway, plays an important role in cell adhesion and gene transcription (Peifer et al., 1991, 1994; Noordermeer et al., 1994). In some cell types, an elevated level of β-catenin helps maintain pluripotency, and it may also promote cell differentiation at the developmental stage (Sokol, 2011). Consistent with the GO term results revealing that neuron differentiation and cell differentiation were downregulated, CTNNB1 was also downregulated in the axon-regenerated neurons. However, previous studies only focused on the promotion of differentiation to facilitate SCI repair (Xu et al., 2017a; Geissler et al., 2018; Li et al., 2018). No studies have described inhibition of the CTNNB1 gene to promote the repair of SCI.

The proto-oncogene tyrosine-protein kinase, YES, is an enzyme encoded by the YES1 gene (Semba et al., 1985), which belongs to the Src protein family. Secondary damage after SCI includes edema formation. Vascular endothelial growth factor, an endothelial mitogen as well as a potent mediator of vascular permeability, contributes to brain formation by binding of the vascular endothelial growth factor receptor, leading to recruitment and stimulation of the catalytic activity of Src (Senger et al., 1983; Cobbs et al., 1998; Schlessinger, 2000). An inhibitor of Src family tyrosine kinases reduces edema and the inflammatory response to SCI, subsequently improving motor function (Akiyama et al., 2003, 2004). Fibroblast growth factor receptor 2 (FGFR2), also known as cluster of differentiation 332 (CD332), plays vital roles in embryonic development and tissue repair (Xu et al., 2017b; Ishiwata, 2018). FGFR2 is involved in anti-apoptosis and nerve repair activated by FGF10 (Chen et al., 2017). At present, the role of FGFR2 in the central nervous system is poorly understood. These genes are involved in inflammation, cell proliferation and apoptosis, but their role in SCI is unclear; therefore, the role of these genes is worth studying.

The pathway analysis of modules showed that they were mainly associated with focal adhesion, tight junctions, regulation of actin cytoskeleton, cytokine-cytokine receptor interaction, TGF-β signaling, melanogenesis, and oxidative phosphorylation. In cell biology, focal adhesions are associated with mechanical force and signal transduction between cells or between cells and the extracellular matrix. In addition to anchoring the cell, they act as signal carriers, informing the cell about the condition of the extracellular matrix and thus influence their behavior (Riveline et al., 2001). In addition, focal adhesions are involved in cell motility, proliferation, migration and survival (Zaidel-Bar et al., 2004; Fu et al., 2010; Li et al., 2012; Liu et al., 2018; Yan et al., 2018). Tight junctions, also known as occluding junctions or zonulae occludentes, are multiprotein junctional complexes whose general function is to prevent leakage of transported solutes and water and to seal the paracellular pathway (Anderson and Van Itallie, 2009). Tight junctions also play a critical role in cell migration, proliferation, and differentiation (Kurasawa et al., 2011; Wu et al., 2011; Gonzalez-Mariscal et al., 2017). Although, tight junctions are mainly associated with epithelial and endothelial cells, no study has elucidated the relationship between focal adhesions or tight junctions and SCI. Nevertheless, the migration, proliferation and differentiation of Schwann cells and neural progenitor cells play an important role in repair after SCI in zebrafish (Reimer et al., 2008; Hui et al., 2010; Briona and Dorsky, 2014). However, based on the current findings, we believe that focal adhesion or tight junctions may play an important role in the repair of SCI in zebrafish.

In the present study, through functional enrichment analysis for DEGs, we have highlighted many known pathways involved in the repair of SCI, including Wnt and Notch signaling pathways. By analyzing the top 10 hub genes, we identified several genes worth studying, including CTNNB1, YES1 and FGFR2. In addition, through the enrichment of DEGs and functional analysis, we found that spectrins may promote the regeneration of SCI axons in zebrafish and mammals, while TGF-β signals may inhibit repair after SCI in zebrafish. In addition, focal adhesion and tight junctions might promote the migration and proliferation of some zebrafish cells after SCI, such as Schwann cells or neural progenitor cells, and promote repair after SCI.

In summary, this study helps us better understand the repair mechanism of SCI in zebrafish and provides new research targets for mammalian SCI. However, pathway or gene functions were not verified in this study, and more in-depth studies are needed to explore the role of identified genes and pathways in the repair of SCI in zebrafish and mammalian species.

Additional files:

Additional file 1: Open peer review report 1 ^{(99.3KB, pdf)} .

Additional Table 1: The full names of the genes in Table 2.

Additional Table 1.

The full names of the genes in Table 2

Gene symbol	Gene title
ATP1B1B	Atpase, Na+/K+ transporting, beta 2b polypeptide
ACSL6	Acyl-coa synthetase long-chain family member 6
ADH5	Alcohol dehydrogenase 5
AK3	Adenylate kinase 3
ANAPC13	Anaphase promoting complex subunit 13
ATP1A3B	Atpase, Na+/K+ transporting, alpha 3b polypeptide
ATP1B2A	Atpase, Na+/K+ transporting, beta 2a polypeptide
ATP1B2B	Atpase, Na+/K+ transporting, beta 2a polypeptide
ATP2B1A	Atpase, Na+/K+ transporting, beta 2a polypeptide
ATP2B2	Atpase, Na+/K+ transporting, beta 2a polypeptide
ATP6V1F	Atpase, Na+/K+ transporting, beta 2a polypeptide
ATPV0E2	Atpase, Na+/K+ transporting, beta 2a polypeptide
BHMT	Betaine-homocysteine methyltransferase
BMPR1AA	Bone morphogenetic protein receptor, type iaa
BMPR1AB	Bone morphogenetic protein receptor, type iab
CAMK2D1	Calcium/calmodulin-dependent protein kinase (cam kinase) II delta 1
CAMK2D2	Calcium/calmodulin-dependent protein kinase (cam kinase) II delta 1
COX4I1L	Cytochrome c oxidase subunit IV isoform 1, like
COX5AB	Cytochrome c oxidase subunit Vab
COX6A1	Cytochrome c oxidase subunit via polypeptide 1
COX6B1	Cytochrome c oxidase subunit vib polypeptide 1
COX7A2A	Cytochrome c oxidase subunit viia polypeptide 2a (liver)
CPT1AB	Carnitine palmitoyltransferase 1Ab (liver)
CYP51	Cytochrome P450, family 51
DCN	Decorin
ELOVL7A	ELOVL fatty acid elongase 7a
ENO1B	Enolase 1b, (alpha)
FDFT1	Farnesyl-diphosphate farnesyltransferase 1
GCDHB	Glutaryl-coa dehydrogenase b
GLULB	Glutamate-ammonia ligase (glutamine synthase) b
GPT2L	Glutamic pyruvate transaminase (alanine aminotransferase) 2, like
HACD2	3-hydroxyacyl-coa dehydratase 2
HADHAA	Hydroxyacyl-coa dehydrogenase/3-ketoacyl-coa thiolase/enoyl-coa hydratase (trifunctional protein), alpha subunit a
HADHB	Hydroxyacyl-coa dehydrogenase/3-ketoacyl-coa thiolase/enoyl-coa hydratase (trifunctional protein), beta subunit
HER6	Hairy-related 6
ID2B	Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein, b
IDH1	Isocitrate dehydrogenase 1 (NADP+), soluble
INHBB	Inhibin, beta B
JAG1B	Jagged 1b
LFNG	LFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase
MAO	Monoamine oxidase
MARCO	Macrophage receptor with collagenous structure
MRC1A	Mannose receptor, C type 1a
MYCB	V-myc avian myelocytomatosis viral oncogene homolog b
NDUFA4	NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4
NDUFB5	NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5
NME5	NME/NM23 family member 5
NOTCH1A	Notch 1a
NOTCH3	Notch 3
PAICS	Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase
PAPSS2B	3’-phosphoadenosine 5’-phosphosulfate synthase 2b
PLCB4	Phospholipase C, beta 4
PPP1CB	Protein phosphatase 1, catalytic subunit, beta isozyme
PPP3CB	Protein phosphatase 3, catalytic subunit, beta isozyme
PPP3R1B	Protein phosphatase 3 (formerly 2B), regulatory s1ubunit B, alpha isoform, b
PRICKLE1A	Prickle homolog 1a
PSAT1	Phosphoserine aminotransferase 1
RHOAB	Ras homolog gene family, member Ab
RHOCB	Ras homolog gene family, member Ab
SDHC	Succinate dehydrogenase complex, subunit C, integral membrane protein
SHMT2	Serine hydroxymethyltransferase 2 (mitochondrial)
SMURF2	SMAD specific E3 ubiquitin protein ligase 2
TDH	L-threonine dehydrogenase
TUBA2	Tubulin, alpha 2
TUBA8L2	Tubulin, alpha 8 like 2
TUBB2	Tubulin, beta 2A class iia
YWHAZ	Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide

Additional Table 2: The full names of the proteins in Figure 4.

Additional Table 2.

The full names of the proteins in Figure 4.

Protein	Protein annotation
Abi1a	Abl-interactor 1a
Arf3a	ADP-ribosylation factor 3a
Arvcfb	Armadillo repeat gene deleted in velocardiofacial syndrome b
Atp1a3a	Atpase, Na+/K+ transporting, alpha 3a polypeptide
Atp2b3a	Atpase, Ca++ transporting, plasma membrane 3a
Bcar1	Breast cancer anti-estrogen resistance 1
Bmpr1aa	Bone morphogenetic protein receptor, type iaa
Bmpr1ab	Bone morphogenetic protein receptor, type iab
Calm1a	Calmodulin 1a
Cox5ab	Cytochrome c oxidase subunit Vab
Cox6a1	Cytochrome c oxidase subunit via polypeptide 1
Cox6b1	Cytochrome c oxidase subunit vib polypeptide 1
Cox7a2	Cytochrome c oxidase subunit viia polypeptide 2a (liver)
Crkl	V-crk avian sarcoma virus CT10 oncogene homolog-like
Ctnnb1	Catenin (cadherin-associated protein), beta 1
Ctnnd2a	Catenin (cadherin-associated protein), delta 2a
Ctnnd2b	Catenin (cadherin-associated protein), delta 2b
Cxcl12a	Chemokine (C-X-C motif) ligand 12a (stromal cell-derived factor 1)
Eef1a1b	Eukaryotic translation elongation factor 1 alpha 1b
ENSDARG00	Fibroblast growth factor receptor 3
000004782
Fgfr2	Fibroblast growth factor receptor 2
Fgfrl1a	Fibroblast growth factor receptor-like 1a
Fkbp1b	FK506 binding protein 1b
Flt4	Fms-related tyrosine kinase 4
Fzd7a	Frizzled class receptor 7a
Fzd7b	Frizzled class receptor 7b
Gad1b	Glutamate decarboxylase 1b
Gng3	Guanine nucleotide binding protein (G protein), gamma 3
Got1	Glutamic-oxaloacetic transaminase 1, soluble
HSPA4L	Heat shock protein 4 like
Idh1	Isocitrate dehydrogenase 1 (NADP+), soluble
Ilvbl	Ilvb (bacterial acetolactate synthase)-like
Inhbb	Inhibin, beta B
Kita	V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog a
Ldhbb	Lactate dehydrogenase Bb
LOC566572	Leucine-rich repeat-containing 4
Lrrn1	Leucine rich repeat neuronal 1
Mapk4	Mitogen-activated protein kinase 4
Musk	Muscle, skeletal, receptor tyrosine kinase
Mycb	V-myc avian myelocytomatosis viral oncogene homolog b
Myo18aa	Myosin xviiiaa
Ndufa4	NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4
Ndufb5	NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5
Nsfa	N-ethylmaleimide-sensitive factor a
OGDHL	Oxoglutarate dehydrogenase-like
Papss2b	3’-phosphoadenosine 5’-phosphosulfate synthase 2b
PKP4	Plakophilin 4
Ptenb	Phosphatase and tensin homolog B
Rasd1	RAS, dexamethasone-induced 1
Rhoab	Ras homolog gene family, member Ab
Rhoad	Ras homolog gene family, member Ad
Rnd3a	Rho family gtpase 3a
S1pr1	Sphingosine-1-phosphate receptor 1
Slc1a2b	Solute carrier family 1 (glial high affinity glutamate transporter), member 2b
Slc32a1	Solute carrier family 32 (GABA vesicular transporter), member 1
Smurf2	SMAD specific E3 ubiquitin protein ligase 2
Snap25b	Synaptosomal-associated protein, 25b
Sypb	Synaptophysin b
Tpm1	Tropomyosin 1 (alpha)
Vav3b	Vav 3 guanine nucleotide exchange factor b
Vegfaa	Vascular endothelial growth factor Aa
Yes1	YES proto-oncogene 1, Src family tyrosine kinase
Ywhaqb	Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide b
Zgc:103663	Solute carrier family 6 member 1b
Zgc:112335	Tubulin, beta 2A class iia

Additional Table 3: The full names of the genes in Table 4.

Additional Table 3.

The full names of the genes in Table 4

Gene symbol	Gene title
CRKL	V-crk avian sarcoma virus CT10 oncogene homolog-like
BCAR1	Breast cancer anti-estrogen resistance 1
PTENB	Phosphatase and tensin homolog B
RHOAB	Ras homolog gene family, member Ab
YES1	YES proto-oncogene 1, Src family tyrosine kinase

Additional Table 4: The full names of the genes in Table 5.

Additional Table 4.

The full names of the genes in Table 5

Gene symbol	Gene title
ATP1A3A	Atpase, Na+/K+ transporting, alpha 3a polypeptide
BMPR1AA	Bone morphogenetic protein receptor, type iaa
BMPR1AB	Bone morphogenetic protein receptor, type iab
CALM1A	Calmodulin 1a
COX5AB	Cytochrome c oxidase subunit Vab
COX6A1	Cytochrome c oxidase subunit via polypeptide 1
COX6B1	Cytochrome c oxidase subunit vib polypeptide 1
CTNNB1	Catenin (cadherin-associated protein), beta 1
CXCL12A	Chemokine (C-X-C motif) ligand 12a (stromal cell-derived factor 1)
FLT4	Fms-related tyrosine kinase 4
FZD7A	Frizzled class receptor 7a
FZD7B	Frizzled class receptor 7b
GOT1	Glutamic-oxaloacetic transaminase 1, soluble
IDH1	Isocitrate dehydrogenase 1 (NADP+), soluble
INHBB	Inhibin, beta B
KITA	V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog a
LDHBB	Lactate dehydrogenase Bb
MYCB	V-myc avian myelocytomatosis viral oncogene homolog b
NDUFA4	NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4
NDUFB5	NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5
OGDHL	Oxoglutarate dehydrogenase-like
PAPSS2B	3’-phosphoadenosine 5’-phosphosulfate synthase 2b
SMURF2	SMAD specific E3 ubiquitin protein ligase 2
TPM1	Tropomyosin 1 (alpha)
VEGFAA	Vascular endothelial growth factor Aa