Adult Gekko japonicus were used as described by Dong et al. (2013). Briefly, adult animals were fed ad libitum with mealworms and housed in an air-conditioned room with a controlled temperature (22–25°C) and saturated humidity. Anesthesia was induced by cooling the animals on ice prior to tail amputation. Amputation was performed at the sixth caudal vertebra, identified based on the special tissue structure present at that position (McLean and Vickaryous, 2011), by placing a slipknot of nylon thread and pulling gently until the tail was detached, thus mimicking the autotomy undergoing for natural defense.

All experiments were conducted in accordance with guidelines of the NIH (Guide for the Care and Use of Laboratory Animal: 1985), and the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience. Experiments were approved according to the Animal Care and Use Committee of Nantong University and the Jiangsu Province Animal Care Ethics Committee. All geckos (n = 15) were anesthetized on ice prior to sacrifice.

To obtain the full length of gecko Snail family members, anti-sense primer for Snail1 (5′-CAT GCG GGA GAA AGT CCG GGA GCA GGT T-3′), Snail2 (5′- TGT TTG TGC AGA AGA GAC ATG CGG GAG A -3′) and Snail3 (5′- GCA CAT CCG CAC CCA CAC GCT GC -3′) ; sense primer for Snail1 (5′- GAA GCC CAA CTA CAG CGA GCT GGA GAG -3′), Snail2 (5′- ACT TCA AGG ACA CAT CAG AAC TCA CAC C -3′) and Snail3 (5′- TCA AGA TGC ACA TCC GCA CCC ACA CGC T -3′) were designed according to genome sequences (Liu et al., 2015). Both 5′-RACE and 3′-RACE were performed using the BD SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. Comparison against the GenBank protein database was performed using the PSI-BLAST network server at the National Center for Biotechnology Information (Altschul et al., 1997). Multiple protein sequences were aligned using the MegAlign program by the CLUSTAL method in the DNASTAR software package (Burland, 2000).

Snail overexpression (LV5-Snail) lentivirus was produced in Shanghai GenePharma Co. Ltd, according to the manufacturer’s procedures. The ORF of snail family members was cloned to the LV5 vector via the Not I and Bam HI sites, respectively. Snail expression was driven by the EF-1α promoter, and the expression of reporter enhanced green fluorescent protein (eGFP) was driven by CMV promoter. Both Snail and eGFP sequence were incorporated into a lentivirus. Lentiviruses were produced using 293T cells, and the viral titers reached 1 × 10⁹ TU/ml for further studies.

Total RNA was prepared with Trizol (Gibco, Gran Island, NY, USA) from different tissues, including the brain, spinal cord, heart, liver, testis and ovary of adult geckos. Total RNAs were also extracted from 0.5 cm spinal cord segments of 20 geckos amputated from the sixth caudal vertebra at 1 day, 3 days, 1 week and 2 weeks, respectively.

For Q-PCR examination of Snail temporal expression, the first-strand cDNA was synthesized using Omniscript Reverse Transcription Kit (QIAGEN) in a 20 μl reaction system containing 2 μg total RNA, 0.2 U/μl M-MLV reverse transcriptase, 0.5 mM dNTP mix, 1 μM Oligo-dT primer. The cDNA was diluted 1:5 before use in Q-PCR assays. The sequence-specific primers were designed and synthesized by Invitrogen (Shanghai, China). Primer pair and probe for Snail1: forward primer 5′- CCG AGA AAT TCC ACT GCA -3′, reverse primer 5′- GGT ATG GCT TCG GAT GTG -3′; for Snail2, forward primer 5′- TAC CTT TAT GAG AGC TAC CCA -3′, reverse primer 5′- TTC CCA AAG ACG AAG GAT ATC -3′; for Snail3, forward primer 5′- TTA GTT GCT CCG TCC AGA -3′, reverse primer 5′- AAA CCA CGT TGC CAT ACA -3′. Q-PCR reactions were performed in a final volume of 20 μl (1 μl cDNA template and 19 μl Q-PCR reaction buffer containing 2.5 mmol/L MgCl₂, 0.2 mmol/L dNTPs, anti-sense and sense primers 0.5 μmol/L, taqman probe 0.4 μmol/L, DNA polymerase 0.2 μl and 1 × DNA polymerase buffer). The Rotor-Gene 5 software (Corbett Research, Rotor-Gene, Australia) was used for real-time PCR analysis. Reactions were processed using one initial denaturation cycle at 94°C for 5 min followed by 40 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. Fluorescence was recorded during each annealing step. At the end of each PCR run, data were automatically analyzed by the system and amplification plots obtained. Snail full-length plasmid was used to prepare standard curves and used as a specificity control for real-time PCR. The expression levels of the Snail cDNA were normalized to an endogenous EF-1α cDNA using forward primer 5′-CCT TCA AAT ATG CCT GGG T-3′, reverse primer 5′-CAG CAC AGT CAG CTT GAG AG-3′ and taqman probe 5′-TTG GAC AAG CTG AAG GCA GAA CGT G-3′. In addition, a negative control without the first-strand cDNA was also performed.

Human neuroblastoma cell line SH-SY5Y (Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences Cell Resource Center) or gecko astrocytes cell line, Gsn1, were grown in Dulbecco’s Modified Eagles Medium (DMEM, Gibco BRL) supplemented with 10% (v/v) fetal bovine serum in a 37°C or 30°C humidified incubator with 5% CO₂. For glucose deprivation (GD)-induced neuronal apoptosis, differentiated SH-SY5Y cells induced with 1 μM all trans-retinoic acid (RA, Sigma) were transfected with LV5-Snail or control lentivirus, and cultured in DMEM with medium supplemented with 5% (v/v) fetal bovine serum. Then, they were subjected to GD insult as described previously (Ferretti et al., 2016). Cells were transferred to the glucose-free DMEM in the incubator chamber for 24 h incubation. At the end of cell treatments, cell culture was subjected to various assessments, or counterstained with annexinV-PE for 10 min at 20–25°C and mounted on slide glasses with mounting medium. Images were captured on a Nikon Diaphot microscope.

Gsn1 cells were treated with 4 ng/ml recombinant TGFβ1 or TGFβ2 (Peprotech) with or without 10 μmol/L TGFβ receptor inhibitor LY2109761 (Selleck) for 2 h, respectively. The cells were then subjected to the determination of Snail transcriptional expression.

Gsn1 cells were resuspended in fresh pre-warmed (30°C) complete medium, counted and plated at a density of 2 × 10⁵ cells/ml on 0.01% poly-L-lysine-coated 96-well plates. At the indicated time point after cell transfection, 50 mM EdU was applied to the cultures and the cells were grown for an additional 2 h. Finally, the cells were fixed with 4% formaldehyde in PBS for 30 min. After labeling, the Gsn1 cells were assayed using Cell-Light EdU DNA Cell Proliferation Kit (Ribobio) according to the manufacturer’s protocol. Analysis of Gsn1 proliferation (ratio of EdU⁺ to all Gsn1 cells) was performed using images of randomly selected fields obtained on a DMR fluorescence microscope (Leica Microsystems, Bensheim, Germany). Assays were performed three times using triplicate wells.

Migration of astrocytes was studied using 6.5 mm transwell chambers with 8 μm pores (Corning Costar) as described previously (Dong et al., 2013). One hundred microliters astrocytes (2 × 10⁵ cells/ml) resuspended in DMEM/F12 were transferred to the top chambers of each transwell and allowed to migrate at 30°C in 5% CO₂ for 30 h, and 600 μl of DMEM/F12 was injected into the lower chambers. The upper surface of each membrane was cleaned with a cotton swab at the indicated time point. Cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet, imaged, and counted using a DMR inverted microscope (Leica Microsystems). Assays were done three times using triplicate wells.

Protein was extracted from cells with a buffer containing 1% SDS, 100 mM Tris–HCl, 1 mM PMSF and 0.1 mM β-mercaptoethanol. Protein concentration of each specimen was detected by the Bradford method to maintain the same loads. Protein extracts were heat denatured at 95°C for 5 min, electrophoretically separated on 10% SDS–PAGE, and transferred to PVDF membranes. The membranes were subjected to the reaction with a 1:1000 dilution of primary antibodies in TBS buffer at 4°C overnight, followed by a reaction with secondary antibody conjugated with goat anti-rabbit or goat anti-mouse HRP (Proteintech) dilution 1:1000 at room temperature for 2 h. After the membrane was washed, the HRP activity was detected using an ECL kit. The image was scanned with a GS800 Densitometer Scanner (Bio-Rad), and the data were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-actin (1:5000) was used as an internal control. Antibodies used in Western blot are: Snail1 (polyclonal antibody prepared from polypeptides), Snail3 (polyclonal antibody prepared from polypeptides); GFAP (Sigma); Cleaved caspase 3 (Asp175), p-ERK1/2, ERK1/2, p-AKT, AKT, Bcl-XL (Cell Signaling), N-cadherin and β-actin (Proteintech).

RhoA, Rac1 or Cdc42 activation was determined using the rhotekin-RBD that specifically binds activated Rho and the PBD-PAK that has a high affinity for both GTP-Rac and GTP-Cdc42 (RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit, Cytoskeleton, Denver, CO, USA). In brief, the cells were lysed with ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 2 mM MgCl₂, 1% NP-40, 10% glycerol, 100 mM NaCl, and Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland) and centrifuged for 5 min at 14,000 g. The equivalent protein amounts of lysate (500 μg total cell protein) were performed pull-down assay with 50 μg rhotekin-RBD beads and 20 μg PAK-PBD beads, and rotated for 60 min at 4°C. The beads were washed three times with lysis buffer and heated for 5 min at 100°C in SDS-PAGE sample buffer, and then analyzed for bound RhoA, Rac1 and Cdc42 molecules by Western blotting using anti-RhoA antibody (1:500, Cytoskeleton), anti-Rac1 antibody (1:500, Cytoskeleton) or anti-Cdc42 antibody (1:500, Cytoskeleton).

The spinal cord segments were harvested, post-fixed and sectioned. Sections were allowed to incubate with ployclonal rabbit anti-gecko Snail1, rabbit anti-gecko Snail3 antibody (1:500 dilution), polyclonal rabbit anti-bovine galactocerebroside antibody (1:200 dilution, Millipore), polyclonal rabbit anti-human GFAP antibody (1:500 dilution, Sigma), or polyclonal rabbit anti-human neuron-specific enolase (NSE) antibody (1:200 dilution, Abcam) at 4°C for 36 h. The sections were further reacted with the Cy3-labeled secondary antibody goat anti-mouse IgG (1:400 dilution, Gibco), or the FITC-labeled secondary antibody goat anti-rabbit IgG (1:400 dilution, Gibco) at 4°C overnight, followed by observation under a confocal laser scanning microscope (Leica, Heidelberg, Germany).

Statistical significance of differences between groups was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc comparisons tests with SPSS 15.0 (SPSS, Chicago, IL, USA). Normality and homoscedasticity of the data were verified before any statistical analysis using levene’s test. Statistical significance was set at p < 0.05 level (significant) and p < 0.01 (highly significant).

By screening genome of gecko, three Snail paralogs, namely Snail1, Snail2 and Snail3, are recovered. The full sequence of Snail1 (GenBank accession number KT032183), Snail2 (KT032184) and Snail3 (KT032185) amplified by 5′- and 3′- RACE, encodes a protein of 259, 268 and 300 amino acid residues, respectively (Figure 1A). All these paralogs contain the N-terminal SNAG domain, a short sequence of nine amino acids (amino acids 1–9) associating with transcriptional repression (Manzanares et al., 2001). The CtBP (C-terminal Binding Protein) interaction motif, which is prevalent in invertebrates to facilitate interaction with the co-repressor C-terminal Binding Protein (Hemavathy et al., 2000), was also present in gecko Snail2 and Snail3 (Figure 1A), in concert with the findings from Xenopus, chicken, mouse and human Snail2 (Hemavathy et al., 2000). Gecko Snail paralogs possess five C2H2 zinc finger motifs at the C-terminal domain (Figure 1A), while only four such motifs are found in human Snail1, suggesting differential binding affinities of gecko Snail1 to target genes (Sefton et al., 1998; Villarejo et al., 2014).

Phylogenetic tree constructed using the PHYML implementation of Maximum-Likelihood demonstrated that gecko Snail paralogs clustered with corresponding homologs of other vertebrates, suggesting an evolutionary conservation of the protein in the phylogeny (Figure 1B).

We first examined the expression of Snail paralogs in different gecko tissues by RT-PCR. Results revealed that Snail1, Snail2 and Snail3 were ubiquitously expressed in the brain, spinal cord, heart, liver, testis and ovary (Figures 2A–C). To understand the potential roles of Snail paralogs in the spontaneously regenerating spinal cord, gecko tail was detached at the sixth caudal vertebra, and 0.5 cm segments at the injured sites were collected at 0 day, 1 day, 3 days, 1 week and 2 weeks, respectively. Expression analysis of Snail paralogs displayed that both Snail1 and Snail3 were markedly upregulated in the cord from 1 day onwards after lesion, while Snail2 decreased at 3 days, 1 week and 2 weeks, (Figures 2D–F). We therefore focused our attention on the roles of Snail1 and Snail3 in the regenerating spinal cord, while ignoring those of Snail2 for its dispensable action in embryonic, especially in neural crest development (Jiang et al., 1998). The expression level of Snail1/3 protein showed a consistency with those of the transcription, except at 2 weeks due to differential translation mechanisms (Figures 3A–C). Subsequent immunostaining in the sections of the same segments demonstrated that both Snail1 and Snail3 located in the nucleus, and colocalized with NSE- and astrocyte-specific GFAP-positive cells, but not with oligodendrocyte-specific galactocerebroside-positive cells (Figure 3D). The staining intensity in the two cell types is also enhanced after SCI (data not shown), indicating that both neurons and astrocytes are potentially regulated by Snail1 and Snail3 during the spinal cord regeneration.

To unveil the physiological roles of increased expression of Snail1 and Snail3 in neurons following spinal cord transection, we overexpressed Snail1 and Snail3 in the differentiated SH-SY5Y cells by lentivirus. Compared with the controls, the length of neurite or cell morphology has not been changed remarkably (Figure 4A). Given that a large amount of neurons survived from axonal injury in gecko spinal cord, we turned to account for its function on anti-apoptosis, which has been mentioned in the hematopoietic progenitor cells and radial glial precursor cells (Wu et al., 2005; Zander et al., 2014). GD is a convictive model for inducing cell apoptosis in vitro (Ferretti et al., 2016). Following glucose free for 24 h in the culture medium, the control cells suffered markedly apoptosis, as evidenced by morphology and Annexin-V staining (Figures 4B–D). Whereas cells transfected with Snail1 or Snail3 lentivirus showed an increased ratio of survival, indicating the important anti-apoptotic roles of both proteins on injured neurons (Figures 4B–D).

We next sought to investigate the mechanism of Snail-expressing cells resistant to GD. Biochemical results showed that the activity of cleaved caspase3 was significantly decreased, and both the MAPK (p-ERK) and PI3K (p-AKT) pathways were highly active in Snail1- and snail3-expressing cells (Figures 4E–J). It has been well known that MEK/Erk and PI3-K/Akt pathways can mediate the upregulation of Bcl-xL, a death-inhibitory member of the Bcl-2 family that blocks the stress-induced release of cytochrome c (Ramljak et al., 2003; Vega et al., 2004). As such, we determined the expression of Bcl-xL in the Snail1- and Snail3-expressing SH-SY5Y cells. As expected, it was significantly upregulated in comparison with those of the control (Figures 4K,L). The data indicate that activation of the MEK/Erk and PI3-K/Akt ascribed to enforced Snail expression, contributes to the survival properties following GD.

Both Snail1 and Snail3 expression were also upregulated in astrocytes following gecko spinal cord amputation. Accordingly, we examined their effects on gecko Gsn1 cell line in vitro following lentivirus overexpression. Snail1- and Snail3-expressing astrocytes have not shown a significant increase in proliferation, as assayed by EDU incorporation (Figures 5A,C). However, transwell experiments demonstrated that strengthening expression of Snail1 and Snail3 facilitated the migration of astrocytes (Figures 5B,D).

Cell migration and morphological changes are implicated in the actin cytoskeleton remodeling, and this process is controlled by RhoA/Cdc42/Rac1 pathways (Pawlak and Helfman, 2001). To determine whether Snail affects the status of RhoA/Cdc42/Rac1 in astrocytes, GTP-bound RhoA/Cdc42/Rac1 was investigated by pull-down assay. The level of active Cdc42 and Rac1 was found to be increased markedly in Snail1- and Snail3-expressing cells (Figures 5E–H), whereas the active GTP-RhoA remained unaltered (Figures 5I,J). The data indicate that Snail1 and Snail3 mediate astrocytic migration through Cdc42/Rac1 signaling.

The most prominent function for the Snail is its involvement in inducing EMT during the development and tumor progression, where it down-regulates epithelial genes and up-regulates mesenchymal genes (Zeisberg and Neilson, 2009). It has been shown that Scratch1 and Scratch2 regulate neuronal migration onset via an EMT-like mechanism (Itoh et al., 2013), whereas EMT-related markers in glia lineages are still unknown. Astrocytes are derived from heterogeneous populations of progenitor cells in the neuroepithelium of the developing CNS (Rowitch and Kriegstein, 2010), hinting at the same ectodermal origin with epithelial cells. Therefore, we began to understand whether Snail participated in the regulation of EMT-related markers in astrocytes. Both Snail1 and Snail3 have not facilitated the expression of GFAP, a marker of reactive astrocytes in the mammalian CNS (Figures 6A,B), but they enhanced the expression of N-cadherin (Figures 6A,C), a similar pattern emerging in the EMT process (Zeisberg and Neilson, 2009). Neither Snail1 nor Snail3 was able to affect the expression level of vimentin, but Snail3 additionally stimulated the transcription of fibronectin (Figure 6D). The data indicate that Snail1 and Snail3 are capable of activation of several EMT-related markers by avoiding reactive gliosis.

TGFβ, a pleiotrophic cytokine, has been shown to mediate Snail expression by binding to the constitutively active type II serine/threonine kinase receptor (TβRII), whereby it heteromerizes with and activates the type I serine/threonine kinase TGFβ receptor (TβRI; Barrallo-Gimeno and Nieto, 2005; Thakur et al., 2014). To ascertain specific TGFβ types involved in the regulation of snail expression in the regenerating spinal cord, we first examined the expression changes of TGFβ1, TGFβ2 and TGFβ3 in response to the cord injury. Both TGFβ1 and TGFβ2 showed an increase in the regenerating spinal cord, which might correlate with the transcriptional activation of Snail1/3 (Figure 7A). Cultured Gsn1 cells were further treated with 0, 1, 2, 4, 8 ng/ml and 16 ng/ml recombinant TGFβ1 or TGFβ2 proteins for 2 h, respectively. Transcriptional analysis revealed that Snail1 was activated by TGFβ1 or TGFβ2 in a concentration-dependent manner (Figures 7B,C). Addition of 10 μmol/L TGFβ receptor inhibitor LY2109761 efficiently blocked these actions (Figure 7D). In contrast, expression of Snail3 remains unchanged following treatment with recombinant TGFβ1 or TGFβ2 protein, suggesting additional factor(s) responsible for its activation.