Transcription factor Sox11b is involved in spinal cord regeneration in adult zebrafish

Animals

Adult zebrafish (Danio rerio), 6 months old, were bought from Aquatica Tropicals (Plant City, FL). The body lengths for all fish were 2–3 cm. Before surgery, the fish were kept in groups of 10 with a light cycle of 14 hours light and 10 hours dark at a temperature of 28°C.

Spinal cord injury

All animal experiments were approved by the Rutgers University Animal Care Committee. Spinal cord transection was performed as previously described (Becker et al., 1997). Briefly, fish were anesthetized by immersion in 0.033% aminobenzoic acid ethylmethylester (MS222, Sigma-Aldrich, St. Louis, MO) for 5 min. A longitudinal incision was made at the side of the fish to expose the vertebral column, which was then cut between two vertebrae at a level halfway between the dorsal fin and the operculum, about 4 mm caudal to the brainstem/spinal cord transitional zone. Wounds were sealed with Histoacryl (B. Braun, Melsungen, Germany), and the fish were kept individually at room temperature for up to 6 weeks following surgery. As control, a sham injury was performed by making the incision but without injury to the spinal cord.

Intraperitoneal BrdU application

Animals were anesthetized and intraperitoneally injected with BrdU (bromodeoxyuridine) (Sigma-Aldrich) solution (2.5 mg/ml) in a volume of 50 μl immediately after the transection.

Microarray analysis

In total, we studied 6 groups of injured or sham-injured control fish for survival times of 4 hrs, 12 hrs, and 11 days. Total RNA was isolated from 5 pooled spinal cords (5 mm caudally away from the lesion site) per time point using Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. A total 200 ng of RNA was used for Agilent Zebrafish Oligo Microarray (Agilent Technologies, Santa Clara, CA) analysis. Samples were labeled using the NuGEN (San Carlos, CA) WT-Ovation kit and hybridized at the Bionomics Research & Technology Center (Rutgers University). Microarray assays were performed on three biological replicate spinal cord pools per time point. Gene expression results were imported and analyzed using Agilent GeneSpring GX software. Data were quantile normalized, filtered for detectable expression in 50% of the samples, and then tested by 2-way ANOVA (treatment, time) at 5% FDR. Hierarchical clustering and gene ontology analysis were performed using Agilent GeneSpring GX software or R/Bioconductor (http://www.bioconductor.org).

Real time quantitative RT-PCR (qPCR)

For qPCR analysis, first-strand cDNA was generated using random primers and Superscript™ II Reverse Transcriptase (Invitrogen, Carlsbad, CA). The PCR primers were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA). The following primer sequences were used:

• Sox11b, 5’-cgagttcccggactattgca-3’ and 5’-tctcccgcgatcatctcact-3’;

• Ascl1a, 5’-cgccaagatggaaataagcg-3’ and 5’-caagcaggtggcatgaactg-3’;

• Nestin, 5’-cggcccttgatccgttataa-3’ and 5’-accgatgtggtgtgaggtttc-3’;

• Ribosomal protein P0, 5’-atcggctacccaactcttgct-3’ and 5’-tgtttcgacagtgacagccag-3’.

Reverse transcribed mRNA was amplified in the presence of SYBR green (Applied Biosystems). Accumulation of fluorescence was monitored during amplification in an Applied Biosystems 7900HT sequence detection system (Applied Biosystems), using 10 μl reactions in 384-well plates. All assays were performed on triplicate samples. All assay products were validated using melting curves to confirm the presence of single PCR products. Ribosomal protein P0 served as the internal control for qPCR.

In situ hybridization for Sox11b, Oct4, Nestin, Ascl1a

In situ hybridization probes (sense and antisense probes) for Sox11b (NM_131337.1), Nestin (XM_001919887.1), Ascl1a (NM_131219.1) and Oct4 (NM_131112) were transcribed in vitro from purified RT-PCR fragments. Each amplified probe was subcloned into pGEM-T Easy vector (Promega, Madison, WI) containing the T7 promoter sequence: 5’-ccctatagtgagtcgtatta-3’ and SP6 promoter sequence: 5’-atttaggtgacactatagaa-3’. All four probes were transcribed in vitro using the SP6 MEGAScript Kit (Ambion, Austin, TX) or T7 MEGAScript Kit (Ambion) as described previously (Tongiorgi et al., 1995).

Non-radioactive detection of mRNA in sections of adult zebrafish CNS was performed as published previously (Bernhardt et al., 1996). The spinal cords were fixed for 6 hrs in 4% paraformaldehyde in PBS (pH 7.0), followed by incubation in 15% sucrose in PBS overnight. Then, 15 μm sections of spinal cords were cut from fresh-frozen tissue on a cryostat. The sections were pre-hybridized for 3 hrs at 55°C, and hybridized with the DIG-labeled probes at 55°C overnight. After extensive washing (at 55°C), alkaline phosphatase-coupled anti-DIG Fab fragment antibodies (Roche, Indianapolis, IN) were applied at 4°C overnight. Antibody binding was detected using an alkaline phosphatase reaction with NBT/BCIP (nitro-blue-tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate; Roche) as substrates. Developing times ranged from several hours to overnight depending on the level of mRNA expression in the spinal cord. Sense probes were developed in parallel under the same conditions as the antisense probes, serving as negative controls. Sham injured control and injured fish were analyzed on the same microscopic slides. Sections were viewed and photographed using an Axiophot microscope (Zeiss, Oberkochen, Germany).

Immunohistochemistry and double staining for in situ hybridization and immunohistochemistry

Immunohistochemical staining was performed following standard methods. Sections were prepared as described above and incubated with primary antibody against HuC/D (1:20, Invitrogen), GFAP (1:200, Invitrogen), NG2 (1:100, Millipore, Billerica, MA), BrdU (1:200, DAKO, Carpinteria, CA) overnight at 4°C, followed by incubation with secondary antibody Alexa Fluor 488 goat anti-mouse (1:500, Invitrogen) for 1 hr at 37°C. Fluorescence images were collected using an Axiophot microscope. Antigen retrieval was performed by incubating the sections for 30 min in 2N HCl for BrdU immunohistochemistry at 37°C. For double staining experiments, in situ hybridization preceded immunohistochemistry as described previously (Chen et al., 2007).

Cell counts

BrdU positive cells in the wall of the central canal were counted in the same region of the spinal cord both in sham-injured control and injured zebrafish. Ten consecutive cross sections (from the lesion site to the caudal part of the spinal cord) per zebrafish were selected for counting by fluorescence microscopy. The average number of cells was used for statistical analysis. The observer was blinded to experimental treatments. The same BrdU pulse labeling assay was applied to the fish that received standard control morpholino treatment and the fish that received Sox11b antisense morpholino treatment.

Morpholino treatment

Sox11b vivo-morpholino (5’-CTGTGCTCCGTCTGCTGCACCATGT-3’, vivo-porter coupled, Gene Tools, Philomath, OR) and Ascl1a vivo-morpholino (5’-TTTCCATCTTGGCGGTGATGTCCAT-3’, vivo-porter coupled) and standard control morpholino (5’-CCTCTTACCTCAGTT-3’, vivo-porter coupled) were dissolved in Danieau solution (Nasevicius and Ekker, 2000) and soaked onto small pieces of Gelfoam (Upjohn, Kalamazoo, MI) in a volume of 6 μl. These pieces of Gelfoam were divided into 30 smaller pieces to yield 600 ng of morpholino per piece and allowed to dry. Per zebrafish, one of these pieces was applied to the spinal transection site immediately after performing the transection. The wound was sealed with Histoacryl and each animal was allowed to survive the operation for 6 weeks.

Swim tracking

Swimming capabilities of injured fish at 6 weeks after Sox11b morpholino or standard control morpholino treatment were analyzed in two trials of 5 min each (intertrial interval, 4 hrs). In each trial, the zebrafish was placed in a brightly illuminated (100 lux) glass tank (50×30×30 cm) filled with aquarium water (5 cm deep; 25°C). A video camera recorded the trials from above the tank. We used an ANY-maze video system (Stoelting Co, Wood Dale, IL) to track swim paths. Mean lengths of the swim paths (“total distance moved”) of the two trials were used for graphical presentation and statistical analysis. The experimenter was blinded to the treatment of individual zebrafish before testing.

Statistical analysis

Student’s t-test was used to compare the Sox11b, Nestin and Ascl1a qPCR mRNA levels between experimental group and sham injured control group and also for analysis of the difference in BrdU positive cell numbers and in the swimming behavior between experimental group and sham injured control group. Two-way ANOVA was used to compare the Sox11b mRNA expression between each group in microarray analysis. Differences were considered statistically significant at p<0.05, marked with an asterisk (*). Data are presented as mean ± S.D. of at least 3 independent experiments.

The expression of Sox11b is increased in the caudal part of spinal cord after injury

To investigate damage-induced gene expression of the injured spinal cord, Agilent Zebrafish Oligo Microarray assays were performed. We chose 4 hrs, 12 hrs and 11 days post-injury for harvesting the spinal cord caudal to the lesion site because these times represent the acute and the more chronic response phases of the spinal cord to the injury. A large list of genes was selected by two-way ANOVA as differentiated by treatment (i.e., injury vs. control, 530 genes), time (3,096 genes) or an interaction of the two (6 genes) (GEO accession number: GSE20460. The link for the GEO dataset: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20460). We explored the literature on many of these genes, and noted that Sox11b (Agilent probe ID A_15_P102878) was of particular interest in the context of regeneration after spinal cord injury. The change in Sox11b expression after injury is shown in Fig. 1A. Microarray analysis indicated that the expression of Sox11b was similar to control during acute injury phase time points (4 hrs or 12 hrs) but had increased significantly by 11 days post injury, with a 2.44 ± 0.117 fold change from sham-injured control at this time (1.00 ± 0.009; p<0.01; Fig. 1A, Table 1). qPCR analysis confirmed the array results, producing 1.66 ± 0.135 fold (Table 2) or 1.83 ± 0.156 fold from control in two separate sets of fish. While this effect of injury is not large, it was highly reproducible and statistically significant in every assay that was performed—perhaps only a subset of cells in the dissected tissue express Sox11b and therefore the effect has been diluted in the dissected tissue samples. Examining ANOVA-selected array results by K-means clustering (not shown), GAP-43 mRNA was found to cluster with Sox11b, suggesting that the pattern of regulation of Sox11b after spinal cord injury was similar to other regeneration-associated genes. We chose to identify cells expressing Sox11b to better understand its function after injury.

Sox11b expression is up-regulated in ependymal cells lining the central canal and in newly-generated neurons in the spinal cord caudal to the lesion site

To determine which cell types express Sox11b, we performed in situ hybridization. At 12 hrs post-injury, expression of Sox11b mRNA increased in the injured spinal cords compared to the sham-injured controls (Fig. 1B-J). Sox11b expression was mainly in ependymal cells lining the central canal and in some cells located in the gray matter and dorsal column. At 3 and 11 days post-injury, the hybridization signal for Sox11b mRNA continued to increase relative to sham-injured controls. In addition, more Sox11b positive cells appeared in the gray matter compared to 12 hrs post-injury samples (Fig. 1B-J). At 6 weeks post-injury, Sox11b expression returned to normal levels (Data not shown). BrdU, a thymidine analog that is incorporated into the DNA of dividing cells during S-phase, labels mitotic cells. We used pulse labeling with BrdU to find newly generated cells in the spinal cord. At 3 days post-injury, the number of BrdU-positive proliferative cells increased compared to sham-injured controls, mainly in ependymal cells lining the central canal (Fig. 2A, 2B). Moreover, double labeling of BrdU and HuC/D, a marker of neuronally committed stem cells or immature neurons (Marusich et al., 1994), showed that the immunofluorescence signal for BrdU co-localized on some ependymal cells (Fig. 2C). These data suggest that ependymal cells could differentiate toward neurons in response to injury.

To investigate the migration of newly generated cells, we applied the same BrdU pulse labeling assay to fish that received Sox11b antisense morpholino treatment and standard control morpholino treatment. At 3 days after BrdU application, BrdU-positive cells were mainly localized in the wall of the central canal (Fig. 2A-C). But, at 11 days after BrdU application, few BrdU-positive cells were in the wall of central canal while more BrdU-positive cells were present in the gray matter of spinal cord (Fig. 3A), indicating that newly generated cells migrated toward gray matter from the wall of central canal. By comparison, fish that received Sox11b antisense morpholino treatment had less BrdU-positive cells in the wall of the central canal (at 3 days after injury) and the gray matter of spinal cord (at 11 days after injury) than fish that had received standard control morpholino treatment (Fig. 3A).

To identify the Sox11b-positive cells in the spinal cord parenchyma, we double stained with in situ hybridization for Sox11b and immunohistochemistry for NG2, GFAP and HuC/D. NG2 is a marker for oligodendrocyte precursor cells. GFAP is a marker for mature and reactive astrocytes. Immunofluorescence signals for NG2 or GFAP did not co-localize with the in situ hybridization signal for Sox11b, indicating the lack of Sox11b expression in glial-restricted precursor cells and astrocytes. However, immunofluorescence signal for HuC/D co-localized with in situ hybridization signal for Sox11b. This result suggests that the Sox11b-positive cells in the spinal cord parenchyma are newly-differentiating neuronal precursors and/or immature neurons (Fig. 3B).

Sox11b regulates the expression of Nestin in the caudal part of spinal cord after injury

Because ependymal cells and newly-differentiating neuronal precursors expressed Sox11b, we hypothesized that Sox11b participates in neurogenesis initiated by endogenous ependyma-derived neural stem cells. Therefore we investigated the relationship between injury-induced Sox11b expression and the neural stem cell associated gene, Nestin. qPCR showed that expression of Nestin mRNA in the injured spinal cords did not differ from sham-injured controls at 4 and 12 hrs post-injury. But at 11 days post-injury, Nestin expression significantly increased in injured spinal cords compared to sham-injured controls (Table 2). The in situ hybridization signal was clearly visible at 3 and 11 days post-injury, while hybridization signals were very weak both at 12 hrs post-injury and in the sham-injured controls (Fig. 4A-F). Significantly, Nestin was also primarily expressed in ependymal cells lining the central canal and in some cells located in the spinal cord parenchyma (Fig. 4A-F), closely resembling the expression pattern of Sox11b. Double staining with in situ hybridization for Nestin and immunohistochemistry for HuC/D showed that only few cells were co-labeled. One possible explanation is that Nestin and HuC/D are markers of different kinds of cells. Nestin is a marker for neural stem cells, whereas HuC/D is a marker for neuronally committed stem cells or immature neurons. Only few neuronally committed stem cells were co-labeled with nestin and HuC/D. Taken together, these results reveal that Sox11b is expressed both in neural stem cells and neuronally committed stem cells after injury.

To evaluate the effect of Sox11b on Nestin expression, we inhibited translation of Sox11b mRNA in the spinal cord by applying antisense morpholino to the injury site at the time of injury. Given the time points that Sox11 is up-regulated post-injury, we chose to assess Nestin expression at 3 and 11 days after morpholino treatment. Interestingly, Sox11b antisense morpholino treatment strongly reduced Nestin expression in the caudal spinal cord (Table 3) compared with standard control morpholino treatment. These results indicate that Sox11b is involved in regulating the Nestin expression in the caudal part of the spinal cord after injury.

Since the HMG domain is known to regulate target gene expression by pairing with specific partner proteins, we searched for a partner protein corresponding to Sox11b. We examined the expression of Oct4 (POU5F1), a POU transcription factor family member, using in situ hybridization. Oct4 expression was clearly visible in the spinal cord after injury (Fig. 5A-E). Oct4 mRNA was primarily expressed in ependymal cells lining the central canal and in some cells of the gray matter, closely resembling the expression pattern of Sox11b (Fig. 1B-J).

Sox11b increases expression of proneural bHLH transcription factor Ascl1a

Since expression of Sox11b is largely restricted to newly-differentiating neuronal precursors or immature neurons in spinal cord parenchyma, and because of its involvement in regulating the Nestin expression in the caudal spinal cord after injury, we hypothesized that Sox11b regulated neurogenesis after spinal cord injury. To test this hypothesis we examined the expression of the proneural bHLH (basic helix-loop-helix) transcription factor Ascl1a (also named Mash1a) in the spinal cord caudal to lesion site. Ascl1a participates in neuronal differentiation and subtype specification during embryogenesis (Helms et al., 2005; Parras et al., 2007; Kim et al., 2007; Fausett et al., 2008). Its expression after spinal cord injury is significantly greater than in sham-injured controls (Fig. 6A, Table 1). qPCR confirmed the microarray results (Table 2). In situ hybridization data showed Ascl1a expression in the caudal part of spinal cord at 12 hrs after injury and continued upregulation of the gene at 3 and 11 days after injury (Fig. 6B-G), with an expression pattern similar to that of Sox11b (Fig. 1B-J). However, as contrast to Sox11b, Ascl1a expression was stronger in cells of the spinal cord parenchyma and weaker in ependymal cells lining the central canal.

To determine whether Sox11b induction participates in up-regulation of Ascl1a expression, we administered Sox11b antisense morpholino, Ascl1a antisense morpholino, or standard control morpholino to acutely injured spinal cords and examined Ascl1a and Sox11b expression by qPCR. At 3 days post-injury, Sox11b antisense morpholino treatment significantly reduced Ascl1a expression compared with standard control morpholino-treated spinal cords (Table 3). At 11 days post-injury, Sox11b antisense morpholino treatment had no effect on Ascl1a mRNA expression (Table 3). At 3 days post-injury, Ascl1a antisense morpholino treatment increased Sox11b expression compared to standard control morpholino (Table 4). These results suggest that Sox11b expression may be necessary for Ascl1a expression after injury. In addition, Ascl1a antisense morpholino-treated spinal cord exhibited an increase in the expression of Nestin (Table 4). Double staining by in situ hybridization for Ascl1a and immunofluorescence staining for HuC/D indicated that Ascl1a was expressed in cells of the spinal cord parenchyma, co-localizing with the immunofluorescence signal for HuC/D (Fig. 6H-K). Taken together, these results indicate that Ascl1a expression induced by Sox11b does not promote the maintenance of neural stem cells, but may promote their differentiation toward a neuronal lineage.

Sox11b promotes locomotor recovery

To examine the effects of reduced Sox11b expression on functional recovery, we quantified locomotor activity by measuring distance moved in 5 min (Becker et al., 2004) in fish treated with Sox11b antisense or standard control morpholino. At 6 weeks after injury the total distance moved was 9.1 ± 5.9 m (n=4) for fish treated with Sox11b antisense morpholino, and 16.9 ± 3.8 m (n=6, p<0.05) for fish treated with standard control morpholino. Thus, fish that had received Sox11b antisense morpholino moved 46% less than fish that had received standard control morpholino (Fig. 7A, B).