The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain
- Caghan Kizil†1,
- Stefanie Dudczig†1,
- Nikos Kyritsis1,
- Anja Machate1,
- Juliane Blaesche1,
- Volker Kroehne1 and
- Michael Brand1Email author
© Kizil et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 20 June 2012
Published: 23 July 2012
Unlike mammals, zebrafish exhibits extensive neural regeneration after injury in adult stages of its lifetime due to the neurogenic activity of the radial glial cells. However, the genes involved in the regenerative neurogenesis response of the zebrafish brain are largely unknown. Thus, understanding the underlying principles of this regeneration capacity of the zebrafish brain is an interesting research realm that may offer vast clinical ramifications.
In this paper, we characterized the expression pattern of cxcr5 and analyzed the function of this gene during adult neurogenesis and regeneration of the zebrafish telencephalon. We found that cxcr5 was upregulated transiently in the RGCs and neurons, and the expression in the immune cells such as leukocytes was negligible during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in altered proliferation and neurogenesis response of the RGCs. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection, we observed outcomes similar to the overexpression of the dominant negative cxcr5 variant.
Thus, based on our results, we propose that cxcr5 imposes a proliferative permissiveness to the radial glial cells and is required for differentiation of the RGCs to neurons, highlighting novel roles of cxcr5 in the nervous system of vertebrates. We therefore suggest that cxcr5 is an important cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.
KeywordsAdult zebrafish telencephalon cxcr5 Radial glia Proliferation Regenerative neurogenesis Adult neurogenesis Differentiation
The zebrafish, unlike mammals, can regenerate its adult brain and therefore has become an excellent system to study adult neurogenesis and brain regeneration in recent years [1–6]. The adult neurogenic capacity of zebrafish is in part due to widespread stem cell niches and the neurogenic regions located in the brain [1, 2, 6–14]. The zebrafish has sixteen ventricular stem cell zones along the entire anterior-posterior brain axis . One of these proliferation zones is located dorsally along the ventricle in the telencephalon [7, 8]. The dorsal telencephalic progenitors, which show radial glial cell (RGC) morphology and are positive for glia markers such as S100β and her4.1, display constitutive neurogenic activity during the life-span of the fish [12, 15]. In addition to the constitutive levels of adult neurogenesis, zebrafish brain can also induce a regenerative response after traumatic injury [3–6]. For instance stab wounds in the adult fish brain induce cell death, inflammation, and proliferation of RGCs . Using a Cre-lox lineage tracing strategy, these cells were shown to give rise to newborn neurons, which migrate to the lesioned site and integrate into the existing brain circuitry . Therefore, zebrafish offers a unique opportunity for understanding the molecular basis of adult neurogenesis and brain regeneration in vertebrates. However, the molecular programs and the genes involved in these processes are largely unknown in zebrafish.
In order to identify genes involved in adult neurogenesis and regeneration response of the adult zebrafish telencephalon, we performed an in situ hybridization (ISH) screen. We identified that the gene cxcr5 was expressed in the ventricular region of the zebrafish telencephalon. cxcr5 encodes for a G-protein-coupled chemokine receptor with seven transmembrane domains . The extracellular domain at the N-terminus is critical for ligand binding whereas the intracellular part is involved in G-protein interaction and activation [16, 17]. The receptor was first isolated from human Burkitt’s lymphoma and is also known as the Burkitt lymphoma receptor-1 (BLR-1) [18, 19]. The ligand for cxcr5 is the B-cell-attracting chemokine 1 (BCA-1), also known as Cxcl13 [20, 21]. In mammals, cxcr5 is expressed by mature B cells and in a subset of T cells in the immune system [22, 23]. CXCR5 and CXCL13 are responsible for the organization of B cell follicles and for directing T-helper cells to the lymphoid follicles in humans [24, 25]. Furthermore, the chemokine CXCL13 attracts and maintains B and T cells during inflammation . Besides the immune system, cxcr5 is found in the central nervous system in adult mice in the granule and Purkinje cell layer of the cerebellum [26–28]. The receptor is also expressed in human neuronal precursor cells, which migrate across the blood vessels in the brain upon exposure to CXCL13 . However, the role of cxcr5 gene in the adult neurogenesis and the regeneration of the central nervous system in vertebrates is unknown.
In our study, we analyzed the expression pattern and the function of cxcr5 during adult neurogenesis and regeneration of the zebrafish telencephalon. We show that cxcr5 is detectable in the RGCs and neurons, during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in reduced and increased proliferation and neurogenesis response of the RGCs, respectively. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection , we also observed reduction of regenerative neurogenesis - an outcome similar to the overexpression of the dominant negative cxcr5 variant using transgenic tools. We propose that cxcr5 is an essential cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon after injury.
Results and discussion
cxcr5 is expressed in radial glial cells and neurons in the adult zebrafish telencephalon
The expression of cxcr5 cells along the ventricle suggests that these are progenitor cells [1, 2, 6]. Therefore, we analyzed whether the cxcr5 gene is expressed in her4.1-positive RGCs, which act as neurogenic progenitors during adult neurogenesis and regeneration [3, 12, 15, 31]. We performed fluorescent in situ hybridization (FISH) for cxcr5 coupled to immunohistochemistry to detect the reporter activity in RGCs of the transgenic line Tg(her4.1:GFP). In the unlesioned (Figure 1D-D”) and lesioned (Figure 1E-E”) telencephalons, cxcr5 is expressed in the her4.1-positive RGCs located along the dorsal ventricle (white asterisks) and the total number of cxcr5-positive radial glial cells increase significantly after inducing the lesion (Figure 1F). We also observed cxcr5 expression in her4.1-negative cells (Figure 1D-E”, yellow asterisks), which are located close to the ventricle in the periventricular region, suggesting that cxcr5 is also expressed in neurons. Based on our quantitative real-time PCR (qRT-PCR) analyses, we found that upregulated cxcr5 expression reduces back to unlesioned levels after 3 dpl (Figure 1G), as the expression pattern of cxcr5 at 7 dpl and 15 dpl resembles the unlesioned brains (Figure 1H-K). Additionally, the expression of cxcr5 around the lesion site diminishes after 7 dpl (Figure 1L). These results suggest that cxcr5 might have a transient early role during the regeneration response of the adult zebrafish telencephalon.
ISH for cxcr5 showed that in addition to the RGCs, cxcr5 was also expressed close to the ventricular surface in cells that are not radial glia (Figure 1, Figure 2C, 2E). To test whether those cxcr5-positive cells are neurons, we used the transgenic line Tg(HuC:GFP) in combination with the FISH for cxcr5. We observed cxcr5 expression in HuC-positive neuronal cells in unlesioned (Additional file 2: Figure S2A) and lesioned telencephalons at 3 days post-lesion (dpl) (Additional file 2: Figure S2B), and a significant increase in the number of cxcr5-positive neurons upon lesion (Additional file 2: Figure S2C). The expression of cxcr5 in non-glial periventricular cells (Figure 1, Figure 2) suggests that cxcr5 is present in precursor cells that have committed to differentiate into neurons. These cells express the transcription factor NeuroD [34, 35]. To determine whether the cxcr5 is present in these cells, we performed a chromogenic in situ for cxcr5 and immunohistochemistry for NeuroD (Additional file 2: Figure S2C, D). We found that cxcr5 can be detected in the vast majority of NeuroD cells in the unlesioned and lesioned telencephalons (white asterisks in Additional file 2: Figure S2C and S2D), indicating that cxcr5 is also expressed in differentiating neurons and thus might have a role in neuronal differentiation.
Cxcr5 is expressed in mature B cells and in a subset of T helper memory cells in the immune system of mammals  and traumatic injury in the vertebrate brain induces acute inflammation that includes these cells [36–39], suggesting that cxcr5 might be expressed in immune cells in the telencephalon. To address this, we performed ISH for cxcr5 and L-Plastin (a marker for microglia and leukocytes,  and GFP immunohistochemistry on telencephlons of Tg(her4.1:GFP) transgenic fish before and after lesioning. In unlesioned and lesioned telencephalons, we found that L-Plastin-positive cells do not express cxcr5 (Additional file 3: Figure S3), indicating that cxcr5 is not expressed in leukocytes in the adult zebrafish telencephalon. These findings suggest that cxcr5 is present only in the RGCs and the neurons in the adult zebrafish telencephalon.
cxcr5 regulates the proliferative capacity of RGCs and neuronal differentiation after injury
Since cxcr5 is expressed in proliferating RGCs, we tested whether cxcr5 has a role in proliferation of these cells in the telencephalon by using the two transgenic conditional misexpression lines: Tg(hsp:EGFP-T2A-dncxcr5) and Tg(hsp:EGFP-T2A-FL-cxcr5). We analyzed proliferating cells in the homeostatic and regenerating telencephalons after one heat shock per day over four days (one before and three after the lesion or sham operations) (Figure 3B). In unlesioned animals, proliferating cell nuclear antigen (PCNA) and GFP immunohistochemistry (Figure 3C-E”) showed that in comparison to the non-transgenic siblings (Figure 3C), transgenic fish for both variants of cxcr5 show no significant difference in the number of proliferating cells at the ventricle (Figure 3D-F). Following injury (Figure 3G-I), overexpression of full-length cxcr5 significantly increased the ventricular cell proliferation (25.1 ± 8.3%; mean ± SD) (Figure 3I,J). However, overexpression of the dominant-negative cxcr5 did not alter the number of PCNA-positive cells (Figure 3H,J). These results indicate that cxcr5 is sufficient to promote cell proliferation of radial glial cells but may not be required, suggesting that the lack of alteration in cell proliferation in the dominant negative transgenic line might be an indication of the specificity of the effect after overexpression of the full length cxcr5. It is therefore possible that whether or not an RGC expressing cxcr5 proliferates, depends not solely on cxcr5 but also on other contextual factors (such as hypothetically, the prevalence of mitogenic factors, alleviation of suppressive mechanisms on neurogenesis, or other molecular programs activated upon the need for newborn neurons). Thus, we suggest a permissive role for cxcr5 in RGC proliferation. This hypothesis is supported by our observations that when the full-length cxcr5 is overexpressed, the proliferation of the RGCs increases only after injury but not during homeostasis. In such a case, even though the cxcr5 overexpression may endow RGCs the ability to proliferate more, the prevailing signaling in unlesioned conditions is not sufficient to enhance the cell proliferation. However, after an injury, the telencephalon induces molecular programs that ultimately converge on the enhanced proliferation of the RGCs . Consequently, when cxcr5 is overexpressed following a lesion, it can increase the cell proliferation because the competent RGCs will be influenced by the injury-dependent mitogenic signals. Alternatively, the temporal dynamics of constitutive neurogenesis may be different, possibly slower, than that of the injury conditions, and this may prevent detection of significant changes in cell proliferation and neurogenesis in unlesioned brains, due to the time window we have used in our analyses. Overall, given that we observed cxcr5 expression in proliferating RGCs and also showed that when overexpressed, cxcr5 can enhance the proliferation of these cells context-dependently, we argue that cxcr5 might be a permissive but not an instructive factor involved in maintaining the regenerative neurogenic capacity of the radial glial cells, by priming them for proliferation as cxcr5 expression is sufficient but not required for proliferation of ventricular progenitors.
We also detected cxcr5 in non-proliferating RGCs (Figure 3). This suggests that either the expression of cxcr5 in non-proliferating glia reflects a physiological stage of the RGC at which it is competent to proliferate but is not actually in the cell cycle, or cxcr5 has a proliferation-independent role in those glial cells. In adult brains, glial cells are known to be heterogeneous in terms of their proliferation and progenitor characteristics [1, 2, 6, 12, 15]. Therefore, cxcr5 may either be expressed in glial cells with different inherent propensities to become neurogenic and proliferative or it can be a part of the mechanism that establishes the functional heterogeneity. It is not clear whether the constitutive neurogenesis and the injury-induced neurogenesis use the same progenitor cells to form neurons by simply enhancing the rate of cell divisions, or whether there are reserve stem cells that are activated after injury. Given that we observed an effect of cxcr5 misexpression on proliferation and neurogenesis only after the injury, it is possible that the roles of cxcr5 we identified are associated with the regenerative response. This suggests that the expression of cxcr5 in unlesioned brains may either be relevant to a permissive proliferative and neurogenic capacity of the RGCs or be a physiological function independent of RGC proliferation. However, it is currently not possible to distinguish between these alternatives with our current level of understanding. Long-term single-cell lineage tracing experiments with a cxcr5-positive non-proliferative RGC will help us understand whether under homeostatic conditions, cxcr5-positive glial cells initiate cell division and form neurons, or whether they are required for an as-yet unidentified role in the glial cells.
We demonstrated that cxcr5 is expressed at different physiological states of glial cells - during quiescence, proliferation and differentiation into neurons. We also demonstrated that cxcr5 function is biologically relevant to the proliferation of the neurogenic progenitor cells and the regenerative neurogenesis response of the adult zebrafish brain (Figure 6). Therefore, we present cxcr5 as an intriguing factor relevant to the proliferative status and differentiation capacity of the RGCs after lesion in the adult zebrafish telencephalon. In mammals, after traumatic lesions in the central nervous system, glial cells initiate proliferation but cannot replenish the lost neurons [37, 41–47]. One of the detrimental cues for neurogenesis is believed to be the acute inflammation [38, 48–50]. Given that zebrafish can regenerate traumatic injuries in the brain and the fish RGCs expresses cxcr5, which we showed to be relevant to RGC proliferation and neuronal differentiation, we believe that cxcr5 can be an interesting candidate that may be involved in the permissive link between the immune and the nervous systems in zebrafish. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.
All animal experiments were carried out in accordance with the recommendations and permits of the Landesdirektion Dresden (Permit numbers: AZ 24D-9168.11-1/2008-2, 4 and 14). All surgery was performed under anesthesia, and all efforts were made to minimize suffering. Fish were raised and kept at 28°C under a 14-hour light, 10 hours dark cycle, and fed with brine shrimp artemia daily as described . Wildtype experimental animals were adult fish from the gol-b1 line with AB genetic background .
Tissue preparation and cryosectioning
Fish were sacrificed with 1% 3-amino-benzoic acid ethyl ester (MESAB, Sigma, Schnelldorf, Germany) and the skull was opened at the hindbrain area with sharp forceps to enable a better fixation of the tissue. The skull was partly removed without damaging or relocating the telencephalon. The head was cut off behind the pectoral fins with a scalpel and the brains were fixed in 2% paraformaldehyde (PFA, Sigma, Schnelldorf, Germany) at 4°C overnight. Heads were washed twice with 0.1 M phosphate buffer, transferred in 20% sucrose-ethylene-diamine-tetra-acetic acid (EDTA, Sigma, Schnelldorf, Germany) solution (in 0.1 M phosphate buffer, pH: 7.5) and incubated overnight at 4°C. Brains were then frozen in 7.5% gelatin/20% sucrose in 0.1 M phosphate buffer and cryosectioned at 14-μm thickness using a cryostat. Heads were sectioned for the telencephalon between olfactory bulb and diencephalon with sequential distribution to three slides, therefore one slide contained every third section. Sections were stored at −20°C.
In situ hybridization, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining
A fragment of the zebrafish cxcr5 gene [NCBI Accession ID: XM_003200482; Ensembl Accession ID: ENSDARG00000010514] was isolated from 2 days post-fertilization (dpf) embryonic total cDNA using the following primers: forward primer: 5′-TACATCTGGCAGTGGCTGAC-3′ and reverse primer: 5′-CAGCAGGTCTCTTCGGAAAC-3′. The amplicon (726 bp) was subcloned into pGEM T-Easy (Promega). A DIG or fluorescein-labeled mRNA probe was generated using an in vitro transcription kit (Roche, Mannheim, Germany). In situ hybridization was performed as described [12, 53]. To combine ISH with IHC, the primary antibody for the immunohistochemistry was added with the anti-DIG or anti-Fluorescein antibody. After the in situ staining reaction, the secondary antibody was applied. IHC was performed as described [3, 8, 11, 12, 30]. Antigen retrieval was performed with Tris–HCl buffer, pH: 8.0 at 100°C for 5 minutes, 10 mM sodium citrate buffer, pH: 6.0 at 85°C for 6 minutes or 2 M HCl for 20 minutes at 37°C. The following antibodies were used: primary antibodies: anti-BrdU (rat IgG2a, Serotec, Düsseldorf, Germany, 1:500), anti-GFP (rabbit, Molecular Probes, Darmstadt, Germany, 1:1,000); anti-GFP (chicken, Abcam, Cambridge, UK, 1:3,000); anti-HuC/D (mouse IgG2b, Molecular Probes, 1:50), anti-neuroD (mouse IgG2a, Abcam, 1:750), Anti-Lcp1 (rabbit, 1:10,000, gift from Michael Redd, University of Utah, USA), anti-PCNA (mouse IgG2a, Dako Cyto, Hamburg, Germany, 1:500), anti-DIG (sheep, Roche, Mannheim, Germany, 1:4,000), anti-fluorescein (sheep, Roche, 1:5,000); secondary antibodies: goat anti-rat, goat anti-rabbit, goat anti-mouse (IgG, IgG2a, IgG2b), goat anti-chicken (coupled to Alexa-488, Alexa-555; Alexa-633, Molecular Probes, 1:500). TUNEL staining was performed using ApopTag® Red in situ apoptosis detection kit (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions.
To label cells in S-phase of the cell cycle, zebrafish were immersed in 10 mM BrdU (Sigma) solution . BrdU was dissolved in E3 medium, the zebrafish were incubated for desired periods indicated throughout the text.
Brain lesions and cerebroventricular microinjection (CVMI)
Fish were anesthetized using MESAB (0.01%). A 30-gauge cannula (Becton Dickinson Biosciences, Heidelberg, Germany), which corresponds to an outer diameter of 200 μm, was inserted through the fish nostril and stabbed along the rostro-caudal axis through the olfactory bulb until reaching the caudal part of the telencephalon as described . CVMI was performed as described , fish were anesthetized using MESAB (0.01%). With the help of a fine needle (Becton Dickinson Biosciences) an incision was made through the skull to the cerebroventricular fluid (CVF) of the adult zebrafish above the optic tectum. This incision generates a slit through the skull with a diameter of approximately 200 μm without damaging the optic tectum underneath. Injected liquid disperses through the CVF rostrally to the forebrain. We generated con-trol (5′-CCTCTTACCTCAGTTACAATTTATA-3′) and translation-blocking antisense (5′-AACCCCTCTCCTCAAGGACTGGCAT-3′) morpholinos for cxcr5, tested these morpholinos in embryos for functionality and specificity (Additional file 5: Figure S5), and used them for CVMI into adult zebrafish brain.
Generation of conditional transgenic lines for cxcr5 gene variants
For full-length cxcr5 transgenics, complete open reading frame of the zebrafish cxcr5 gene [NCBI accession: XM_003200482, Cxcr5: 402 aminoacids] was used (Figure 5A). For the dominant negative variant, the 5th, 6th and the 7th transmembrane domains and the intracellular C-terminal domain were deleted (Δ243-402-Cxcr5 (dnCxcr5), 243 aminoacids) (Figure 5A). The transgenesis cassette contains two flanking Tol2 elements [54, 55], and in between these are the heat-inducible zebrafish hsp70 promoter, the EGFP-T2A-dnCxcr5 or EGFP-T2A-FL-Cxcr5 gene sequence (See Figure 5). To integrate the construct into the zebrafish genome, transgenesis constructs were co-injected with Tol2 transposase mRNA to the one-cell stage of the embryos [54–57]. Founder animals were outcrossed to wild type strains and the progeny was screened for GFP fluorescence after single heat shock at early gastrulation stages. The fish carrying the transgenes heterozygously were outcrossed for two generations to segregate multiple insertions. The nomenclature for the transgenic animals are as follows: Tg(hsp:EGFP-T2A-dncxcr5) for the dominant-negative variant, and Tg(hsp:EGFP-T2A-FL-Cxcr5) for the full-length variant. We also used Tg(her4.1:GFP) and Tg(HuC:GFP) transgenic lines, which were kindly provided by the relevant laboratories.
Heat shock paradigm
Heat-shock experiments were performed on embryos and adults. Embryos (at least 50% epiboly stage) were transferred into 2 ml tubes. After removal of the excess embryo medium (E3, 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2.2H2O, 0.33 mM MgSO4.7H2O, 0.0002% methylene blue, pH: 6.5), E3 preheated to 39°C was added in the tubes, which were incubated for 30 minutes at 38°C. After the heat shock, embryos were returned to Petri dishes filled with E3 and placed in a 28°C incubator. The embryos were analyzed with a fluorescent microscope at least 4 hours after the heat induction. Adult fish were transferred into the heat shock aquarium and received in total, four heat shocks on four consecutive days. Each day the water was heated to 37°C, then the heating elements switched off and the water passively cooled down to 28°C (this takes approximately 5 to 6 hours after the heat shock). The fish were analyzed with an Olympus MVX10 fluorescent microscope after anesthetization with MESAB (0.01%). Embryos were sorted and used for desired purposes.
Imaging, quantification and statistical analysis
Fluorescence images were taken using a structured illumination microscope (Zeiss Apotome AxioImager.Z1) or a laser scanning confocal microscope (Zeiss 510 META). Quantifications were performed in an unbiased manner by counting the absolute number of cells in a minimum of eight different sections of the brain of every fish. For simplicity, results are depicted as the relative amount of cells. For statistical analysis, two-way analysis of variance (ANOVA) and Tukey’s post hoc test or paired t-test were used after determining whether the sample datasets conform to a normal distribution. P-values are indicated as follows: *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001. The number of fish used for every experiment is indicated in the corresponding figure legends.
analysis of variance
B-cell-attracting chemokine 1
Burkitt lymphoma receptor-1
chemokine (C-X-C motif) receptor 5
enhanced green fluorescent protein
fluorescent in situ hybridization
green fluorescent protein
hairy-related 4.1 basic helix-loop-helix transcription factor
in situ hybridization
3-amino-benzoic acid ethyl ester
proliferating cell nuclear antigen
quantitative real-time polymerase chain reaction
radial glial cell
terminal deoxynucleotidyl transferase dUTP nick end labeling.
We would like to thank to Dr Stefan Hans and Dr. Christopher L Antos for discussion and critical reading of the manuscript; and to Dr. Michael Redd for the L-Plastin antibody. This work was supported by research grants to MB from Deutsche Forschungsgemeinschaft SFB-655, European Union (ZF Health, EC Grant Agreement HEALTH-F4-2010-242048) and Technical University of Dresden.
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