Regeneration In Adult Zebrafish Research Articles

Sex hormone receptors, calcium-binding protein and Yap1 signaling regulate sex-dependent liver cell proliferation following partial hepatectomy.

Partial hepatectomy (PH) is commonly used to treat patients with hepatocellular carcinoma. The recovery of patients from PH depends on the initiation of liver regeneration, a process that mainly relies on liver cell proliferation. As sex affects the human liver regeneration progress, we investigated sex disparity in PH-induced liver regeneration in adult zebrafish. We found that, after PH, males began liver regeneration earlier than females in terms of liver cell proliferation and liver mass recovery, and this was associated with earlier activation of Yap1 signaling in male than female livers. We also found that androgen receptors regulated the sex-biased liver regeneration in a Yap1-dependent manner and that activated estrogen receptors are responsible for the later onset of female hepatocyte proliferation. Furthermore, we identified that S100A1, a calcium-binding protein, regulates the sex disparity in liver regeneration, as heterozygous S100A1 knockout inhibited Yap1 activity in male livers and delayed hepatocyte proliferation in males following PH. Thus, multiple pathways and/or their interplays contribute to the sex disparity in liver regeneration, suggesting that sex-biased therapeutic strategies are required for patients who have received PH-based therapies.

Ethyl butyrate inhibits caudal fin regeneration in adult zebrafish by disrupting extracellular matrix remodeling

Wound healing and tissue regeneration are influenced by a variety of factors. Adverse lifestyle habits, such as excessive alcohol consumption, delay wound healing and increase the risk of secondary infections. Ethyl butyrate is a common food additive widely used to enhance the aroma of alcoholic beverages. This additive is generally considered harmless to human health in both industrial and domestic settings. However, the ecotoxicity and its effects on wound healing have not been elucidated. In this study, we used zebrafish as the experimental animal, and the caudal fins were amputated to explore the effects of ethyl butyrate on wound healing and tissue regeneration. The effect of ethyl butyrate on blastema and bone regeneration and its impact on the transcriptional levels of regeneration-related genes and inflammation-related genes were evaluated. RNA-seq was conducted to determine the differentially expressed genes (DEGs) between the treatment and the control groups. KEGG and GO analysis was conducted to explore the functions of DEGs. Significantly enriched GO terms and KEGG pathways were identified to explore the molecular mechanism underlying the inhibition of zebrafish caudal fin regeneration by ethyl butyrate. The results demonstrated that ethyl butyrate significantly inhibited the regeneration of zebrafish caudal fins, including blastema and bone regeneration. Ethyl butyrate exposure significantly downregulated the expression of genes associated with bone and blastema regeneration and inflammation response. KEGG and GO functional analyses revealed that the DEGs were associated with significant enrichment of extracellular matrix-receptor interactions. Ethyl butyrate treatment downregulated the expression of most extracellular matrix-related genes. These findings indicate that ethyl butyrate potentially modulates pathways associated with the structure, adhesion, modification, and degradation of the extracellular matrix, thereby disrupting extracellular matrix remodeling, inhibiting wound inflammation, impairing blastema and bone regeneration and ultimately hindering caudal fin regeneration. In summary, the findings demonstrate that ethyl butyrate disrupts extracellular matrix remodeling and inhibits the regeneration of zebrafish caudal fins. These results provide valuable insights into the rational use of ethyl butyrate and further investigation of wound healing mechanisms.

Keratin 8/18a.1 Expression Influences Embryonic Neural Crest Cell Dynamics and Contributes to Postnatal Corneal Regeneration in Zebrafish.

A complete understanding of neural crest cell mechanodynamics during ocular development will provide insight into postnatal neural crest cell contributions to ophthalmic abnormalities in adult tissues and inform regenerative strategies toward injury repair. Herein, single-cell RNA sequencing in zebrafish during early eye development revealed keratin intermediate filament genes krt8 and krt18a.1 as additional factors expressed during anterior segment development. In situ hybridization and immunofluorescence microscopy confirmed krt8 and krt18a.1 expression in the early neural plate border and migrating cranial neural crest cells. Morpholino oligonucleotide (MO)-mediated knockdown of K8 and K18a.1 markedly disrupted the migration of neural crest cell subpopulations and decreased neural crest cell marker gene expression in the craniofacial region and eye at 48 h postfertilization (hpf), resulting in severe phenotypic defects reminiscent of neurocristopathies. Interestingly, the expression of K18a.1, but not K8, is regulated by retinoic acid (RA) during early-stage development. Further, both keratin proteins were detected during postnatal corneal regeneration in adult zebrafish. Altogether, we demonstrated that both K8 and K18a.1 contribute to the early development and postnatal repair of neural crest cell-derived ocular tissues.

The ion channel Trpc6a regulates the cardiomyocyte regenerative response to mechanical stretch.

Myocardial damage caused, for example, by cardiac ischemia leads to ventricular volume overload resulting in increased stretch of the remaining myocardium. In adult mammals, these changes trigger an adaptive cardiomyocyte hypertrophic response which, if the damage is extensive, will ultimately lead to pathological hypertrophy and heart failure. Conversely, in response to extensive myocardial damage, cardiomyocytes in the adult zebrafish heart and neonatal mice proliferate and completely regenerate the damaged myocardium. We therefore hypothesized that in adult zebrafish, changes in mechanical loading due to myocardial damage may act as a trigger to induce cardiac regeneration. Based on this notion we sought to identify mechanosensors which could be involved in detecting changes in mechanical loading and triggering regeneration. Here we show using a combination of knockout animals, RNAseq and in vitro assays that the mechanosensitive ion channel Trpc6a is required by cardiomyocytes for successful cardiac regeneration in adult zebrafish. Furthermore, using a cyclic cell stretch assay, we have determined that Trpc6a induces the expression of components of the AP1 transcription complex in response to mechanical stretch. Our data highlights how changes in mechanical forces due to myocardial damage can be detected by mechanosensors which in turn can trigger cardiac regeneration.

Inflammation is a critical factor for successful regeneration of the adult zebrafish retina in response to diffuse light lesion.

Inflammation can lead to persistent and irreversible loss of retinal neurons and photoreceptors in mammalian vertebrates. In contrast, in the adult zebrafish brain, acute neural inflammation is both necessary and sufficient to stimulate regeneration of neurons. Here, we report on the critical, positive role of the immune system to support retina regeneration in adult zebrafish. After sterile ablation of photoreceptors by phototoxicity, we find rapid response of immune cells, especially monocytes/microglia and neutrophils, which returns to homeostatic levels within 14days post lesion. Pharmacological or genetic impairment of the immune system results in a reduced Müller glia stem cell response, seen as decreased reactive proliferation, and a strikingly reduced number of regenerated cells from them, including photoreceptors. Conversely, injection of the immune stimulators flagellin, zymosan, or M-CSF into the vitreous of the eye, leads to a robust proliferation response and the upregulation of regeneration-associated marker genes in Müller glia. Our results suggest that neuroinflammation is a necessary and sufficient driver for retinal regeneration in the adult zebrafish retina.

P-aminobenzoic acid promotes retinal regeneration through activation of Ascl1a in zebrafish.

JOURNAL/nrgr/04.03/01300535-202408000-00040/figure1/v/2023-12-16T180322Z/r/image-tiff The retina of zebrafish can regenerate completely after injury. Multiple studies have demonstrated that metabolic alterations occur during retinal damage; however to date no study has identified a link between metabolites and retinal regeneration of zebrafish. Here, we performed an unbiased metabolome sequencing in the N-methyl-D-aspartic acid-damaged retinas of zebrafish to demonstrate the metabolomic mechanism of retinal regeneration. Among the differentially-expressed metabolites, we found a significant decrease in p-aminobenzoic acid in the N-methyl-D-aspartic acid-damaged retinas of zebrafish. Then, we investigated the role of p-aminobenzoic acid in retinal regeneration in adult zebrafish. Importantly, p-aminobenzoic acid activated Achaetescute complex-like 1a expression, thereby promoting Müller glia reprogramming and division, as well as Müller glia-derived progenitor cell proliferation. Finally, we eliminated folic acid and inflammation as downstream effectors of PABA and demonstrated that PABA had little effect on Müller glia distribution. Taken together, these findings show that PABA contributes to retinal regeneration through activation of Achaetescute complex-like 1a expression in the N-methyl-D-aspartic acid-damaged retinas of zebrafish.

Hdac1-deficiency affects the cell cycle axis Cdc25-Cdk1 causing impaired G2/M phase progression and reduced cardiomyocyte proliferation in zebrafish

Zebrafish have the ability to fully regenerate their hearts after injury since cardiomyocytes subsequently dedifferentiate, re-enter cell cycle, and proliferate to replace damaged myocardial tissue. Recent research identified the reactivation of dormant developmental pathways during cardiac regeneration in adult zebrafish, suggesting pro-proliferative pathways important for developmental heart growth to be also critical for regenerative heart growth after injury.Histone deacetylase 1 (Hdac1) was recently shown to control both, embryonic as well as adult regenerative cardiomyocyte proliferation in the zebrafish model. Nevertheless, regulatory pathways controlled by Hdac1 are not defined yet. By analyzing RNA-seq-derived transcriptional profiles of the Hdac1-deficient zebrafish mutant baldrian, we here identified DNA damage response (DDR) pathways activated in baldrian mutant embryos. Surprisingly, although the DDR signaling pathway was transcriptionally activated, we found the complete loss of protein expression of the known DDR effector and cell cycle inhibitor p21. Consequently, we observed an upregulation of the p21-downstream target Cdk2, implying elevated G1/S phase transition in Hdac1-deficient zebrafish hearts. Remarkably, Cdk1, another p21-but also Cdc25-downstream target was downregulated. Here, we found the significant downregulation of Cdc25 protein expression, explaining reduced Cdk1 levels and suggesting impaired G2/M phase progression in Hdac1-deficient zebrafish embryos. To finally prove defective cell cycle progression due to Hdac1 loss, we conducted Cytometer-based cell cycle analyses in HDAC1-deficient murine HL-1 cardiomyocytes and indeed found impaired G2/M phase transition resulting in defective cardiomyocyte proliferation.In conclusion, our results suggest a critical role of Hdac1 in maintaining both, regular G1/S and G2/M phase transition in cardiomyocytes by controlling the expression of essential cell cycle regulators such as p21 and Cdc25.

Puromycin-induced kidney injury and subsequent regeneration in adult zebrafish

ABSTRACT Puromycin treatment can cause glomerular injury to the kidney, leading to proteinuria. However, the pathogenesis of acute kidney injury and subsequent regeneration after puromycin administration in animal models remain unclear. In this work, we examined the characteristics of kidney injury and subsequent regeneration following puromycin treatment in adult zebrafish. We intraperitoneally injected 100 μg of puromycin into zebrafish; sacrificed them at 1, 3, 5, 7, or 14 days post-injection (dpi); and examined the morphological, functional, and molecular changes in the kidney. Puromycin-treated zebrafish presented more rapid clearance of rhodamine dextran than control animals. Morphological changes were observed immediately after the puromycin injection (1–7 dpi) and had recovered by 14 dpi. The mRNA production of lhx1a, a renal progenitor marker, increased during recovery from kidney injury. Levels of NFκB, TNFα, Nampt, and p-ERK increased significantly during nephron injury and regeneration, and Sirt1, FOXO1, pax2, and wt1b showed an increasing tendency. However, TGF-β1 and smad5 production did not show any changes after puromycin treatment. This study provides evidence that puromycin-induced injury in adult zebrafish kidneys is a potential tool for evaluating the mechanism of nephron injury and subsequent regeneration.

Biliary epithelial cells are facultative liver stem cells during liver regeneration in adult zebrafish.

The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR-dependent manner.

Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

A regulatory network of Sox and Six transcription factors initiate a cell fate transformation during hearing regeneration in adult zebrafish

SUMMARYUsing adult zebrafish inner ears as a model for sensorineural regeneration, we ablated the mechanosensory receptors and characterized the single-cell epigenome and transcriptome at consecutive time points during hair cell regeneration. We utilized deep learning on the regeneration-induced open chromatin sequences and identified cell-specific transcription factor (TF) motif patterns. Enhancer activity correlated with gene expression and identified potential gene regulatory networks. A pattern of overlapping Sox- and Six-family TF gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudotime analysis of single-cell transcriptomic data suggested that support cells within the sensory epithelium changed cell identity to a “progenitor” cell population that could differentiate into hair cells. We identified a 2.6 kb DNA enhancer upstream of the sox2 promoter that, when deleted, showed a dominant phenotype that resulted in a hair-cell-regeneration-specific deficit in both the lateral line and adult inner ear.

High-Intensity Low Frequency Pulsed Electromagnetic Fields treatment stimulates fin regeneration in adult zebrafish. A pre-liminary report

High-Intensity Low Frequency Pulsed Electromagnetic Fields treatment stimulates fin regeneration in adult zebrafish. A pre-liminary report

Visual Function is Gradually Restored During Retina Regeneration in Adult Zebrafish.

In comparison to mammals, zebrafish are able to regenerate many organs and tissues, including the central nervous system (CNS). Within the CNS-derived neural retina, light lesions result in a loss of photoreceptors and the subsequent activation of Müller glia, the retinal stem cells. Müller glia-derived progenitors differentiate and eventually restore the anatomical tissue architecture within 4 weeks. However, little is known about how light lesions impair vision functionally, as well as how and to what extent visual function is restored during the course of regeneration, in particular in adult animals. Here, we applied quantitative behavioral assays to assess restoration of visual function during homeostasis and regeneration in adult zebrafish. We developed a novel vision-dependent social preference test, and show that vision is massively impaired early after lesion, but is restored to pre-lesion levels within 7 days after lesion. Furthermore, we employed a quantitative optokinetic response assay with different degrees of difficulty, similar to vision tests in humans. We found that vision for easy conditions with high contrast and low level of detail, as well as color vision, was restored around 7–10 days post lesion. Vision under more demanding conditions, with low contrast and high level of detail, was regained only later from 14 days post lesion onwards. Taken together, we conclude that vision based on contrast sensitivity, spatial resolution and the perception of colors is restored after light lesion in adult zebrafish in a gradual manner.

A Comparative Analysis of Gene and Protein Expression Throughout a Full 28-Day Retinal Regeneration Time-Course in Adult Zebrafish

Following photoreceptors ablation by intense light exposure, adult zebrafish are capable of complete regeneration due to the ability of their Müller glia (MG) to re-enter the cell cycle, creating progenitors that differentiate into new photoreceptors. The majority of previous reports on retinal regeneration focused on the first few days of the regenerative response, which include MG cell-cycle re-entry and progenitor cell proliferation. With this study, we analyzed the full 28-day time-course of regeneration by pairing a detailed morphological/immunological analysis with RNA-seq transcriptional profiling at 8 key time points during retinal regeneration. We observed several novel findings. First, we provide evidence for two separate peaks of MG gliosis, with the secondary gliotic peak occurring after MG cell-cycle re-entry. Second, we highlight a distinct transcriptional shift between 5- and 10-days post lesion that highlights the transition from progenitor proliferation to differentiation into new photoreceptors. Third, we show distinctly different patterns of transcriptional recovery of the photoreceptor opsins at 28 days post lesion. Finally, using differential gene expression analysis, we revealed that the established functional recovery of the retina at 28 days post lesion does not, in fact, return to an undamaged transcriptional state, potentially redefining what the field considers complete regeneration. Together, to our knowledge, this work represents the first histological and transcriptomic map of a 28-day time-course of retinal regeneration in adult zebrafish.

Development and characterization of a chronic photoreceptor degeneration model in adult zebrafish that does not trigger a regenerative response

Zebrafish (Danio rerio) have become a highly-utilized model system in the field of regenerative biology because of their endogenous ability to regenerate many tissues and organs, including the retina. The vast majority of previous research on retinal regeneration in adult zebrafish utilizes acute methodologies for retinal damage. Acute retinal cell death triggers a reactive gliosis response of Müller glia (MG), the resident macroglia of the retina. In addition, each activated MG undergoes asymmetric cell division to produce a neuronal progenitor, which continues to divide and ultimately gives rise to new retinal neurons. Studies using these approaches have uncovered many crucial mechanisms by which MG respond to acute damage. However, they may not adequately mimic the chronic neuronal degeneration observed in many human retinal degenerative diseases. The current study aimed to develop a new long-term, chronic photoreceptor damage and degeneration model in adult zebrafish. Comparing the subsequent cellular responses to that of the commonly-used acute high-intensity model, we found that low, continuous light exposure damaged the outer segments of both rod and cone photoreceptors, but did not result in significant apoptotic cell death, MG gliosis, or MG cell-cycle re-entry. Instead, chronic light nearly completely truncated photoreceptor outer segments and resulted in a recruitment of microglia to the area. Together, these studies present a chronic photoreceptor model that can be performed in a relatively short time frame (21 days), that may lend insight into the cellular events underlying non-regenerative photoreceptor degeneration observed in other model systems.

Expression analysis of Hsp90α and cytokines in zebrafish caudal fin regeneration

Zebrafish (Danio rerio) is an ideal model organism for exploring the ability and mechanism of tissue regeneration in the vertebrate. However, the specific cellular and molecular mechanism of caudal fin regeneration in zebrafish remains largely unclear. Therefore, we first confirmed the crucial period of fin regeneration in adult zebrafish by morphological and histological analysis. Then we performed RNA-Seq analysis of the caudal fin regeneration at three key stages, which provided some clues for exploring the mechanism of caudal fin regeneration. Moreover, we also determined the expressions of inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, TGF-β, and the immune-related pathway JAK2α and STAT1b in the caudal fin of zebrafish following fin amputation by quantitative real time PCR (qPCR). Particularly, Hsp90α expression at mRNA and protein level determined by qPCR and Western blotting, respectively, and whole-mount in situ hybridization of Hsp90α were also performed in this study. The results showed that inflammatory cytokines were mainly expressed in the early period of caudal fin regeneration (1–3 days post amputation, dpa), indicating that fish immune system was involved in the fin regeneration. Furthermore, the high expression of Hsp90α in the vicinity of blastema and blood vessels of the regenerating fin suggests that Hsp90α may play a role in the initiation and promotion of caudal fin regeneration. Overall, our results provide a framework for further understanding the cellular and molecular mechanism in caudal fin regeneration.

Role of the kcnk2 ion channel in zebrafish heart regeneration

Myocardial infarction leads to a loss of cardiac tissue, which is then replaced by fibrosis. Following this loss in adult mammals, pathological hypertrophy takes place and leads to heart failure. Zebrafish are able to regenerate their heart after an injury at all stages of life. Mammals also have this ability during the early stages of life but this process seems to be inhibited during adulthood in favour of an antagonist pathway: the hypertrophic response. Based on this paradigm, we believe that factors which induce hypertrophy in mammals will most likely induce cardiomyocyte proliferation and regeneration in adult zebrafish. One of these factors is KCNK2, a member of the two-pore-domain potassium (K2P) channels, which was recently associated with cardiac ischemia and pathological hypertrophy. This study aims to characterise the zebrafish orthologs of KCNK2 and determine their role during heart regeneration in zebrafish. Chemical inhibition combined with a zebrafish conditional line expressing a dominant negative isoform of Kcnk2 in the cardiomyocytes were used. The hearts were extracted at different time points after ventricular amputation to be fixed, sectioned and stained, using BrdU to assess proliferation, or AFOG to assess the presence/absence of a scar. Both channels were characterised in HEK-293 cells using patch clamp technic. Electrophysiological analysis of zebrafish Kcnk2 orthologs showed that, like their human counterpart, they are activated by mechanical stretch, polyunsaturated fatty acids and intracellular acidification and they are expressed in cardiomyocytes. Using a specific antagonist and our transgenic line, we also showed that inhibition of Kcnk2 function leads to impaired heart regeneration. We identified two zebrafish orthologs of KCNK2 sharing the same properties as human KCNK2. During heart regeneration in vivo, Kcnk2 inhibition seems to inhibit regeneration in zebrafish.

Her9/Hes4 is required for retinal photoreceptor development, maintenance, and survival

The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete. Her9, the zebrafish ortholog of human HES4, is a basic helix-loop-helix-orange transcriptional repressor that regulates neurogenesis in several developmental contexts. We have previously shown that her9 is upregulated during chronic rod photoreceptor degeneration and regeneration in adult zebrafish, but little is known about the role of her9 during retinal development. To better understand the function of Her9 in the retina, we generated zebrafish her9 CRISPR mutants. Her9 homozygous mutants displayed striking retinal phenotypes, including decreased numbers of rods and red/green cones, whereas blue and UV cones were relatively unaffected. The reduction in rods and red/green cones correlated with defects in photoreceptor subtype lineage specification. The remaining rods and double cones displayed abnormal outer segments, and elevated levels of apoptosis. In addition to the photoreceptor defects, her9 mutants also possessed a reduced proliferative ciliary marginal zone, and decreased and disorganized Müller glia. Mutation of her9 was larval lethal, with no mutants surviving past 13 days post fertilization. Our results reveal a previously undescribed role for Her9/Hes4 in photoreceptor differentiation, maintenance, and survival.

Midkine-a functions as a universal regulator of proliferation during epimorphic regeneration in adult zebrafish.

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.

Expression of dlx genes in the normal and regenerating brain of adult zebrafish.

Dysfunctions in the GABAergic system lead to various pathological conditions and impaired inhibitory function is one of the causes behind neuropathies characterized by neuronal hyper excitability. The Dlx homeobox genes are involved in the development of nervous system, neural crest, branchial arches and developing appendages. Dlx genes also take part in neuronal migration and differentiation during development, more precisely, in the migration and differentiation of GABAergic neurons. Functional analysis of dlx genes has mainly been carried out in developing zebrafish embryos and larvae, however information regarding the expression and roles of these genes in the adult zebrafish brain is still lacking. The extensive neurogenesis that takes place in the adult zebrafish brain, makes them a good model for the visualization of mechanisms involving dlx genes during adulthood in physiological conditions and during regeneration of the nervous system. We have identified the adult brain regions where transcripts of dlx1a, dlx2a, dlx5a and dlx6a genes are normally found and have confirmed that within telencephalic domains, there is high overlapping expression of the four dlx paralogs with a marker for GABAergic neurons. Co-localization analyses carried with the Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) reporter line have also shown that in some areas of the diencephalon, cells expressing the dlx5a/6a bigene may have a neural stem cell identity. Furthermore, investigations in a response to stab wound lesions, have demonstrated a possible participation of the dlx5a/6a bigene, most likely of dlx5a, during regeneration of the adult zebrafish brain. These observations suggest a possible participation of dlx-expressing cells during brain regeneration in adult zebrafish and also provide information on the role of dlx genes under normal physiological conditions in adults.