The Promising Role of a Zebrafish Model Employed in Neural Regeneration Following a Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating event that results in a wide range of physical impairments and disabilities. Despite the advances in our understanding of the biological response to injured tissue, no effective treatments are available for SCIs at present. Some studies have addressed this issue by exploring the potential of cell transplantation therapy. However, because of the abnormal microenvironment in injured tissue, the survival rate of transplanted cells is often low, thus limiting the efficacy of such treatments. Many studies have attempted to overcome these obstacles using a variety of cell types and animal models. Recent studies have shown the utility of zebrafish as a model of neural regeneration following SCIs, including the proliferation and migration of various cell types and the involvement of various progenitor cells. In this review, we discuss some of the current challenges in SCI research, including the accurate identification of cell types involved in neural regeneration, the adverse microenvironment created by SCIs, attenuated immune responses that inhibit nerve regeneration, and glial scar formation that prevents axonal regeneration. More in-depth studies are needed to fully understand the neural regeneration mechanisms, proteins, and signaling pathways involved in the complex interactions between the SCI microenvironment and transplanted cells in non-mammals, particularly in the zebrafish model, which could, in turn, lead to new therapeutic approaches to treat SCIs in humans and other mammals.

Advancing Spinal Cord Injury Treatment through Stem Cell Therapy: A Comprehensive Review of Cell Types, Challenges, and Emerging Technologies in Regenerative Medicine.

Spinal cord injuries (SCIs) can lead to significant neurological deficits and lifelong disability, with far-reaching physical, psychological, and economic consequences for affected individuals and their families. Current treatments for SCIs are limited in their ability to restore function, and there is a pressing need for innovative therapeutic approaches. Stem cell therapy has emerged as a promising strategy to promote the regeneration and repair of damaged neural tissue following SCIs. This review article comprehensively discusses the potential of different stem cell types, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and neural stem/progenitor cells (NSPCs), in SCI treatment. We provide an in-depth analysis of the unique advantages and challenges associated with each stem cell type, as well as the latest advancements in the field. Furthermore, we address the critical challenges faced in stem cell therapy for SCIs, including safety concerns, ethical considerations, standardization of protocols, optimization of transplantation parameters, and the development of effective outcome measures. We also discuss the integration of novel technologies such as gene editing, biomaterials, and tissue engineering to enhance the therapeutic potential of stem cells. The article concludes by emphasizing the importance of collaborative efforts among various stakeholders in the scientific community, including researchers, clinicians, bioengineers, industry partners, and patients, to overcome these challenges and realize the full potential of stem cell therapy for SCI patients. By fostering such collaborations and advancing our understanding of stem cell biology and regenerative medicine, we can pave the way for the development of groundbreaking therapies that improve the lives of those affected by SCIs.

Multipotent Mesenchymal Stem Cell-Based Therapies for Spinal Cord Injury: Current Progress and Future Prospects.

Spinal cord injury (SCI) represents a significant medical challenge, often resulting in permanent disability and severely impacting the quality of life for affected individuals. Traditional treatment options remain limited, underscoring the need for novel therapeutic approaches. In recent years, multipotent mesenchymal stem cells (MSCs) have emerged as a promising candidate for SCI treatment due to their multifaceted regenerative capabilities. This comprehensive review synthesizes the current understanding of the molecular mechanisms underlying MSC-mediated tissue repair in SCI. Key mechanisms discussed include neuroprotection through the secretion of growth factors and cytokines, promotion of neuronal regeneration via MSC differentiation into neural cell types, angiogenesis through the release of pro-angiogenic factors, immunomodulation by modulating immune cell activity, axonal regeneration driven by neurotrophic factors, and glial scar reduction via modulation of extracellular matrix components. Additionally, the review examines the various clinical applications of MSCs in SCI treatment, such as direct cell transplantation into the injured spinal cord, tissue engineering using biomaterial scaffolds that support MSC survival and integration, and innovative cell-based therapies like MSC-derived exosomes, which possess regenerative and neuroprotective properties. As the field progresses, it is crucial to address the challenges associated with MSC-based therapies, including determining optimal sources, intervention timing, and delivery methods, as well as developing standardized protocols for MSC isolation, expansion, and characterization. Overcoming these challenges will facilitate the translation of preclinical findings into clinical practice, providing new hope and improved treatment options for individuals living with the devastating consequences of SCI.

Macrophage-Neuroglia Interactions in Promoting Neuronal Regeneration in Zebrafish.

The human nervous system exhibits limited regenerative capabilities following damage to the central nervous system (CNS), leading to a scarcity of effective treatments for nerve function recovery. In contrast, zebrafish demonstrate remarkable regenerative abilities, making them an ideal model for studying the modulation of inflammatory processes after injury. Such research holds significant translational potential to enhance our understanding of recovery from damage and disease. Macrophages play a crucial role in tissue repair and regeneration, with their subpopulations indirectly promoting axonal regeneration through developmental signals. The AP-1 signaling pathway, mediated by TNF/Tnfrsf1a, can elevate HDAC1 expression and facilitate regeneration. Furthermore, following spinal cord injury (SCI), pMN progenitors have been observed to switch between oligodendrocyte and motor neuron fates, with macrophage-secreted TNF-α potentially regulating the differentiation of ependymal-radial glia progenitors and oligodendrocytes. Radial glial cells (RGs) are also essential for CNS regeneration in zebrafish, as they perform neurogenesis and gliogenesis, with specific RG subpopulations potentially existing for the generation of neurons and oligodendrocytes. This review article underscores the critical role of macrophages and their subpopulations in tissue repair and regeneration, focusing on their secretion of TNF-α, which promotes axonal regeneration in zebrafish. We also offer insights into the molecular mechanisms underlying TNF-α's ability to facilitate axonal regeneration and explore the potential of pMN progenitor cells and RGs following SCI in zebrafish. The review concludes with a discussion of various unresolved questions in the field, and ideas are suggested for future research. Studying innate immune cell interactions with neuroglia following injury may lead to the development of novel strategies for treating the inflammatory processes associated with regenerative medicine, which are commonly observed in injury and disease.

Genomic Structure, Protein Character, Phylogenic Implication, and Embryonic Expression Pattern of a Zebrafish New Member of Zinc Finger BED-Type Gene Family.

We reported a new member of the C2H2-zinc-finger BED-type (ZBED) protein family found in zebrafish (Danio rerio). It was previously assigned as an uncharacterized protein LOC569044 encoded by the Zgc:161969 gene, the transcripts of which were highly expressed in the CNS after the spinal cord injury of zebrafish. As such, this novel gene deserves a more detailed investigation. The 2.79-kb Zgc:161969 gene contains one intron located on Chromosome 6 at 16,468,776-16,475,879 in the zebrafish genome encoding a 630-aa protein LOC569044. This protein is composed of a DNA-binding BED domain, which is highly conserved among the ZBED protein family, and a catalytic domain consisting of an α-helix structure and an hAT dimerization region. Phylogenetic analysis revealed the LOC569044 protein to be clustered into the monophyletic clade of the ZBED protein family of golden fish. Specifically, the LOC569044 protein was classified as closely related to the monophyletic clades of zebrafish ZBED4-like isoforms and ZBED isoform 2. Furthermore, Zgc:161969 transcripts represented maternal inheritance, expressed in the brain and eyes at early developmental stages and in the telencephalon ventricular zone at late developmental stages. After characterizing the LOC569044 protein encoded by the Zgc:161969 gene, it was identified as a new member of the zebrafish ZBED protein family, named the ZBEDX protein.

Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos.

After spinal cord injury (SCI) in mammals, neuronal regeneration is limited; in contrast, such regeneration occurs quickly in zebrafish. Member A of the acidic nuclear phosphoprotein 32 (ANP32a) family is involved in neuronal development, but its function is controversial, and its involvement in zebrafish SCI remains unknown. To determine the role of zebrafish ANP32a in the neuronal regeneration of SCI embryos, we microinjected ANP32a mRNA into embryos from zebrafish transgenic line Tg(mnx1:GFP) prior to SCI. Compared to control SCI embryos, the results showed that the regeneration of spinal cord and resumption of swimming capability were promoted by the overexpression of ANP32a mRNA but reduced by its knockdown. We next combined fluorescence-activated cell sorting with immunochemical staining of anti-GFAP and immunofluorescence staining against anti-PH3 on Tg(gfap:GFP) SCI embryos. The results showed that ANP32a promoted the proliferation and cell number of radial glial cells at the injury epicenter at 24 h post-injury (hpi). Moreover, when we applied BrdU labeling to SCI embryos derived from crossing the Tg(gfap:GFP) and Tg(mnx1:TagRFP) lines, we found that both radial glial cells and motor neurons had proliferated, along with their increased cell numbers in Anp32a-overexpression SCI-embryos. On this basis, we conclude that ANP32a plays a positive role in the regeneration of zebrafish SCI embryos.

Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

Hypoxia-Responsive Subtype Cells Differentiate Into Neurons in the Brain of Zebrafish Embryos Exposed to Hypoxic Stress.

Severe hypoxia results in complete loss of central nervous system (CNS) function in mammals, while several other vertebrates, such as zebrafish, can regenerate after hypoxia-induced injury of CNS. Since the cellular mechanism involved in this remarkable feature of other vertebrates is still unclear, we studied the cellular regeneration of zebrafish brain, employing zebrafish embryos from transgenic line huORFZ exposed to hypoxia and then oxygen recovery. GFP-expressing cells, identified in some cells of the CNS, including some brain cells, were termed as hypoxia-responsive recovering cells (HrRCs). After hypoxia, HrRCs did not undergo apoptosis, while most non-GFP-expressing cells, including neurons, did. Major cell types of HrRCs found in the brain of zebrafish embryos induced by hypoxic stress were neural stem/progenitor cells (NSPCs) and radial glia cells (RGs), that is, subtypes of NSPCs (NSPCs-HrRCs) and RGs (RGs-HrRCs) that were induced by and sensitively responded to hypoxic stress. Interestingly, among HrRCs, subtypes of NSPCs- or RGs-HrRCs could proliferate and differentiate into early neurons during oxygen recovery, suggesting that these subtype cells might play a critical role in brain regeneration of zebrafish embryos after hypoxic stress.

Injury-induced Cavl-expressing cells at lesion rostral side play major roles in spinal cord regeneration.

The extent of cellular heterogeneity involved in neuronal regeneration after spinal cord injury (SCI) remains unclear. Therefore, we established stress-responsive transgenic zebrafish embryos with SCI. As a result, we found an SCI-induced cell population, termed SCI stress-responsive regenerating cells (SrRCs), essential for neuronal regeneration post-SCI. SrRCs were mostly composed of subtypes of radial glia (RGs-SrRCs) and neuron stem/progenitor cells (NSPCs-SrRCs) that are able to differentiate into neurons, and they formed a bridge across the lesion and connected with neighbouring undamaged motor neurons post-SCI. Compared to SrRCs at the caudal side of the SCI site (caudal-SrRCs), rostral-SrRCs participated more actively in neuronal regeneration. After RNA-seq analysis, we discovered that caveolin 1 (cav1) was significantly upregulated in rostral-SrRCs and that cav1 was responsible for the axonal regrowth and regenerative capability of rostral-SrRCs. Collectively, we define a specific SCI-induced cell population, SrRCs, involved in neuronal regeneration, demonstrate that rostral-SrRCs exhibit higher neuronal differentiation capability and prove that cav1 is predominantly expressed in rostral-SrRCs, playing a major role in neuronal regeneration after SCI.

The Neuronal Regeneration of Adult Zebrafish After Spinal Cord Injury Is Enhanced by Transplanting Optimized Number of Neural Progenitor Cells.

Cell transplantation is commonly used to study the regeneration and repair of the nervous system in animals. However, a technical platform used to evaluate the optimum number of transplanted cells in the recipient's spinal cord is little reported. Therefore, to develop such platform, we used a zebrafish model, which has transparent embryos, and transgenic line huORFZ, which generates green fluorescent protein (GFP)-expressing cells in the central nervous system under hypoxic stress. After GFP-expressing cells, also termed as hypoxia-responsive recovering cells, were obtained from hypoxia-exposed huORFZ embryos, we transplanted these GFP-(+) cells into the site of spinal cord injury (SCI) in adult wild-type zebrafish, followed by assessing the relationship between number of transplanted cells and the survival rate of recipients. When 100, 300, 500, and 1,000 GFP-(+) donor cells were transplanted into the lesion site of SCI-treated recipients, we found that recipient adult zebrafish transplanted with 300 donor cells had the highest survival rate. Those GFP-(+) donor cells could undergo proliferation and differentiation into neuron in recipients. Furthermore, transplantation of GFP-(+) cells into adult zebrafish treated with SCI was able to enhance the neuronal regeneration of recipients. In contrast, those fish transplanted with over 500 cells showed signs of inflammation around the SCI site, resulting in higher mortality. In this study, we developed a technological platform for transplanting cells into the lesion site of SCI-treated adult zebrafish and defined the optimum number of successfully transplanted cells into recipients, as 300, and those GFP-(+) donor cells could enhance recipient's spinal cord regeneration. Thus, we provided a practical methodology for studying cell transplantation therapy in neuronal regeneration of zebrafish after SCI.

A new member of the forkhead box protein family in zebrafish: Domain composition, phylogenetic implication and embryonic expression pattern.

In this study, we reported a novel member of Forkhead box (Fox) proteins found in model zebrafish (Danio rerio). This new gene we cloned was primarily assigned as zgc:162612, which locates on Chromosome 3 at 26,108,033-26,109,322 in the zebrafish genome, but encodes an uncharacterized protein, LOC100037333. After we determined the nucleotide and deduced amino acid sequences of zgc:162612, we found that zgc:162612 is an intronless gene and contains 1290 base pairs encoding 308 amino acid residues. Zgc:162612 protein is composed of a highly conserved DNA-binding domain similar to that of the Fox protein family, but with variable terminal domains. Based on phylogenetic analysis of all known members within the zebrafish Fox protein family, zgc:162612 was clustered into the zebrafish FoxD isoform subfamily. Thus, we confirmed zgc:162612 as the zebrafish FoxD7 gene. The deduced amino acid sequence of zebrafish FoxD7 shared 49, 49, 74, 63 and 74% identities with that of zebrafish FoxD1, FoxD2, FoxD3, FoxD4 and FoxD6, respectively. Compared with all known FoxD proteins in invertebrate and vertebrate species, the zebrafish FoxD7 is categorized in the same monophyletic group along with FoxD of cephalochordate and sea urchin. Whole-mount in situ hybridization demonstrated that zebrafish FoxD7 transcripts represented maternal inheritance and were ubiquitously expressed throughout the whole embryo at 12hpf. Moreover, while FoxD7 transcripts were expressed in the brain, spinal cord, fins and eyes at early developmental stages, they were mainly presented in the telencephalon ventricular zone at late developmental stages, suggesting that FoxD7 may play roles in neurogenesis and organogenesis during development of zebrafish. Taken together, we have defined a previously uncharacterized gene in the zebrafish genome, zgc:162612, and revealed that Zgc:162612 encodes a novel putative transcription factor, thus becoming a new member of the zebrafish FoxD isoform subfamily.

microRNA-206 modulates an Rtn4a/Cxcr4a/Thbs3a axis in newly forming somites to maintain and stabilize the somite boundary formation of zebrafish embryos.

Although microRNA-206 (miR-206) is known to regulate proliferation and differentiation of muscle fibroblasts, the role of miR-206 in early-stage somite development is still unknown. During somitogenesis of zebrafish embryos, reticulon4a (rtn4a) is specifically repressed by miR-206. The somite boundary was defective, and actin filaments were crossing over the boundary in either miR-206-knockdown or rtn4a-overexpressed embryos. In these treated embryos, C-X-C motif chemokine receptor 4a (cxcr4a) was reduced, while thrombospondin 3a (thbs3a) was increased. The defective boundary was phenocopied in either cxcr4a-knockdown or thbs3a-overexpressed embryos. Repression of thbs3a expression by cxcr4a reduced the occurrence of the boundary defect. We demonstrated that cxcr4a is an upstream regulator of thbs3a and that defective boundary cells could not process epithelialization in the absence of intracellular accumulation of the phosphorylated focal adhesion kinase (p-FAK) in boundary cells. Therefore, in the newly forming somites, miR-206-mediated downregulation of rtn4a increases cxcr4a. This activity largely decreases thbs3a expression in the epithelial cells of the somite boundary, which causes epithelialization of boundary cells through mesenchymal-epithelial transition (MET) and eventually leads to somite boundary formation. Collectively, we suggest that miR-206 mediates a novel pathway, the Rtn4a/Cxcr4a/Thbs3a axis, that allows boundary cells to undergo MET and form somite boundaries in the newly forming somites of zebrafish embryos.

Embryonic expression patterns of Eukaryotic EndoU ribonuclease family gene endouC in zebrafish.

Endou proteins belong to the Eukaryotic EndoU ribonuclease family of enzymes that present high sequence homology with the founding member XendoU domain. The enzymatic activity and three-dimensional structure of some Endou proteins have been previously reported. However, their molecular structure and gene expression patterns during embryogenesis remain to be elucidated. Therefore, we took zebrafish (Danio rerio) endouC as the model to study molecular structure and gene expression dynamics at different developmental stages. Zebrafish endouC cDNA contains 930 base pairs encoding 309 amino acid residues, sharing 27%, 27%, 27%, and 25% identity with that of human, mouse, chicken and frog, respectively. A phylogenetic tree showed that zebrafish EndouA was clustered with vertebrate Endou groups, while zebrafish EndouB and EndouC were found to belong to a unique monophyletic group. Furthermore, the endouC transcript was detected in one-cell embryos, suggesting that it is a maternal gene. While the endouC transcript was only weakly present at early developmental stages, its expression was greatly increased in embryos from 18 to 48 h post-fertilization (hpf) and then decreased after 72 hpf. Finally, endouC was ubiquitously expressed throughout the whole embryo during early embryogenesis, but its expression was enriched in brain, eyes and fin buds from 24 to 96 hpf.

Subtypes of hypoxia-responsive cells differentiate into neurons in spinal cord of zebrafish embryos after hypoxic stress.

BACKGROUND INFORMATION: Neuron stem/progenitor cells (NSPCs) of zebrafish central nervous system (CNS) are known to thrive during oxygen recovery after hypoxia, but not all cell types have been fully characterised due to their heterogeneities. In addition, an in vivo model system is not available that can help us to identify what type-specific cell populations that are involved in neural regeneration and to track their cell fate after regeneration. To solve these issues, we employed a zebrafish transgenic line, huORFZ, which harbours an inhibitory upstream open reading frame of human chop mRNA fused downstream with GFP reporter and driven by cytomegalovirus promoter. When huORFZ embryos were exposure to hypoxic stress, followed by oxygen recovery, GFP was exclusively expressed in some particular cells of CNS. Unlike GFP-negative cells, all GFP-expressing cells were not apoptotic, indicating that cell populations that are able to survive after hypoxia can be identified through this approach. RESULTS: When GFP-expressing cells of spinal cord were studied, we found mostly NSPCs and radial glia cells (RGs), along with a few oligodendrocyte progenitor cells and oligodendrocytes, all termed as hypoxia-responsive recovering cells (HrRCs). After hypoxic stress, these GFP-positive HrRCs did not undergo apoptosis, but GFP-negative neurons did. Prolonged recovery time after hypoxia was correlated with higher proportions of GFP(+)-NSPCs and GFP(+)-RGs, in contrast to lower proportions of proliferating/differentiating GFP(-)-NSPCs and GFP(-)-RGs. Among HrRCs subtypes, only GFP(+)-NSPCs and GFP(+)-RGs proliferated, migrated and differentiated into functional neurons during oxygen recovery. When some HrRCs were ablated in the spinal cord of hypoxia-exposed huORFZ embryos, swimming performance was impaired, suggesting that HrRCs are involved in neuronal regeneration. CONCLUSION: We demonstrated type-specific cell populations able to respond sensitively to hypoxic stress in the spinal cord of zebrafish embryos and that these type-specific populations play a role in neural regeneration. SIGNIFICANCE: Among heterogeneous cell types that exist in the spinal cord of zebrafish embryos after hypoxia, the particular cells that are resistant to hypoxia and also involved in neuronal regeneration can be clearly identified and dynamically traced using a transgenic model fish.