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Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Animal models are necessary to investigate the pathogenic features underlying motor neuron degeneration and for therapeutic development in amyotrophic lateral sclerosis (ALS). Measures of model validity allow for a critical interpretation of results from each model and caution from over-interpretation of experimental models. Face and construct validity refer to the similarity in phenotype and the proposed causal factor to the human disease, respectively. More recently developed models are restricted by limited phenotype characterization, yet new models hold promise for novel disease insights, thus highlighting their importance. In this article, we evaluate the features of face and construct validity of our new zebrafish model of environmentally-induced motor neuron degeneration and discuss this in the context of current environmental and genetic ALS models, including C9orf72, mutant Cu/Zn superoxide dismutase 1 and TAR DNA-binding protein 43 mouse and zebrafish models. In this mini-review, we discuss the pros and cons to validity criteria in each model. Our zebrafish model of environmentally-induced motor neuron degeneration displays convincing features of face validity with many hallmarks of ALS-like features, and weakness in construct validity. However, the value of this model may lie in its potential to be more representative of the pathogenic features underlying sporadic ALS cases, where environmental factors may be more likely to be involved in disease etiology than single dominant gene mutations. It may be necessary to compare findings between different strains and species modeling specific genes or environmental factors to confirm findings from ALS animal models and tease out arbitrary strain- and overexpression-specific effects.

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.

Background Cystinosis is a rare, incurable lysosomal storage disease caused by mutations in the CTNS gene encoding the cystine transporter cystinosin, which leads to lysosomal cystine accumulation in all cells of the body. Patients with cystinosis display signs of podocyte damage characterized by extensive loss of podocytes into the urine at early disease stages, glomerular proteinuria, and the development of focal segmental glomerulosclerosis (FSGS) lesions. Although standard treatment with cysteamine decreases cellular cystine levels, it neither reverses glomerular injury nor prevents the loss of podocytes. Thus, pathogenic mechanisms other than cystine accumulation are involved in podocyte dysfunction in cystinosis. Methods We used immortalized patient-derived cystinosis, healthy, and CTNS knockdown podocytes to investigate podocyte dysfunction in cystinosis. The results were validated in our newly in-house developed fluorescent ctns −/− [Tg(fabp10a:gc-EGFP)] zebrafish larvae model. To understand impaired podocyte functionality, static and dynamic permeability assays, tracer-metabolomic analysis, flow cytometry, western blot, and chemical and dynamic redox-sensing fluorescent probes were used. Results In the current study, we discovered that cystinosis podocytes demonstrate increased ferroptotic cell death caused by mitochondrial reactive oxygen species (ROS)-driven membrane lipid peroxidation. Moreover, cystinosis cells present a fragmented mitochondrial network with impaired tricarboxylic acid cycle (TCA) cycle and energy metabolism. Targeting mitochondrial ROS and lipid peroxidation improved podocyte function in vitro and rescued proteinuria in vivo in cystinosis zebrafish larvae. Conclusions Mitochondrial ROS contribute to podocyte injury in cystinosis by driving lipid peroxidation and ferroptosis, which in turn lead to podocyte detachment. This finding adds cystinosis to the list of podocytopathies associated with mitochondrial dysfunction. The identified mechanisms reveal new therapeutic targets and highlight lipid peroxidation as an exploitable vulnerability of cystinosis podocytes.

The Hippo signaling pathway is a kinase cascade that plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated the Hippo pathway in kidney development and kidney diseases. To further illustrate its roles in vertebrate kidney, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes. The stk3−/− mutant exhibited edema, formation of glomerular cysts and pronephric tubule dilation during the larval stage. Interestingly, disruption of wwtr1, but not yap1, significantly alleviated the renal phenotypes of the stk3−/− mutant, and overexpression of Wwtr1 with the CMV promoter also induced pronephric phenotypes, similar to those of the stk3−/− mutant, during larval stage. Notably, adult fish with Wwtr1 overexpression developed phenotypes similar to those of human polycystic kidney disease (PKD). Overall, our analyses revealed roles of Stk3 and Wwtr1 in renal cyst formation. Using a pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development.

With the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models, such as mice, is time consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model for evaluating the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified as possessing anti-TB activity in vitro. To aid solubilization, compounds were formulated in biocompatible polymeric micelles (PMs). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating the in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited for pinpointing promising compounds for further development.

Tissue-resident macrophages of the brain, including microglia, are implicated in the pathogenesis of various CNS disorders and are possible therapeutic targets by their chemical depletion or replenishment by hematopoietic stem cell therapy. Nevertheless, a comprehensive understanding of microglial function and the consequences of microglial depletion in the human brain is lacking. In human disease, heterozygous variants in CSF1R, encoding the Colony-stimulating factor 1 receptor, can lead to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) possibly caused by microglial depletion. Here, we investigate the effects of ALSP-causing CSF1R variants on microglia and explore the consequences of microglial depletion in the brain. In intermediate- and late-stage ALSP post-mortem brain, we establish that there is an overall loss of homeostatic microglia and that this is predominantly seen in the white matter. By introducing ALSP-causing missense variants into the zebrafish genomic csf1ra locus, we show that these variants act dominant negatively on the number of microglia in vertebrate brain development. Transcriptomics and proteomics on relatively spared ALSP brain tissue validated a downregulation of microglia-associated genes and revealed elevated astrocytic proteins, possibly suggesting involvement of astrocytes in early pathogenesis. Indeed, neuropathological analysis and in vivo imaging of csf1r zebrafish models showed an astrocytic phenotype associated with enhanced, possibly compensatory, endocytosis. Together, our findings indicate that microglial depletion in zebrafish and human disease, likely as a consequence of dominant-acting pathogenic CSF1R variants, correlates with altered astrocytes. These findings underscore the unique opportunity CSF1R variants provide to gain insight into the roles of microglia in the human brain, and the need to further investigate how microglia, astrocytes, and their interactions contribute to white matter homeostasis.

Zebrafish is a well-recognized model for studying human genetic disorders. Recently, we proposed the homozygous cdkl5sa21938 mutant zebrafish as a model of CDKL5 deficiency disorder (CDD), a developmental epileptic encephalopathy with diverse symptoms. This study aimed to explore Cdkl5-associated molecular mechanisms in zebrafish and assess their similarity to those in mammals. We conducted RNA sequencing on whole cdkl5−/− zebrafish and wild-type siblings at 5 and 35 days post-fertilization (dpf) to compare their gene expression profiles. Most significant differentially expressed genes (DEGs) were related to muscle, neuronal, and visual systems which are affected in CDD. Gene Ontology analysis revealed downregulated DEGs enriched in muscle development, extracellular matrix, and actin cytoskeleton functions at both stages, while upregulated DEGs were enriched in eye development functions at 35 dpf. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment of downregulated DEGs in focal adhesion and extracellular matrix (ECM)-receptor interaction pathways at both stages. Neuronal development DEGs were mainly downregulated at both stages, while synaptic signaling DEGs were upregulated at 35 dpf. Crossing cdkl5−/− mutants with the Hb9:GFP transgenic line showed fewer motor neuron cells with shorter axons compared to the wild type, which may explain the impaired motor phenotype observed in zebrafish and CDD patients. Moreover, we identified key downregulated DEGs related to cartilage development at both stages and bone development at 35 dpf, potentially explaining the skeletal defects seen in zebrafish and CDD individuals. In conclusion, Cdkl5 loss in zebrafish leads to dysregulation of genes involved in CDKL5-associated functions in mammals, providing new insights into its less studied functions and phenotypes.

Inflammatory bowel disease (IBD) is a chronic and relapsing immune-mediated disorder characterized by intestinal inflammation and epithelial injury. The underlying causes of IBD are not fully understood, but genetic factors have been implicated in genome-wide association studies, including CTLA-4, an essential negative regulator of T cell activation. However, establishing a direct link between CTLA-4 and IBD has been challenging due to the early lethality of CTLA-4 knockout mice. In this study, we identified zebrafish Ctla-4 homolog and investigated its role in maintaining intestinal immune homeostasis by generating a Ctla-4-deficient ( ctla-4 -/- ) zebrafish line. These mutant zebrafish exhibited reduced weight, along with impaired epithelial barrier integrity and lymphocytic infiltration in their intestines. Transcriptomics analysis revealed upregulation of inflammation-related genes, disturbing immune system homeostasis. Moreover, single-cell RNA-sequencing analysis indicated increased Th2 cells and interleukin 13 expression, along with decreased innate lymphoid cells and upregulated proinflammatory cytokines. Additionally, Ctla-4-deficient zebrafish exhibited reduced diversity and an altered composition of the intestinal microbiota. All these phenotypes closely resemble those found in mammalian IBD. Lastly, supplementation with Ctla-4-Ig successfully alleviated intestinal inflammation in these mutants. Altogether, our findings demonstrate the pivotal role of Ctla-4 in maintaining intestinal homeostasis. Additionally, they offer substantial evidence linking CTLA-4 to IBD and establish a novel zebrafish model for investigating both the pathogenesis and potential treatments.

Pyridoxine-dependent epilepsy (PDE) is a severe neonatal seizure disorder and is here modeled in aldh7a1 -/- zebrafish. Mutant larvae display spontaneous.. Pyridoxine-dependent epilepsy (PDE) is a rare disease characterized by mutations in the lysine degradation gene ALDH7A1 leading to recurrent neonatal seizures, which are uniquely alleviated by high doses of pyridoxine or pyridoxal 5′-phosphate (vitamin B6 vitamers). Despite treatment, neurodevelopmental disabilities are still observed in most PDE patients underlining the need for adjunct therapies. Over 60 years after the initial description of PDE, we report the first animal model for this disease: an aldh7a1-null zebrafish (Danio rerio) displaying deficient lysine metabolism and spontaneous and recurrent seizures in the larval stage (10 days postfertilization). Epileptiform electrographic activity was observed uniquely in mutants as a series of population bursts in tectal recordings. Remarkably, as is the case in human PDE, the seizures show an almost immediate sensitivity to pyridoxine and pyridoxal 5′-phosphate, with a resulting extension of the life span. Lysine supplementation aggravates the phenotype, inducing earlier seizure onset and death. By using mass spectrometry techniques, we further explored the metabolic effect of aldh7a1 knockout. Impaired lysine degradation with accumulation of PDE biomarkers, B6 deficiency, and low γ-aminobutyric acid levels were observed in the aldh7a1−/− larvae, which may play a significant role in the seizure phenotype and PDE pathogenesis. This novel model provides valuable insights into PDE pathophysiology; further research may offer new opportunities for drug discovery to control seizure activity and improve neurodevelopmental outcomes for PDE.