Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

Regeneration and developmental enhancers are differentially compatible with minimal promoters.

Enhancers and promoters are cis-regulatory elements that control gene expression. Enhancers are activated in a cell type-, tissue-, and condition-specific manner to stimulate promoter function and transcription. Zebrafish have emerged as a powerful animal model for examining the activities of enhancers derived from various species through transgenic enhancer assays, in which an enhancer is coupled with a minimal promoter. However, the efficiency of minimal promoters and their compatibility with multiple developmental and regeneration enhancers have not been systematically tested in zebrafish. Thus, we assessed the efficiency of six minimal promoters and comprehensively interrogated the compatibility of the promoters with developmental and regeneration enhancers. We found that the fos minimal promoter and Drosophila synthetic core promoter (DSCP) yielded high rates of leaky expression that may complicate the interpretation of enhancer assays. Notably, the adenovirus E1b promoter, the zebrafish lepb 0.8-kb (P0.8) and lepb 2-kb (P2) promoters, and a new zebrafish synthetic promoter (ZSP) that combines elements of the E1b and P0.8 promoters drove little or no ectopic expression, making them suitable for transgenic assays. We also found significant differences in compatibility among specific combinations of promoters and enhancers, indicating the importance of promoters as key regulatory elements determining the specificity of gene expression. Our study provides guidelines for transgenic enhancer assays in zebrafish to aid in the discovery of functional enhancers regulating development and regeneration.

Decoding an organ regeneration switch by dissecting cardiac regeneration enhancers.

Heart regeneration in regeneration-competent organisms can be accomplished through the remodeling of gene expression in response to cardiac injury. This dynamic transcriptional response relies on the activities of tissue regeneration enhancer elements (TREEs); however, the mechanisms underlying TREEs are poorly understood. We dissected a cardiac regeneration enhancer in zebrafish to elucidate the mechanisms governing spatiotemporal gene expression during heart regeneration. Cardiac lepb regeneration enhancer (cLEN) exhibits dynamic, regeneration-dependent activity in the heart. We found that multiple injury-activated regulatory elements are distributed throughout the enhancer region. This analysis also revealed that cardiac regeneration enhancers are not only activated by injury, but surprisingly, they are also actively repressed in the absence of injury. Our data identified a short (22 bp) DNA element containing a key repressive element. Comparative analysis across Danio species indicated that the repressive element is conserved in closely related species. The repression mechanism is not operational during embryogenesis and emerges when the heart begins to mature. Incorporating both activation and repression components into the mechanism of tissue regeneration constitutes a new paradigm that might be extrapolated to other regeneration scenarios.

leptin b and its regeneration enhancer illustrate the regenerative features of zebrafish hearts.

BACKGROUND: Zebrafish possess a remarkable regenerative capacity, which is mediated by the induction of various genes upon injury. Injury-dependent transcription is governed by the tissue regeneration enhancer elements (TREEs). Here, we utilized leptin b (lepb), an injury-specific factor, and its TREE to dissect heterogeneity of noncardiomyocytes (CMs) in regenerating hearts. RESULTS: Our single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the endothelium/endocardium(EC) is activated to induce distinct subpopulations upon injury. We demonstrated that lepb can be utilized as a regeneration-specific marker to subset injury-activated ECs. lepb+ ECs robustly induce pro-regenerative factors, implicating lepb+ ECs as a signaling center to interact with other cardiac cells. Our scRNA-seq analysis identified that lepb is also produced by subpopulation of epicardium (Epi) and epicardium-derived cells (EPDCs). To determine whether lepb labels injury-emerging non-CM cells, we tested the activity of lepb-linked regeneration enhancer (LEN) with chromatin accessibility profiles and transgenic lines. While nondetectable in uninjured hearts, LEN directs EC and Epi/EPDC expression upon injury. The endogenous LEN activity was assessed using LEN deletion lines, demonstrating that LEN deletion abolished injury-dependent expression of lepb, but not other nearby genes. CONCLUSIONS: Our integrative analyses identify regeneration-emerging cell-types and factors, leading to the discovery of regenerative features of hearts.

Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

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Transcriptional Programs and Regeneration Enhancers Underlying Heart Regeneration.

The heart plays the vital role of propelling blood to the entire body, which is essential to life. While maintaining heart function is critical, adult mammalian hearts poorly regenerate damaged cardiac tissue upon injury and form scar tissue instead. Unlike adult mammals, adult zebrafish can regenerate injured hearts with no sign of scarring, making zebrafish an ideal model system with which to study the molecular mechanisms underlying heart regeneration. Investigation of heart regeneration in zebrafish together with mice has revealed multiple cardiac regeneration genes that are induced by injury to facilitate heart regeneration. Altered expression of these regeneration genes in adult mammals is one of the main causes of heart regeneration failure. Previous studies have focused on the roles of these regeneration genes, yet the regulatory mechanisms by which the expression of cardiac regeneration genes is precisely controlled are largely unknown. In this review, we will discuss the importance of differential gene expression for heart regeneration, the recent discovery of cardiac injury or regeneration enhancers, and their impact on heart regeneration.

Protein nanovaccine confers robust immunity against Toxoplasma.

We designed and produced a self-assembling protein nanoparticle. This self-assembling protein nanoparticle contains five CD8+ HLA-A03-11 supertypes-restricted epitopes from antigens expressed during Toxoplasma gondii's lifecycle, the universal CD4+ T cell epitope PADRE, and flagellin as a scaffold and TLR5 agonist. These CD8+ T cell epitopes were separated by N/KAAA spacers and optimized for proteasomal cleavage. Self-assembling protein nanoparticle adjuvanted with TLR4 ligand-emulsion GLA-SE were evaluated for their efficacy in inducing IFN-γ responses and protection of HLA-A*1101 transgenic mice against T. gondii. Immunization, using self-assembling protein nanoparticle-GLA-SE, activated CD8+ T cells to produce IFN-γ. Self-assembling protein nanoparticle-GLA-SE also protected HLA-A*1101 transgenic mice against subsequent challenge with Type II parasites. Hence, combining CD8+ T cell-eliciting peptides and PADRE into a multi-epitope protein that forms a nanoparticle, administered with GLA-SE, leads to efficient presentation by major histocompatibility complex Class I and II molecules. Furthermore, these results suggest that activation of TLR4 and TLR5 could be useful for development of vaccines that elicit T cells to prevent toxoplasmosis in humans.

Point-of-care testing for Toxoplasma gondii IgG/IgM using Toxoplasma ICT IgG-IgM test with sera from the United States and implications for developing countries.

BACKGROUND: Congenital toxoplasmosis is a serious but preventable and treatable disease. Gestational screening facilitates early detection and treatment of primary acquisition. Thus, fetal infection can be promptly diagnosed and treated and outcomes can be improved. METHODS: We tested 180 sera with the Toxoplasma ICT IgG-IgM point-of-care (POC) test. Sera were from 116 chronically infected persons (48 serotype II; 14 serotype I-III; 25 serotype I-IIIa; 28 serotype Atypical, haplogroup 12; 1 not typed). These represent strains of parasites infecting mothers of congenitally infected children in the U.S. 51 seronegative samples and 13 samples from recently infected persons known to be IgG/IgM positive within the prior 2.7 months also were tested. Interpretation was confirmed by two blinded observers. A comparison of costs for POC vs. commercial laboratory testing methods was performed. RESULTS: We found that this new Toxoplasma ICT IgG-IgM POC test was highly sensitive (100%) and specific (100%) for distinguishing IgG/IgM-positive from negative sera. Use of such reliable POC tests can be cost-saving and benefit patients. CONCLUSIONS: Our work demonstrates that the Toxoplasma ICT IgG-IgM test can function reliably as a point-of-care test to diagnose Toxoplasma gondii infection in the U.S. This provides an opportunity to improve maternal-fetal care by using approaches, diagnostic tools, and medicines already available. This infection has serious, lifelong consequences for infected persons and their families. From the present study, it appears a simple, low-cost POC test is now available to help prevent morbidity/disability, decrease cost, and make gestational screening feasible. It also offers new options for improved prenatal care in low- and middle-income countries.

Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.