Cell-type-specific mRNA transcription and degradation kinetics in zebrafish embryogenesis from metabolically labeled single-cell RNA-seq.

During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.

Microtubule polarity determines the lineage of embryonic neural precursor in zebrafish spinal cord.

The phenomenal diversity of neuronal types in the central nervous system is achieved in part by the asymmetric division of neural precursors. In zebrafish neural precursors, asymmetric dispatch of Sara endosomes (with its Notch signaling cargo) functions as fate determinant which mediates asymmetric division. Here, we found two distinct pools of neural precursors based on Sara endosome inheritance and spindle-microtubule enrichment. Symmetric or asymmetric levels of spindle-microtubules drive differently Sara endosomes inheritance and predict neural precursor lineage. We uncover that CAMSAP2a/CAMSAP3a and KIF16Ba govern microtubule asymmetry and endosome motility, unveiling the heterogeneity of neural precursors. Using a plethora of physical and cell biological assays, we determined the physical parameters and molecular mechanisms behind microtubule asymmetries and biased endosome motility. Evolutionarily, the values of those parameters explain why all sensory organ precursor cells are asymmetric in flies while, in zebrafish spinal cord, two populations of neural precursors (symmetric vs asymmetric) are possible.

Polystyrene microplastics alter the trophic transfer and biotoxicity of fluoxetine in an aquatic food chain.

Microplastics (MPs) and fluoxetine are ubiquitous emerging pollutants in aquatic environments that may interact with each other due to the carrier effects of MPs, posing unpredictable risks to non-target organisms. However, limited studies have focused on the carrier effects of MPs in the aquatic food chain. This study evaluated the influences of polystyrene MPs on the trophic transfer and biotoxicity of fluoxetine in a simple food chain composed of brine shrimp (Artemia nauplii) and zebrafish (Danio rerio). The finding reveals that carrier effects of MPs enhanced the accumulation of waterborne fluoxetine in brine shrimp, but suppressed that in zebrafish due to the distinct retention times. The accumulated fluoxetine in shrimp was further transferred to fish through the food chain, which was alleviated by MPs due to their cleaning effects. In addition, the specific neurotransmission biotoxicity in fish induced by fluoxetine was mitigated by MPs, whilst the oxidative damage, apoptosis, and immune responses in zebrafish were reversely enhanced by MPs due to the stimulating effect. These findings highlight the alleviating effects of MPs on the trophic transfer and specific biotoxicity of fluoxetine in the food chain, providing new insights into the carrier effects of MPs in aquatic environments in the context of increasing global MP pollution.

Butylparaben induced zebrafish (Danio rerio) kidney injury by down-regulating the PI3K-AKT pathway.

Butylparaben, a common endocrine disruptor in the environment, is known to be toxic to the reproductive system, heart, and intestines, but its nephrotoxicity has rarely been reported. In order to study the nephrotoxicity and mechanism of butylparaben, we examined the acute and chronic effects on human embryonic kidney cells (HEK293T) and zebrafish. Additionally, we assessed the potential remedial effects of salidroside against butylparaben-induced nephrotoxicity. Our in vitro findings demonstrated oxidative stress and cytotoxicity to HEK293T cells caused by butylparaben. In the zebrafish model, the concentration of butylparaben exposure ranged from 0.5 to 15 µM. An assortment of experimental techniques was employed, including the assessment of kidney tissue morphology using Hematoxylin-Eosin staining, kidney function analysis via fluorescent dextran injection, and gene expression studies related to kidney injury, development, and function. Additionally, butylparaben caused lipid peroxidation in the kidney, thereby damaging glomeruli and renal tubules, which resulted from the downregulation of the PI3K-AKT signaling pathway. Furthermore, salidroside ameliorated butylparaben-induced nephrotoxicity through the PI3K-AKT signaling pathway. This study reveals the seldom-reported kidney toxicity of butylparaben and the protective effect of salidroside against toxicological reactions related to nephrotoxicity. It offers valuable insights into the risks to kidney health posed by environmental toxins.

Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species.

During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation.

BACKGROUND: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. METHODS: The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. RESULTS: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. CONCLUSIONS: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

ptx3a+ fibroblast/epicardial cells provide a transient macrophage niche to promote heart regeneration.

Macrophages conduct critical roles in heart repair, but the niche required to nurture and anchor them is poorly studied. Here, we investigated the macrophage niche in the regenerating heart. We analyzed cell-cell interactions through published single-cell RNA sequencing datasets and identified a strong interaction between fibroblast/epicardial (Fb/Epi) cells and macrophages. We further visualized the association of macrophages with Fb/Epi cells and the blockage of macrophage response without Fb/Epi cells in the regenerating zebrafish heart. Moreover, we found that ptx3a+ epicardial cells associate with reparative macrophages, and their depletion resulted in fewer reparative macrophages. Further, we identified csf1a expression in ptx3a+ cells and determined that pharmacological inhibition of the csf1a pathway or csf1a knockout blocked the reparative macrophage response. Moreover, we found that genetic overexpression of csf1a enhanced the reparative macrophage response with or without heart injury. Altogether, our studies illuminate a cardiac Fb/Epi niche, which mediates a beneficial macrophage response after heart injury.

Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.

Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.

A 3D-Printed and Freely Available Device to Measure the Zebrafish Optokinetic Response Before and After Injury.

Zebrafish eyes are anatomically similar to humans and have a higher percentage of cone photoreceptors more akin to humans than most rodent models, making them a beneficial model organism for studying vision. However, zebrafish are different in that they can regenerate their optic nerve after injury, which most other animals cannot. Vision in zebrafish and many other vertebrate animals, including humans, can be accessed using the optokinetic response (OKR), which is an innate eye movement that occurs when tracking an object. Because fish cannot use an eye chart, we utilize the OKR that is present in virtually all vertebrates to determine if a zebrafish has vision. To this end, we have developed an inexpensive OKR setup that uses 3D-printed and off-the-shelf parts. This setup has been designed and used by undergraduate researchers and is also scalable to a classroom laboratory setup. We demonstrate that this setup is fully functional for assessing the OKR, and we use it to illustrate the return of the OKR following optic nerve injury in adult zebrafish.

Differential Roles of Diet on Development and Spinal Cord Regeneration in Larval Zebrafish.

The zebrafish is a powerful model organism for studying development and regeneration. However, there is a lack of a standardized reference diet for developmental and regeneration experiments. Most studies evaluate the rate of growth, survival, and fecundity. In this study, we compare three diets and their effects on growth and regeneration after a spinal cord injury (SCI). Fish were fed daily for 1 week with daily measurements of overall length and width of spinal injury. Fish fed a live rotifer diet grew 32%, whereas a commercially available diet only led to a 4% increase in body length. Similarly, differences in rate of regeneration were observed with over 80% of rotifer-fed larvae forming a glial bridge after injury compared to <10% of zebrafish fed with the commercial diet. Our data highlight the need for establishing a standardized diet for regeneration studies to improve research reproducibility.

Bringing Real Inquiry-Based Science to Diverse Secondary Educational Environments: A Virtual Zebrafish Laboratory to Investigate Environmental Health.

The goal of the University of Wisconsin-Milwaukee WInSTEP SEPA program is to provide valuable and relevant research experiences to students and instructors in diverse secondary educational settings. Introducing an online experience allows the expansion of a proven instructional research program to a national scale and removes many common barriers. These can include lack of access to zebrafish embryos, laboratory equipment, and modern classroom facilities, which often deny disadvantaged and underrepresented students from urban and rural school districts valuable inquiry-based learning opportunities. An online repository of zebrafish embryo imagery was developed in the Carvan laboratory to assess the effects of environmental chemicals. The WInSTEP SEPA program expanded its use as an accessible online tool, complementing the existing classroom experience of our zebrafish module. This virtual laboratory environment contains images of zebrafish embryos grown in the presence of environmental toxicants (ethanol, caffeine, and nicotine), allowing students to collect data on 19 anatomical endpoints and generate significant amounts of data related to developmental toxicology and environmental health. This virtual laboratory offers students and instructors the choice of data sets that differ in the independent variables of chemical concentration and duration of postfertilization exposure. This enables students considerable flexibility in establishing their own experimental design to match the curriculum needs of each instructor.

Exploring the Hawaiian Ala Wai Watershed with Zebrafish.

The Ala Wai Canal is an artificial waterway in the tourist district of Waikiki in Honolulu, HI. Originally built to collect runoff from industrial, residential, and green spaces dedicated to recreation, the Ala Wai Canal has since experienced potent levels of toxicity due to this runoff entering the watershed and making it hazardous for both marine life and humans at current concentration, including Danio rerio (zebrafish). A community of learners at educations levels from high school to postbaccalaureate from Oahu, HI was connected through the Consortium for Increasing Research and Collaborative Learning Experiences (CIRCLE) distance research program. This team conducted research with an Investigator and team from Mayo Clinic in Rochester, MN, with the Ala Wai Canal as its primary subject. Through CIRCLE, research trainees sent two 32 oz bottles of Ala Wai- acquired water to a partnered laboratory at the Mayo Clinic in which zebrafish embryos were observed at differing concentrations of the sampled water against a variety of developmental and behavioral assays. Research trainees also created atlases of developmental outcomes in zebrafish following exposure to environmental toxins and tables of potential pesticide contaminants to enable the identification of the substances linked to structural defects and enhanced stress during Ala Wai water exposure experiments.

The InSciEdRS View: A User-Friendly and Accessible Microscope with Easy-to-Follow Companion Curricula.

Microscopes are essential for research and education in science. Unlike computers and online learning tools, however, microscopes are not currently a fixed element in K-12 classrooms, due to steep cost, needless complexity, and often requiring a prohibitive level of staff training to effectively deploy. In a collaboration with Area 10 Labs, Integrated Science Education Outreach (InSciEd Out) developed a state-of-the-art alternative microscope, the InSciEdRS View, to reduce the financial barrier, prohibitive per-student cost, unnecessary complexity, and extensive staff training. Utilizing a 1080p camera and a lunchbox-style case, this Wi-Fi- and USB-connectable microscope comes with all necessary components for visualization of microscopic specimens (10 × -50 × magnification). While built to handle the rigors of classroom use, its imaging capability and battery-operation can make it flexible for a laboratory or fieldwork as well. We further highlight here K-12 curricula that we have developed using larval zebrafish to enable teachers, science outreach leaders, and parents to support active hands-on science observations. The InSciEdRS View microscope and the InSciEd Out curricula are readily scalable, translatable, and accessible for traditional and neurodiverse students and integrating these in various settings can be an efficient way to achieve better outcomes in science education.

Lectins As Effective Tools in the Study of the Biliary Network and the Parenchymal Architecture of the Zebrafish (Danio rerio) Liver.

Lectins are carbohydrate-binding proteins with specific affinity to glycoconjugates expressed in various tissues. Lectins are of substantial utility as research, histochemical, and diagnostic tools in mammalian systems. Reactivity of 12 commonly used plant-based lectins was studied in zebrafish liver. Four lectins, tomato lectin (TL), wheat germ agglutinin, concanavalin A, and Jacalin showed strong reactivity to hepatic parenchymal structures. Importantly, TL reacted to glycoconjugates within segments of the larval and adult intrahepatic biliary network, from canaliculi to bile ducts. We provide evidence that lectins can serve as important histochemical tools to investigate the structural and functional characteristics of the zebrafish liver.

Growing Project BioEYES: A Reflection on 20 Years of Developing and Replicating a K-12 Science Outreach Program.

Project BioEYES celebrated 20 years in K12 schools during the 2022-2023 school year. Using live zebrafish (Danio rerio) during week-long science experiments, sparks the interest of students and teachers from school districts, locally and globally. Over the past two decades, BioEYES has been replicated in different ways based on the interest and capacity of our partners. This article discusses several of the successful models, the common challenges, and how each BioEYES site has adopted guiding principles to help foster their success. The core principles of (a) reinforcing content that students are expected to learn in schools, while focusing on the students BECOMING scientists through hands-on experimentation and (b) establishing trust and buy-in from collaborating teachers and partners are what has led to BioEYES being sustained and replicated over the past two decades.

Dual modifying of MAVS at lysine 7 by SIRT3-catalyzed deacetylation and SIRT5-catalyzed desuccinylation orchestrates antiviral innate immunity.

To effectively protect the host from viral infection while avoiding excessive immunopathology, the innate immune response must be tightly controlled. However, the precise regulation of antiviral innate immunity and the underlying mechanisms remain unclear. Here, we find that sirtuin3 (SIRT3) interacts with mitochondrial antiviral signaling protein (MAVS) to catalyze MAVS deacetylation at lysine residue 7 (K7), which promotes MAVS aggregation, as well as TANK-binding kinase I and IRF3 phosphorylation, resulting in increased MAVS activation and enhanced type I interferon signaling. Consistent with these findings, loss of Sirt3 in mice and zebrafish renders them more susceptible to viral infection compared to their wild-type (WT) siblings. However, Sirt3 and Sirt5 double-deficient mice exhibit the same viral susceptibility as their WT littermates, suggesting that loss of Sirt5 in Sirt3-deficient mice may counteract the increased viral susceptibility displayed in Sirt3-deficient mice. Thus, we not only demonstrate that SIRT3 positively regulates antiviral immunity in vitro and in vivo, likely via MAVS, but also uncover a previously unrecognized mechanism by which SIRT3 acts as an accelerator and SIRT5 as a brake to orchestrate antiviral innate immunity.

Kalium channelrhodopsins effectively inhibit neurons.

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)-recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditis elegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

Biological toxicity of sulfamethoxazole in aquatic ecosystem on adult zebrafish (Danio rerio).

This study evaluated the impacts of sulfamethoxazole (SMX) on antioxidant, immune, histopathological dynamic changes, and gut microbiota of zebrafish. SMX was carried out five groups: 0 (C), 3 mg/L (T3), 6 mg/L (T6), 12 mg/L (T12), and 24 mg/L (T24), with 5 replicates per group for an 8-weeks chronic toxicity test. It was found that SMX is considered to have low toxicity to adult zebrafish. SMX with the concentration not higher than 24 mg/L has no obvious inhibitory effect on the growth of fish. Under different concentrations of SMX stress, oxidative damage and immune system disorder were caused to the liver and gill, with the 12 and 24 mg/L concentration being the most significant. At the same time, it also causes varying degrees of pathological changes in both intestinal and liver tissues. As the concentration of SMX increases, the composition and abundance of the gut microbiota in zebrafish significantly decrease.