Applications & Publications
Technical Notes
D. rerio Analysis and Sorting: High throughput screening of innate immune responses in zebrafish embryos
Profiling of live D. reriolarvae using the COPAS XL instrument. Work performed in collaboration with the Spaink Laboratory, University of Leiden (ANXL-02)
Publications
m6A-mRNA Reader YTHDF2 Identified as a Potential Risk Gene in Autism With Disproportionate Megalencephaly
Nishizaki et al. January 30, 2025 https://doi.org/10.1002/aur.3314
View Abstractm6A-mRNA Reader YTHDF2 Identified as a Potential Risk Gene in Autism With Disproportionate Megalencephaly
Among autistic individuals, a subphenotype of disproportionate megalencephaly (ASD-DM) seen at three years of age is associated with co-occurring intellectual disability and poorer prognoses later in life. However, many of the genes contributing to ASD-DM have yet to be delineated. In this study, we identified additional ASD-DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD-DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection, totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD-DM genes harboring de novo protein-impacting variants. Our findings include 14 high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM, including CHD8 and PTEN. By performing functional network analysis, we expanded to additional candidate genes, including one previously implicated in ASD-DM (PIK3CA) as well as 184 additional genes connected with ASD or DM alone. Using zebrafish, we modeled a de novo tandem duplication impacting YTHDF2, encoding an N6-methyladenosine (m6A)-mRNA reader, in an ASD-DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size, while overexpressing YTHDF2 resulted in increased head/brain size matching that of the proband. Single-cell transcriptomes of YTHDF2 gain-of-function larvae point to reduced expression of Fragile-X-syndrome-associated FMRP-target genes globally and in the developing brain, providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader, YTHDC1, in our ASD-DM cohort. Though we highlight only two cases to date, our study provides support for the m6A-RNA modification pathway as potentially contributing to this severe form of autism.
Burn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry
Smoot et al. April 14, 2024 Heliyon. 2024 Apr 30; 10(8): e29675. Published online 2024 Apr 14. doi: 10.1016/j.heliyon.2024.e29675
View AbstractBurn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry
Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.
Adaptive Optics in an Oblique Plane Microscope
McFadden et al. March 22, 2024 Version 1. bioRxiv. Preprint. 2024 Mar 22. doi: 10.1101/2024.03.21.586191Published in: Biomed Opt Express. 2024 Aug 1; 15(8): 4498–4512.
View AbstractAdaptive Optics in an Oblique Plane Microscope
Adaptive optics (AO) can restore diffraction limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both the illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.
Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival
Tuttle et al. February 29, 2024 J Neurosci. 2024 Apr 17; 44(16): e1600232024. Prepublished online 2024 Feb 29. doi: 10.1523/JNEUROSCI.1600-23.2024
View AbstractSingle-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival
Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon–Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.
A retroviral link to vertebrate myelination through retrotransposon-RNA-mediated control of myelin gene expression
Ghosh et al. February 15, 2024 Cell, Volume 187, Issue 4p814-830.e23,February 15, 2024
View AbstractA retroviral link to vertebrate myelination through retrotransposon-RNA-mediated control of myelin gene expression
Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.
Macrophage mediated mesoscale brain mechanical homeostasis mechanically imaged via optical tweezers and Brillouin microscopy in vivo
So et al. December 27, 2023 Version 2. bioRxiv. Preprint. 2023 Dec 27 [revised 2024 Mar 6]. doi: 10.1101/2023.12.27.573380
View AbstractMacrophage mediated mesoscale brain mechanical homeostasis mechanically imaged via optical tweezers and Brillouin microscopy in vivo
Tissues are active materials where epithelial turnover, immune surveillance, and remodeling of stromal cells such as macrophages all regulate form and function. Scattering modalities such as Brillouin microscopy (BM) can non-invasively access mechanical signatures at GHz. However, our traditional understanding of tissue material properties is derived mainly from modalities which probe mechanical properties at different frequencies. Thus, reconciling measurements amongst these modalities remains an active area. Here, we compare optical tweezer active microrheology (OT-AMR) and Brillouin microscopy (BM) to longitudinally map brain development in the larval zebrafish. We determine that each measurement is able to detect a mechanical signature linked to functional units of the brain. We demonstrate that the corrected BM-Longitudinal modulus using a density factor correlates well with OT-AMR storage modulus at lower frequencies. We also show that the brain tissue mechanical properties are dependent on both the neuronal architecture and the presence of macrophages. Moreover, the BM technique is able to delineate the contributions to mechanical properties of the macrophage from that due to colony stimulating factor 1 receptor (CSF1R) mediated stromal remodeling. Here, our data suggest that macrophage remodeling is instrumental in the maintenance of tissue mechanical homeostasis during development. Moreover, the strong agreement between the OT-AM and BM further demonstrates that scattering-based technique is sensitive to both large and minute structural modification in vivo.
Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity
Nöth et al. December 21, 2023 Reproductive Toxicology, Open access, Published: 21 December 2023, Volume 98, pages 537–549, (2024)
View AbstractAnalysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity
Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.
A mechanistic understanding of the effects of polyethylene terephthalate nanoplastics in the zebrafish (Danio rerio) embryo
Bashirova et al. February 02, 2023 Sci Rep. 2023; 13: 1891. Published online 2023 Feb 2. doi: 10.1038/s41598-023-28712-y
View AbstractA mechanistic understanding of the effects of polyethylene terephthalate nanoplastics in the zebrafish (Danio rerio) embryo
Plastic pollution, especially by nanoplastics (NPs), has become an emerging topic due to the widespread existence and accumulation in the environment. The research on bioaccumulation and toxicity mechanism of NPs from polyethylene terephthalate (PET), which is widely used for packaging material, have been poorly investigated. Herein, we report the first use of high-resolution magic-angle spinning (HRMAS) NMR based metabolomics in combination with toxicity assay and behavioural end points to get systems-level understanding of toxicity mechanism of PET NPs in intact zebrafish embryos. PET NPs exhibited significant alterations on hatching and survival rate. Accumulation of PET NPs in larvae were observed in liver, intestine, and kidney, which coincide with localization of reactive oxygen species in these areas. HRMAS NMR data reveal that PET NPs cause: (1) significant alteration of metabolites related to targeting of the liver and pathways associated with detoxification and oxidative stress; (2) impairment of mitochondrial membrane integrity as reflected by elevated levels of polar head groups of phospholipids; (3) cellular bioenergetics as evidenced by changes in numerous metabolites associated with interrelated pathways of energy metabolism. Taken together, this work provides for the first time a comprehensive system level understanding of toxicity mechanism of PET NPs exposure in intact larvae.
Grouping of chemicals into mode of action classes by automated effect pattern analysis using the zebrafish embryo toxicity test
Teixidó et al. March 07, 2022 Reproductive Toxicology, Open Access, Volume 96, pages 1353–1369, Published: 07 March 2022
View AbstractGrouping of chemicals into mode of action classes by automated effect pattern analysis using the zebrafish embryo toxicity test
Morphometric analysis of developing zebrafish embryos allow predicting teratogenicity modes of action in higher vertebrates
Jarque et al. August 19, 2020 Reproductive Toxicology 96 (2020) 337-348; https://doi.org/10.1016/j.reprotox.2020.08.004
Morphometric analysis of developing zebrafish embryos allow predicting teratogenicity modes of action in higher vertebrates
An automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos’
Jarque et al. August 29, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0203087 August 29, 2018
An automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos’
Semi-automated detection of goitrogenic compounds using transgenic zebrafish embryos and the VAST BioImager platform
SETAC Europe 25th Annual Meeting
Sergio Jarque¹, Eva Fetter², Marek Pípal¹, Marie Smutná¹, Ludek Blaha¹, Stefan Scholz².
May 03, 2015
Semi-automated detection of goitrogenic compounds using transgenic zebrafish embryos and the VAST BioImager platform
1RECETOX, Masaryk University, Faculty of Science, Kamenice 753/5, 625 00, Brno
2Department of Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany
Robotic injection of zebrafish embryos for high-throughput screening in disease models
Herman P. Spainka, Chao Cuib, Malgorzata I. Wiwegera, b, Hans J. Jansenb, Wouter J. Venemana, Rubén Marín-Juezb, Jan de Sonnevillec, Anita Ordasa, Vincenzo Torracaa, Wietske van der Enta, William P. Leendersd, Annemarie H. Meijer,a, B. Ewa Snaar-Jagalskaa, Ron P. Dirksb, August 15, 2013 Volume 62, Issue 3, 15 August 2013, Methods 62: 246–254
View AbstractRobotic injection of zebrafish embryos for high-throughput screening in disease models
a Department of Molecular Cell Biology, Institute of Biology, Leiden University, The Netherlands, b ZF-screens B.V., Leiden, The Netherlands, c Life Science Methods B.V., Leiden, The Netherlands, d Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands