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opencc-zeroOct 2021View details →

A 3D Adult Zebrafish Brain Atlas (AZBA) for the Digital Age

Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online (azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

opencc-zeroDec 2022View details →

Data for: Impact of alpha crystallin loss on zebrafish lens development

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three a-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated aBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

opencc-zeroJan 2023View details →

Drugs prescribed in Phelan-McDermid Syndrome differentially impact sensory behaviors in shank3 zebrafish models

Altered sensory processing is a pervasive symptom in individuals with Autism Spectrum Disorders (ASD). Especially in cases of profound autism, individuals are often medicated to manage behaviors like aggression and/or self-harm and/or epilepsy, and it remains unclear how these medications might impact perception/sensory processing. To test this, we screened three medications, risperidone, Lithium Chloride (LiCl), and carbamazepine (CBZ), prescribed to individuals with Phelan-McDermid Syndrome (PMS) and one drug, 2-Methyl-6-(phenylethynyl) pyridine (MPEP) tested in rodent models of PMS, for their effects on a sensory-induced behavior in two zebrafish PMS models with frameshift mutations in the N- or C- termini. PMS is caused by deletions of the terminal end of chromosome 22 or point mutations in Shank3. People with PMS can present with an array of symptoms including ASD, epilepsy, gastrointestinal distress, and reduced responses to sensory stimuli. Our zebrafish mutant shank3ab models likewise show reduced responses in a Visual Motor Response (VMR) assay, in which increased locomotion is triggered by light-to-dark transitions. To test how pharmacological treatments affect the VMR, we exposed larvae to selected drugs for twenty-four hours and then quantified their locomotion during four ten-minute cycles of lights on-to-off stimuli. We found that risperidone normalized the VMR in shank3 models, LiCl and CBZ had no effect on the VMR in any of the three genotypes. MPEP reduced the VMR in WT to levels seen in shank3 models but caused no changes in either shank3 model. Our work shows that the effects of drugs on sensory processing is varied in a way that can be highly genotype- and drug-dependent.

opencc-zeroJan 2023View details →

Data for: Inhibition drives habituation of a larval zebrafish visual response

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). Using Ca2+ imaging during repeated dark flashes, we identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt, or that potentiated their response. To identify molecular players, we used a small molecule-screening approach to search for compounds that alter habituation learning. Among the pathways we identified were Melatonin and Estrogen signaling, as well as GABAergic inhibition. By analyzing which functional classes of neurons are GABAergic, and the result of pharmacological manipulations of the circuit, we propose that GABAergic inhibitory motifs drive habituation, perhaps through the potentiation of GABAergic synapses. Our results have identified multiple molecular pathways and cell types underlying a form of long-term plasticity in a vertebrate brain, and allow us to propose the first iteration of a model for how and where this learning process occurs.

opencc-zeroJan 2023View details →

Amacrine cells differentially balance zebrafish colour circuits in the central and peripheral retina

In vertebrate vision, the feature-extracting circuits of the inner retina are driven by photoreceptors whose outputs are already pre-processed. In zebrafish, for example, outer retinal circuits split "colour" from "greyscale" information across all four cone-photoreceptor types. How does the inner retina process this incoming spectral information while also combining cone signals to shape new greyscale functions? We address this question by imaging the light-driven responses of amacrine cells (ACs) and bipolar cells (BCs) in larval zebrafish, in the presence and pharmacological absence of inner retinal inhibition. We find that amacrine cells exert distinct effects on greyscale processing depending on retinal region, as well as contributing to the generation of colour opponency in the central retina. However, in the peripheral retina amacrine cells enhanced opponency in some bipolar cells while at the same time suppressing pre-existing opponency in others, such that the net change in the number of colour-opponent units was essentially zero. To achieve this 'dynamic balance' ACs counteracted intrinsic colour opponency of BCs via the On-channel. Consistent with these observations, Off-stratifying ACs were exclusively achromatic, while all colour opponent ACs stratified in the On-sublamina. This study reveals that the central and peripheral retina of larval zebrafish employ fundamentally distinct inhibitory circuits to control the interaction between greyscale- and colour-processing. Differential actions on the On- and Off-channels control the transmission of colour-opponent signals in the periphery.

opencc-zeroMay 2023View details →

Research data of Env38 protein functions in SVCV infection in zebrafish

Endogenous retroviruses (ERVs) are the relics of ancient retroviruses occupying a substantial fraction of vertebrate genomes. However, knowledge about the functional association of ERVs with cellular activities remains limited. Recently, we have identified approximately 3,315 ERVs from zebrafish at genome-wide level, among which 421 ERVs were actively expressed in response to the infection of Spring viraemia of carp virus (SVCV). These findings demonstrated the previously unrecognized activity of ERVs in zebrafish immunity, thereby making zebrafish an attractive model organism for deciphering the interplay among ERVs, exogenous invading viruses, and host immunity. In the present study, we investigated the functional role of an envelope protein (Env38) derived from an ERV-E5.1.38-DanRer element in zebrafish adaptive immunity against SVCV in view of its strong responsiveness to SVCV infection. This Env38 is a glycosylated membrane protein mainly distributed on MHC-II+ antigen-presenting cells (APCs). By performing blockade and knockdown/knockout assays, we found that the deficiency of Env38 markedly impaired the activation of SVCV-induced CD4+ T cells and thereby led to the inhibition of IgM+/IgZ+ B cell proliferation, IgM/IgZ Ab production, and zebrafish defense against SVCV challenge. Mechanistically, Env38 activates CD4+ T cells by promoting the formation of pMHC-TCR-CD4 complex via cross-linking MHC-II and CD4 molecules between APCs and CD4+ T cells, wherein the surface subunit (SU) of Env38 associates with the second immunoglobin domain of CD4 (CD4-D2) and the first α1 domain of MHC-IIα (MHC-IIα1). Notably, the expression and functionality of Env38 were strongly induced by zebrafish IFNφ1, indicating that env38 acts as an IFN-stimulating gene (ISG) regulated by IFN signaling. To the best of our knowledge, this study is the first to identify the involvement of an Env protein in host immune defense against an exogenous invading virus by promoting the initial activation of adaptive humoral immunity. It improved the current understanding of the cooperation between ERVs and host adaptive immunity.

opencc-zeroFeb 2023View details →

Data from: Whole-body variational modularity in the zebrafish: An inside-out story of a model species

Actinopterygians are the most diversified clade of extant vertebrates. Their impressive morphological disparity bears witness to tremendous ecological diversity. Modularity, the organization of biological systems into quasi-independent anatomical/morphological units, is thought to increase evolvability of organisms and facilitate morphological diversification. Our study aims to quantify patterns of variational modularity in a model actinopterygian, the zebrafish (Danio rerio), using 3D-geometric morphometrics on osteological structures isolated from micro-CT scans. 72 landmarks were digitised along cranial and postcranial ossified regions of 30 adult zebrafishes. Two methods were used to test modularity hypotheses, the covariance ratio and the distance matrix approach. We find strong support for two modules, one comprised of paired fins and the other comprised of median fins, that are best explained by functional properties of subcarangiform swimming. While the skull is tightly integrated with the rest of the body, its intrinsic integration is relatively weak supporting previous findings that the fish skull is a modular structure. Our results provide additional support for the recognition of similar hypotheses of modularity identified based on external morphology in various teleosts, and at least two variational modules are proposed. Thus, our results hint at the possibility that internal and external modularity patterns may be congruent.

opencc-zeroMar 2023View details →

Behavior and brain size of larval zebrafish exposed to environmentally relevant concentrations of betamethylamino- L-alanine (BMAA)

Harmful algal blooms (HABs) release toxic compounds in water and are increasing in frequency worldwide. The neurotoxin β-methylamino-L-alanine (BMAA) is released by HABs and has garnered much attention over the past twenty years due to its association with human neurodegenerative disorders, but its effects on wildlife are still largely unknown. This study characterized the effects of chronic exposure to environmentally relevant concentrations of BMAA on the behavior and brain size of developing zebrafish (Danio rerio). Zebrafish were continuously exposed to 0, 1, 10, or 100 μg/L waterborne BMAA between 0- and 5-days post-fertilization (dpf) before the onset of exogenous feeding. At 5 dpf, locomotion and responses to vibrational and visual stimuli were assessed. Following behavioral testing, larvae body and brain size were measured. Survival between 0 and 5 dpf did not differ between treatments. Moreover, BMAA exposure did not affect thigmotaxis, startle response magnitude, habituation to repeated presentation of vibrational startling stimuli, or relative brain size. A moderate increase in overall activity was observed in larvae exposed to 10 ug/L BMAA under light, but this effect was not seen in dark conditions, indicating that visual processing may have been affected by chronic BMAA exposure. Thus, our results show that passive continuous exposure to environmentally relevant concentrations of BMAA prior to first feeding in zebrafish does not affect overall brain development, locomotion, anxiety, and motor neuron-mediated reflexes, but suggest targeted neurotoxicity within the visual system.

opencc-zeroMar 2023View details →

Data from: The contribution of mutation to variation in temperature-dependent sprint speed in zebrafish, Danio rerio

The contribution of new mutations to phenotypic variation, and the consequences of this variation for individual fitness, are fundamental concepts for understanding genetic variation and adaptation. Here, we investigated how mutation influenced variation in a complex trait in zebrafish, Danio rerio. Typical of many ecologically relevant traits in ectotherms, swimming speed in fish is temperature-dependent, with evidence of adaptive evolution of thermal performance. We chemically induced novel germline point mutations in males and measured sprint speed in their sons at six temperatures (between 16°C and 34°C). Heterozygous mutational effects on speed were strongly positively correlated among temperatures, resulting in statistical support for only a single axis of mutational variation, reflecting temperature-independent variation in speed (faster-slower mode). These results suggest pleiotropic effects on speed across different temperatures, however, spurious correlations arise via linkage, or heterogeneity in mutation number when mutations have consistent directional effects on each trait. Here, mutation did not change mean speed, indicating no directional bias in mutational effects. The results contribute to emerging evidence that mutations may predominantly have synergistic cross-environment effects, in contrast to conditionally neutral or antagonistic effects which underpin thermal adaptation. We discuss several aspects of experimental design that may affect resolution of mutations with non-synergistic effects.

opencc-zeroMar 2023View details →

Fasting increases investment in soma upon refeeding at the cost of gamete quality in zebrafish

Fasting increases lifespan in invertebrates, improves biomarkers of health in vertebrates, and is increasingly proposed as a promising route to improve human health. Nevertheless, little is known about how fasted animals use resources upon refeeding, and how such decisions affect putative trade-offs between somatic growth and repair, reproduction, and gamete quality. Such fasting-induced trade-offs are based on strong theoretical foundations and have been recently discovered in invertebrates, but the data on vertebrates is lacking. Here we report that fasted female zebrafish, Danio rerio, increase investment in soma upon refeeding, but it comes at a cost of egg quality. Specifically, an increase in fin re-growth was accompanied by a reduction in 24-hours post-fertilization offspring survival. Refed males showed a reduction in sperm velocity and impaired 24-hour post-fertilisation offspring survival. These findings underscore the necessity of considering the impact on eggs and sperm when assessing evolutionary and biomedical implications of lifespan-extending treatments in females and males and call for careful evaluation of the effects of fasting (both during and post) on fertilisation.

Crosstalk assessment for multi-colour immunofluorescence of Histone 3 and Polymerase II post-translational modifications in pluripotent zebrafish embryos

Microscopy images recorded to assess the extent of crosstalk from the detection channels of H3K27ac and recruited RNA polymerase II (Serine 5 phosphorylation of the C-terminal domain heptad repeat of subunit 1) to the detection channel of elongating RNA polymerase II (Serine 5 phosphorylation of the C-terminal domain heptad repeat of subunit 1). Sample preparation and image recording was carried out jointly by Süheyla Eroğlu-Kayikci, Elisa Kämmer, and Lennart Hilbert.

Label-free adaptive optics single-molecule localization microscopy for whole zebrafish

The specimen-induced aberration has been a major factor limiting the imaging depth of single-molecule localization microscopy (SMLM). Here, we report the application of label-free wavefront sensing adaptive optics to SMLM for deep-tissue super-resolution imaging. The proposed system measures complex tissue aberrations from intrinsic reflectance rather than fluorescence emission and physically corrects the wavefront distortion more than three-fold stronger than the previous limit. This enables us to resolve sub-diffraction morphologies of cilia and oligodendrocytes in whole zebrafish as well as dendritic spines in thick mouse brain tissues at the depth of up to 102 μm with localization number enhancement by up to 37 times and localization precision comparable to aberration-free samples. The proposed approach can expand the application range of SMLM to whole zebrafish that cause the loss of localization points owing to severe tissue aberrations.

Zebrafish with lyz:EGFP expressing neutrophils: Mesh Well Inserts Z-stack 3

96-well plate z-stack of zebrafish with lyz:EGFP expressing neutrophils acquired with a multi-camera array microscope (MCAM)(Ramona Optics Inc., Durham, NC, USA). Mesh well inserts are used and half of the zebrafish on the plate were injected with csf3r morpholino. The overall z-stack is broken into four files.   HDF5 files can be opened using open source Python software: https://docs.xarray.dev/

Zebrafish with lyz:EGFP expressing neutrophils: Mesh Well Inserts Z-stack 2

Zebrafish with lyz:EGFP expressing neutrophils: csf3r_MO injected stack

96-well plate z-stack of zebrafish with lyz:EGFP expressing neutrophils acquired with a multi-camera array microscope (MCAM)(Ramona Optics Inc., Durham, NC, USA). Zebrafish larvae have been injected with csf3r morpholino.   HDF5 files can be opened using open source Python software: https://docs.xarray.dev/

Zebrafish WT: Z-stack

96-well plate z-stack of wild type zebrafish acquired with a multi-camera array microscope (MCAM)(Ramona Optics Inc., Durham, NC, USA).   HDF5 files can be opened using open source Python software: https://docs.xarray.dev/

opencc-by-4.0Jul 2023View details →

Zebrafish with lyz:EGFP expressing neutrophils: Dibutyl phthalate Stack

96-well plate z-stack of zebrafish with lyz:EGFP expressing neutrophils acquired with a multi-camera array microscope (MCAM)(Ramona Optics Inc., Durham, NC, USA). Fish were treated with Dibutyl pthalate or DMSO prior to imaging.   HDF5 files can be opened using open source Python software: https://docs.xarray.dev/

Zebrafish with lyz:EGFP expressing neutrophils: Mesh Well Inserts Z-stack 1

Zebrafish with lyz:EGFP expressing neutrophils: Non-injected stack

96-well plate z-stack of zebrafish with lyz:EGFP expressing neutrophils acquired with a multi-camera array microscope (MCAM)(Ramona Optics Inc., Durham, NC, USA).   HDF5 files can be opened using open source Python software: https://docs.xarray.dev/