Biology subjects

Gagnon, J. A.

Publications and source records attributed to Gagnon, J. A..

18 recordsLinked to original sources

bioRxiv2026

Gadusol in epidermal mucus protects larval zebrafish from UVB-induced DNA damage

Ultraviolet B (UVB) radiation causes DNA damage and poses a threat to organisms exposed to sunlight. Zebrafish embryos are protected from UVB by maternally deposited gadusol, a small-molecule sunscreen, but it has been unclear how protection is maintained as maternal gadusol is depleted during larval development. Here, we show that gadusol synthesis in the developing zebrafish skin is required for UVB defense throughout larval development. Maternal gadusol persists through early larval stages, but juvenile fish depend solely on zygotic gadusol for protection against DNA damage and death after UVB exposure. Melanin provides a more modest contribution to survival. Gadusol is present in the epidermal mucus layer at the body's outermost surface, positioning it to absorb UVB before it reaches underlying cells. Depletion of epidermal mucocytes increases UVB-induced DNA damage and activates a DNA damage response. Together, these findings identify a maternal-to-zygotic handoff in gadusol-based photoprotection and support a model in which gadusol in surface mucus intercepts UVB before it reaches living tissues.

developmental biology↗

bioRxiv2026

Grazing protists foster zebrafish virus transmission

Virus transmission is facilitated by diverse means, ranging from particles hitchhiking on dust and other fomites to vector-borne replication. Aquatic ecosystems are rich with interactions between species at various trophic levels, but how these interactions influence virus transmission and infection biology is largely unknown. By mixing zebrafish picornavirus 1 (ZfPV-1) with a panel of freshwater microbial eukaryotes, we found that phagocytic cells promote virus infections in larval zebrafish, even though the virus is unable to replicate in these microbes. Ciliates, which act as crucial concentrators of micronutrients via continuous grazing behavior, stood out as promoters of robust and accelerated virus infections. We found that simply adding the model ciliate Tetrahymena thermophila to mixed populations of infected and uninfected zebrafish facilitated chains of experimental ZfPV-1 transmission between fish. This process depends on Tetrahymena phagocytosis and is likely due to the ability of the ciliates to deliver a concentrated bolus of viruses when consumed by larval fish. Notably, the pathogenic snakehead rhabdovirus was not transmitted after mixing with Tetrahymena, revealing selectivity, potentially through coevolution with ZfPV-1, that is not evident in all RNA fish viruses. We propose that non-host ciliates can act as virus vessels that increase virus transmission between hosts while potentially altering courses of infection by boosting zebrafish immunity. Virus vessels fill a crucial space on the continuum of virus transmission dynamics for viruses lacking the ability to replicate in intermediate species. Given the tools available to manipulate these genetically accessible systems, this paradigm opens new avenues for investigating otherwise hidden features of infection biology with experiments better approximating natural complexity in the wild.

evolutionary biology↗

bioRxiv2026

Clonal dynamics deviate from neutral drift in zebrafish spermatogenesis

Spermatogonial stem cells (SSCs) maintain male fertility, but how their clonal dynamics change with age remains poorly understood. Here, we use in vivo CRISPR barcoding in zebrafish to label SSCs and track their contributions to sperm production through monthly sampling across the fertile lifespan. We find that only a subset of embryonic germ cells contributes to adult sperm production and that the relative contributions of individual clones shift dramatically over time. To interpret these dynamics, we developed a mathematical model that quantifies clonal drift rates and formally tests neutral versus non-neutral hypotheses. Most SSC clones show significant evidence of drift, yet their dynamics are inconsistent with a neutral model where clones have equal fitness and their dynamics are driven solely by random chance. Instead, we observe heterogeneous clonal dynamics, including a positive relationship between clone size and drift rate. Together, these findings demonstrate that SSC clonal dynamics are non-neutral across the zebrafish reproductive lifespan with important implications for allele transmission.

developmental biology↗

bioRxiv2026

PGE2 modulation of stimulus valence directs zebrafish behavioral fever

Animals seek warmth during fever, but whether this behavior reflects altered temperature perception or interpretation remains unresolved. A prevailing view holds that fever reduces perceived temperature, making the environment feel colder. Here, we use behavior, modeling, and calcium imaging in larval zebrafish to test these hypotheses. We find that viral immune signals drive larval zebrafish to seek warmer temperatures in a PGE2-dependent manner. We localized PGE2 receptors to thermoregulatory brain regions that are conserved across vertebrates. Through comparative behavioral modeling we show that contrary to the prevailing view, fever does not reduce the perception of warmth. Instead, PGE2 alters the valence of temperature changes, such that heating becomes attractive while cooling turns aversive. Calcium imaging reveals that modulation of medullary activity by immune signals directs these changes in stimulus valence. Together, these results identify PGE2-driven changes in stimulus valence as a key principle of the sensorimotor basis of fever.

neuroscience↗

bioRxiv2026

A fast-to-faithful transition shapes the DNA repair landscape during embryogenesis

Accurate and timely repair of DNA double-strand breaks (DSBs) is essential for genome maintenance in all cells. Embryos are particularly vulnerable to DSBs. During zebrafish development, a single fertilized cell undergoes rapid divisions to form an embryo of 50,000 cells in the first 24 hours, subjecting its genome to intense replication stress and the inevitable formation of genomic DSBs. While we know that failure to repair these breaks can result in embryonic lethality, the kinetics, fidelity, and pathway choice of DSB repair during embryogenesis are not well understood. Here, we used light-activated CRISPR to generate targeted genomic DSBs across zebrafish embryo development. Importantly, DSB induction occurs within seconds after light stimulation, enabling precise measurements of repair kinetics within a single cell cycle. We found that DSBs were repaired within 15 minutes during the early, rapid-division stages. At later stages, the pace of DNA repair declines as the cell cycle slows. By leveraging mathematical modeling and mutants that disrupt DNA repair pathways, we uncovered a developmental transition from error-prone microhomology-mediated end joining to more faithful non-homologous end joining that correlates with the gradual shift in repair kinetics. To our knowledge, this is the first study to resolve DSB repair dynamics with high temporal resolution during embryo development. Our study establishes a framework for systematically interrogating the cellular responses to DNA damage in living model organisms. SIGNIFICANCE STATEMENTThis study reveals how developing embryos rapidly repair dangerous DNA breaks while balancing speed and accuracy. Using a light-activated CRISPR system, the authors precisely control when DNA damage occurs and measure repair in real time in living zebrafish embryos. They show that early embryos prioritize extremely fast but error-prone repair, then transition to slower, more accurate mechanisms as development progresses. This "fast-to-faithful" shift explains how embryos protect their genomes during rapid cell division. The work provides a new framework for studying DNA repair in vivo and has implications for understanding developmental disorders, genome editing outcomes, and how cells manage DNA damage under changing biological conditions.

genetics↗

bioRxiv2025

Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury

Heart injury triggers a robust cellular response in zebrafish, characterized by neovascularization, immune cell activation, and the infiltration of proliferative cardiomyocytes that collectively lead to scarless regeneration. Upon injury, damage-associated molecular patterns are released by dying cells and injured extracellular matrix. These molecules bind to pattern recognition receptors on various cell types, promoting inflammation and immune cell recruitment by upregulating chemokines and pro-inflammatory cytokines. We previously identified the activation of injury-induced interferon signaling as a distinguishing feature between the regenerative zebrafish and non-regenerative medaka. Here, we establish interferon-{Phi}1 (IFN{square}1) as the primary driver of interferon signaling after zebrafish heart injury. IFN{square}1 expression is induced hours after injury and directs interferon-stimulated gene expression, which peaks at 72 hours post-injury. This response is lost in ifnphi1 mutants, which disrupt IFN{square}1 expression. ifnphi1 mutants have reduced neutrophil recruitment to the injured myocardium, while macrophages and neovascularization are unaffected. By studying later stages of regeneration, we find that ifnphi1 mutant hearts have a modest defect in scar resolution. Collectively, these findings uncover a critical early signaling cascade during the inflammatory response to heart injury and provide new insights into the mechanisms that choreograph zebrafish heart regeneration.

developmental biology↗

bioRxiv2025

MIC-Drop-seq: Scalable single-cell phenotyping of mutant vertebrate embryos

Advances in genome engineering and single-cell RNA sequencing (scRNAseq) have revolutionized the ability to precisely map gene functions, yet scaling these techniques for large-scale genetic screens in animals remains challenging. We combined high-throughput gene disruption in zebrafish embryos via Multiplexed Intermixed CRISPR Droplets with phenotyping by multiplexed scRNAseq (MIC-Drop-seq). In one MIC-Drop-seq experiment, we intermixed and injected droplets targeting 50 transcriptional regulators into 1,000 zebrafish embryos, followed by pooled scRNAseq. Tissue-specific gene expression and cell abundance analysis of demultiplexed mutant cells recapitulated many known phenotypes, while also uncovering novel functions in brain and mesoderm development. We observed pervasive cell-extrinsic effects among these phenotypes, highlighting how whole-embryo sequencing captures complex developmental interactions. Thus, MIC-Drop-seq provides a powerful and scalable platform for mapping gene functions in vertebrate development with cellular resolution.

genomics↗

bioRxiv2023

Germ cells do not progress through spermatogenesis in the infertile zebrafish testis

Vertebrate spermatogonial stem cells maintain sperm production over the lifetime of an animal but fertility declines with age. While morphological studies have greatly informed our understanding of typical spermatogenesis, the molecular and cellular mechanisms underlying spermatogenesis are not yet understood, particularly with respect to the onset of fertility. We used single-cell RNA sequencing to generate a developmental atlas of the zebrafish testis. Using 5 timepoints across the adult life of a zebrafish, we described cellular profiles in the testis during and after fertility. While all germ cell stages of spermatogenesis are detected in testes from fertile adult zebrafish, testes from older infertile males only contained spermatogonia and a reduced population of spermatocytes. These remaining germ cells are transcriptionally distinct from fertile spermatogonia. Immune cells including macrophages and lymphocytes drastically increase in abundance in infertile testes. Our developmental atlas reveals the cellular changes as the testis ages and defines a molecular roadmap for the regulation of male fertility.

developmental biology↗

bioRxiv2023

Microbe transmission from pet shop to lab-reared zebrafish reveals a pathogenic birnavirus

Zebrafish are popular research organisms selected for laboratory use due in part to widespread availability from the pet trade. Many contemporary colonies of laboratory zebrafish are maintained in aquaculture facilities that monitor and aim to curb infections that can negatively affect colony health and confound experiments. The impact of laboratory control on the microbial constituents associated with zebrafish in research environments compared to the pet trade are unclear. Diseases of unknown causes are common in both environments. We conducted a metagenomic survey to broadly compare the zebrafish-associated microbes in pet trade and laboratory environments. We detected many microbes in animals from the pet trade that were not found in laboratory animals. Co-housing experiments revealed several transmissible microbes including a newly described non-enveloped, double-stranded RNA virus in the Birnaviridae family we name Rocky Mountain birnavirus (RMBV). Infections were detected in asymptomatic animals from the pet trade, but when transmitted to laboratory animals RMBV was associated with pronounced antiviral responses and hemorrhagic disease. These experiments highlight the pet trade as a distinct source of diverse microbes that associate with zebrafish and establish a paradigm for the discovery of newly described pathogenic viruses and other infectious microbes that can be developed for study in the laboratory.

microbiology↗

bioRxiv2023

Distinct features of the regenerating heart uncovered through comparative single-cell profiling

Adult humans respond to heart injury by forming a permanent scar, yet other vertebrates are capable of robust and complete cardiac regeneration. Despite progress towards characterizing the mechanisms of cardiac regeneration in fish and amphibians, the large evolutionary gulf between mammals and regenerating vertebrates complicates deciphering which cellular and molecular features truly enable regeneration. To better define these features, we compared cardiac injury responses in zebrafish and medaka, two fish species that share similar heart anatomy and common teleost ancestry but differ in regenerative capability. We used single-cell transcriptional profiling to create a time-resolved comparative cell atlas of injury responses in all major cardiac cell types across both species. With this approach, we identified several key features that distinguish cardiac injury response in the non-regenerating medaka heart. By comparing immune responses to injury, we found altered cell recruitment and a distinct pro-inflammatory gene program in medaka leukocytes, and an absence of the injury-induced interferon response seen in zebrafish. In addition, we found a lack of pro-regenerative signals, including nrg1 and retinoic acid, from medaka endothelial and epicardial cells. Finally, we identified alterations in the myocardial structure in medaka, where they lack embryonic-like primordial layer cardiomyocytes, and fail to employ a cardioprotective gene program shared by regenerating vertebrates. Our findings reveal notable variation in injury response across nearly all major cardiac cell types in zebrafish and medaka, demonstrating how evolutionary divergence influences the hidden cellular features underpinning regenerative potential in these seemingly similar vertebrates.

developmental biology↗

bioRxiv2023

Gadusol is a maternally provided sunscreen that protects fish embryos from DNA damage

Ultraviolet radiation (UVR) and its deleterious effects on living cells selects for UVR-protective mechanisms. Organisms across the tree of life evolved a variety of natural sunscreens to prevent UVR-induced cellular damage and stress. However, in vertebrates, only melanin is known to act as a sunscreen. Here we demonstrate that gadusol, a transparent compound discovered over 40 years ago in fish eggs, is a maternally provided sunscreen required for survival of embryonic and larval zebrafish exposed to UVR. Mutating an enzyme involved in gadusol biosynthesis increases the formation of cyclobutane pyrimidine dimers, a hallmark of UVB-induced DNA damage. Compared to the contributions of melanin and the chorion, gadusol is the primary sunscreening mechanism in embryonic and larval fish. The gadusol biosynthetic pathway is retained in the vast majority of teleost genomes but is repeatedly lost in species whose young are no longer exposed to UVR. Our data demonstrate that gadusol is a maternally provided sunscreen that is critical for early-life survival in the most species-rich branch of the vertebrate phylogeny.

developmental biology↗

bioRxiv2021

Functional and ultrastructural analysis of reafferent mechanosensation in larval zebrafish

All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animals proximity, which need to be distinguished from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body. In this study, we characterize in detail how the hair cells, which are the receptor cells of the lateral line, discriminate between such reafferent and exafferent signals in zebrafish larvae. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies that cancel out self-generated reafferent stimulation during locomotion, and that dopaminergic signals from the hypothalamus may have a role in threshold modulation both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts. We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.

neuroscience↗

bioRxiv2020

Orthogonal CRISPR-Cas genome editing and efficient inhibition with anti-CRISPRs in zebrafish embryos

The CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is the most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1), are orthogonal systems capable of operating simultaneously in zebrafish. CRISPR systems from Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) were also active in embryos. We implemented multichannel CRISPR recording using three CRISPR systems and show that LbaCas12a may provide superior information density compared to previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. Our results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.

genetics↗

bioRxiv2019

Discovery of a prevalent picornavirus by visualizing zebrafish immune responses

The discovery of new viruses currently outpaces our capacity for experimental examination of infection biology. To better couple virus discovery with immunology, we genetically modified zebrafish to visually report on virus infections. After generating a strain that expresses green fluorescent protein (GFP) under an interferon-stimulated gene promoter, we repeatedly observed transgenic larvae spontaneously expressing GFP days after hatching. RNA sequencing of GFP-positive zebrafish revealed a picornavirus distantly related to known viruses. We detected this virus in publicly available sequencing data from seemingly asymptomatic zebrafish in research institutes world-wide. These data revealed widespread tissue tropism and identified the clonal CG2 strain as notably vulnerable to picornavirus infection. Infection induces the expression of numerous antiviral immune defense genes in zebrafish, an effect also evident in public data. Our study describes a prevalent picornavirus that infects zebrafish, and provides new strategies for simultaneously discovering viruses and their impact on vertebrate hosts.

immunology↗

bioRxiv2019

Zebrafish Dscaml1 is Essential for Retinal Patterning and Function of Oculomotor Subcircuits

Down Syndrome Cell Adhesion Molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the role of dscaml1 in the zebrafish oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Loss of zebrafish dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses showed that mutants have abnormal gaze stabilization, impaired fixation, disconjugation, and faster fatigue. Notably, the saccade and fatigue phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, for which no animal model exists. Two-photon calcium imaging showed that loss of dscaml1 leads to impairment in the saccadic premotor pathway but not the pretectum-vestibular premotor pathway, indicating a subcircuit requirement for dscaml1. Together, we show that dscaml1 has both broad and specific roles in oculomotor circuit function, providing a new animal model to investigate the development of premotor pathways and their associated human ocular disorders.

neuroscience↗

bioRxiv2018

Accurate analysis of genuine CRISPR editing events with ampliCan

We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, off-target read detection and quantifies insertions, deletions, HDR repair as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated datasets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.

bioinformatics↗

bioRxiv2017

Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain by scGESTALT

Hundreds of cell types are generated during development, but their lineage relationships are largely elusive. Here we report a technology, scGESTALT, which combines cell type identification by single-cell RNA sequencing with lineage recording by cumulative barcode editing. We sequenced ~60,000 transcriptomes from the juvenile zebrafish brain and identified more than 100 cell types and marker genes. We engineered an inducible system that combines early and late barcode editing and isolated thousands of single-cell transcriptomes and their associated barcodes. The large diversity of edited barcodes and cell types enabled the generation of lineage trees with hundreds of branches. Inspection of lineage trajectories identified restrictions at the level of cell types and brain regions and helped uncover gene expression cascades during differentiation. These results establish scGESTALT as a new and widely applicable tool to simultaneously characterize the molecular identities and lineage histories of thousands of cells during development and disease.

developmental biology↗

bioRxiv2016

Zebrafish Nanog is not required in embryonic cells

SUMMARY STATEMENTThe study of nanog mutants reveals that Nanog is required only for extraembryonic tissue development, not in embryonic cells.\n\nABSTRACTThe role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the formation of the extraembryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. By contrast, a more recent study has proposed that Nanog directly regulates transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. While zygotic nanog mutants survive to adulthood, maternal-zygotic and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, the YSL fails to form and embryonic tissue detaches from the yolk. Zygotic transcription of a subset of embryonic genes is affected in nanog mutants but both the YSL and embryonic phenotype can be rescued by providing nanog mRNA in YSL precursors. Notably, nanog mutant cells transplanted into wild-type hosts proliferate and contribute to embryonic tissues from all germ layers. These results indicate that zebrafish Nanog is necessary for YSL formation but is not directly required for embryonic cell differentiation.

developmental biology↗