Biology subjects

Levraud, J.-P.

Publications and source records attributed to Levraud, J.-P..

10 recordsLinked to original sources

bioRxiv2026

I. Phantom: a multiplex CRISPR-Cas9 gRNA toolkit for efficient generation of fully unpigmented F0 zebrafish

Zebrafish are an increasingly popular model organism, valued for their natural transparency during embryonic stages. However, imaging at later developmental stages is affected by several types of pigments distributed over the body surface. These pigments partially or completely absorb or reflect light, and some also exhibit fluorescence, thereby interfering with imaging. Chemical approaches, such as phenylthiourea (PTU), are available to address this problem. However, PTU only inhibits melanin production and can affect development, making its use controversial. Many mutant zebrafish lines lacking single pigments have already been described, and combinations of melanin and iridophore deficient lines such as the casper and crystal fish greatly improved in vivo imaging at late stages. However, breeding multiple recessive mutations with a desired reporter transgene is laborious, and xanthophores remain in these lines. Here, we address these limitations by developing and optimising a multiplex CRISPR-Cas9 gRNA set that simultaneously targets the three major pigment types, while minimising mortality. This approach generates a new phenotype, termed phantom, characterised by the complete absence of visible pigmentation directly in F0 zebrafish larvae, while retaining the ability to survive to adulthood and reproduce.

molecular biology↗

bioRxiv2026

Pdgfrβ signaling orchestrates meningeal repair via the mobilization of arachnoid cells.

Zebrafish possess remarkable regeneration abilities, including the capacity to repair their central nervous system (CNS). Leveraging the optical accessibility of the zebrafish brain, we investigated the mechanisms underlying meningeal repair following laser-induced brain injuries to the optic tectum. In previous work, using live imaging of laser-induced surface injuries to the optic tectum in juvenile zebrafish, we identified a population of flat PDGFR{beta}+ cells that rapidly migrate to the wound site and contribute to meningeal repair. Here, using pharmacological inhibition or genetic perturbation of PDGFR{beta}, we show that recruitment of these PDGFR{beta}+ meningeal cells is strongly dependent on PDGFR{beta} signaling, unlike recruitment of PDGFR{beta}+ pericytes deeper in the wound. Furthermore, PDGFR{beta} inhibition diminishes neurite regrowth and macrophage recruitment. Using photoconversion assays, we traced the origin of PDGFR{beta}+ meningeal cells that migrated to the wound in response to injury, in the midbrain-forebrain and midbrain-hindbrain sulci. Our findings highlight PDGFR{beta}s pivotal role in orchestrating meningeal repair and reveal novel cellular dynamics during CNS regeneration. These results provide insights into potential therapeutic strategies for enhancing brain repair and mitigating fibrosis in mammals, where meningeal scarring remains a barrier to CNS regeneration. HighlightsPDGFR{beta} signaling orchestrates the rapid accumulation of meningeal cells at CNS injury sites to drive tissue remodeling. PDGFR activity acts as a critical regulator, co-recruiting PDGFR{beta}+ meningeal cells and mfap4+ macrophages to the lesion. Meningeal cells are essential for neural repair, with PDGFR inhibition leading to significantly reduced neurite density. Zebrafish maintain locomotor resilience post-injury, identifying a clear distinction between meningeal-driven axonal regrowth and basic motor circuitry recovery. Establishes a high-resolution zebrafish platform to identify conserved meningeal targets for mammalian CNS regeneration.

cell biology↗

bioRxiv2025

The two groups of zebrafish type I interferons target different tissues, paralleling the mammalian type I: type III IFN functional division

Interferons (IFNs) are ancient cytokines that arose in jawed vertebrates [~]400-500 million years ago. IFN systems are present with conserved antiviral functions across vertebrate lineages, including zebrafish (Danio rerio). In mammals, antiviral IFNs are divided between type I interferons (IFN-I), which drive systemic responses, and type III interferons (IFN-III), which protect barrier mucosal epithelia, owing to the specific distribution of their respective receptors. Although zebrafish lack IFN-III, they have IFN-Is which subdivide into 2 groups with distinct receptors, providing a unique opportunity to study how antiviral immunity has evolved in the absence of IFN-III. Whilst previous work has suggested complementary, non-redundant roles for IFNs from these groups, the tissue specificity has not yet been resolved. As larvae, zebrafish only express one group 1 (IFN{varphi}1) and one group 2 IFN (IFN{varphi}3). Using viral infection assays and reporter transgenics, we found that IFNs from group 1 (IFN{varphi}1) and group 2 (IFN{varphi}3) are produced by distinct subsets of cells, with no detectable co-expression. To assess tissue and cell-type-specific responses to these two IFNs, we used ISG reporter fish imaging and whole-larva single cell RNA sequencing after injection of recombinant IFN{varphi}1 and IFN{varphi}3. Despite a similar core ISG response, distinct downstream ISG programs across multiple tissues and organ systems were found. In particular, barrier epithelial cells, such as enterocytes, responded more strongly to IFN{varphi}1, while myeloid cells responded more strongly to IFN{varphi}3. Our results indicate that zebrafish IFN-I families have functionally diversified their antiviral immune responses by tissue context, driven by cellular partitioning of both IFN-I production and response. These results mirror the division of labour between mammalian IFN-I and IFN-III, emphasising the evolutionary importance of tissue division of immune responses, as well as deepening our understanding of the zebrafish as a model for host-pathogen interactions.

immunology↗

bioRxiv2025

Interleukin-22 in enteroendocrine cells controls early-life gut motility through interactions with the microbiota

The gut microbiota, immune system, and enteric nervous system tightly interact to regulate adult gut physiology. Yet the mechanisms establishing gut physiology during development remain unknown. Here, we report that in developing zebrafish, enteroendocrine cells produce IL-22 in response to microbial signals before lymphocytes populate the gut. In larvae, IL-22 is crucial to set gut microbiota composition, and ghrelin hormone expression to promote gut motility. IL-22 developmental function depends on its ability to modulate gut microbiota, as bacterial transfer from wild-type zebrafish restored gut motility in il22-/- by reestablishing ghrelin hormone expression. Additionally, IL-22-deficient mice show impaired gut motility and reduced ghrelin expression in early life, indicating a conserved function. Altogether, we identify a circuit where enteroendocrine cells regulate gut function via cytokine control of the microbiota, showing how gut physiology is set prior to immune system maturation.

immunology↗

bioRxiv2024

Spatial dynamics of peripheral and central nervous system infection by an interferon-inducing neuroinvasive virus

Organ-to-organ dissemination of viruses is a critical feature of host-virus interactions. In particular, neuroinvasive viruses are able to enter the central nervous systems (CNS), which may result in death or permanent neurological impairment. The complex mechanisms underpinning this spread are poorly understood, as they depend on a variety of parameters, including initial site of entry, route of access to the CNS, and immune responses. To better understand these phenomena, we analyzed the spatial dynamics of Sindbis virus (SINV) dissemination in transparent zebrafish larvae. Using fluorescent reporter viruses, we observed that SINV readily invaded the CNS after inoculation at various peripheral sites. From tail muscle, the virus used dorsal root ganglia (DRG) sensory neurons as a gateway to the spinal cord and further propagation to the brain. While peripheral infection was systematically transient, due to the key protective role of the strong and rapid type I interferon (IFN) response, CNS infection was persistent and more variable. Within the CNS, viral dissemination resulted both from long-distance axonal transport and short distance shedding, and IFN response was local, while it was systemic in the periphery. A mathematical model was built on this quantitative imaging foundation, that provided additional insight on the parameters of this infection, such as the rate of new virion production, estimated around 1 to 2 infective virions per productively infected cell per hour; the occurrences of CNS entry events, which was 2 to 3 per larva; or the impact of the IFN response, which did not only prevent new infections but accelerated the death of infected cells.

microbiology↗

bioRxiv2021

Exploring zebrafish larvae as a COVID-19 model: probable SARS-COV-2 replication in the swim bladder

Animal models are essential to understand COVID-19 pathophysiology and for pre-clinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, bloodstream, or yolk, did not result in detectable SARS-CoV-2 replication in wild-type larvae. However, when the virus was inoculated in the swim bladder, a modest increase in viral RNA was observed after 24 hours, suggesting a successful infection in some animals. This was confirmed by immunohistochemistry, with cells positive for SARS-CoV-2 nucleoprotein observed in the swim bladder. Several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. Mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.

microbiology↗

bioRxiv2021

A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus.

Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases.

cell biology↗

bioRxiv2020

A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2

The COVID-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with SARS-CoV-2 and allow rapid and high throughput testing of drugs and vaccines. Exposure of larvae to SARS-CoV-2 Spike (S) receptor binding domain (RBD) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ACE inhibitor, reverted this effect. Intranasal administration of SARS-CoV-2 S RBD in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. Zebrafish intranasally treated with SARS-CoV-2 S RBD became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. Single cell RNA-Seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. Combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of SARS-CoV-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model.

immunology↗

bioRxiv2019

Interferon-stimulated genes in zebrafish and human define an ancient arsenal of antiviral immunity

The evolution of the interferon (IFN) system, the major innate antiviral mechanism of vertebrates, remains poorly understood. According to the detection of type I IFN genes in cartilaginous fish genomes, the system appeared 500My ago. However, the IFN system integrates many other components, most of which are encoded by IFN-stimulated genes (ISGs). To shed light on its evolution, we have used deep RNA sequencing to generate a comprehensive list of ISGs of zebrafish, taking advantage of the high quality genome annotation in this species. We analyzed larvae after inoculation of recombinant zebrafish type I IFN, or infection with chikungunya virus, a potent IFN inducer. We identified more than 400 zebrafish ISGs, defined as being either directly induced by IFN or induced by the virus in an IFN receptor-dependent manner. Their human orthologues were highly enriched in ISGs, particularly for highly-inducible genes. We identified 72 orthology groups containing ISGs in both zebrafish and human, revealing a core ancestral ISG repertoire, which includes most of the known signaling components of the IFN system. Many downstream effectors were also already present 450 My ago in the common ancestor of tetrapods and bony fish, and diversified as multi-gene families independently in the two lineages. A large proportion of the ISG repertoire is lineage-specific; around 40% of protein-coding zebrafish ISGs had no human orthologue. We identified 14 fish-specific gene families containing multiple ISGs, including finTRIMs. This work illuminates the evolution of the IFN system and provides a rich resource to explore new antiviral mechanisms.\n\nKey pointsO_LIWe established an exhaustive list of larval zebrafish ISGs.\nC_LIO_LIOrthologous ISGs in fish and human identify a large ancestral ISG repertoire.\nC_LI

immunology↗

bioRxiv2018

Single-cell transcriptional analysis reveals naïve helper ILC-like cells in zebrafish

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates is poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and following immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient fish which lack T and B cells, and discovered diverse populations of helper ILC-like cells. Unexpectedly, fish displayed a rorc-positive, naive subset that established a Type 3 or Type 2 response only upon immune challenge. Specifically, naive ILC-like cells expressed il22 and tnfa following exposure to inactivated bacteria, or il13 following exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.

immunology↗