Biology subjects

Del Bene, F.

Publications and source records attributed to Del Bene, F..

17 recordsLinked to original sources

bioRxiv2026

Coupling of tubulin acetylation to microtubule stabilization through molecular mimicry

Microtubules support diverse cellular functions through regulation by microtubule-associated proteins and tubulin post-translational modification, yet how these two layers are mechanistically integrated remains unclear. -tubulin acetylation marks mechanically resilient microtubules, and its incorporation in defined microtubule sub-populations is not well understood. Here, we identify MTCL1 as a molecular link between microtubule stabilization and post-translational modification installation. We find that MTCL1 stabilizes microtubules and alters the luminal surface when copolymerized with tubulin, remodeling -tubulin and enhancing TAT-mediated tubulin acetylation through molecular mimicry. This effect depends on assembly history and is not observed in pre-assembled microtubules. Targeted deletion of MTCL1 in zebrafish impacts axonal organization, leading to motor defects and increased seizure susceptibility. These findings establish MTCL1 as a licensing factor that couples microtubule stabilization with acetylation to regulate neuronal function.

biophysics↗

bioRxiv2026

Repressed mTORC1 signaling and transient dendritic pruning support axonal regeneration

Dendrite degeneration is an early pathological feature following axonal damage, yet its role during successful axonal regeneration remains poorly understood. Using sparse labeling of retinal ganglion cells in adult zebrafish, we show that axonal regeneration is accompanied by a transient phase of dendritic pruning following optic nerve crush. Dendritic pruning occurs during axon elongation and is reversed after brain reinnervation. Although insulin-mTOR signaling is rapidly upregulated immediately after injury, it is subsequently suppressed, coinciding with dendritic pruning and sustained axonal growth. Prolonging mTOR activation through insulin treatment or direct pathway stimulation reduces dendritic pruning in an mTORC1-dependent manner but severely delays axonal regrowth. Together, these findings identify dendritic remodeling as a key temporally regulated component of successful axonal regeneration and reveal a functional trade-off between dendritic preservation and axon regrowth. This balance may represent a critical constraint for promoting CNS repair in mammals.

neuroscience↗

bioRxiv2026

Error-free and efficient prime editing in absence of maternal Polθ

Prime editing is a versatile genome-editing technology developed to make small edits while minimising the unwanted mutations associated with approaches based on double-strand breaks. In zebrafish embryos, however, prime editing is highly error-prone, typically yielding more unwanted mutations than precise edits. Here, we show that most unwanted mutations are deletions flanked by microhomologies and insertions templated from neighbouring sequences, both hallmarks of microhomology-mediated end-joining of double-strand breaks. These breaks are unlikely to result from direct cleavage of both genomic strands by the prime editor. Instead, we propose that the rapid cell divisions of early zebrafish embryos promote the conversion of nicks into double-strand breaks by replication forks. Loss of maternally deposited DNA polymerase {theta} (Pol{theta}), the core factor in microhomology-mediated end-joining, abolishes unwanted mutations and can increase edit rates above 50%, resulting in error-free and efficient prime editing. Our work identifies the source of unwanted prime-editing mutations in zebrafish and establishes a practical strategy to achieve high edit rates and near-perfect precision.

genetics↗

bioRxiv2025

Lrrns define a visual circuit underlying brightness and contrast perception

Brightness and contrast are fundamental features of vision, crucial for object detection, environmental navigation, and feeding. Here, we identify a brightness-and contrast-processing circuit in the zebrafish visual system and uncover the role of Leucine-rich repeat neuronal (Lrrn) cell adhesion molecules (CAMs) in regulating its assembly. Deep-projecting retinal ganglion cells (RGCs) serve as the first synaptic relay to the brain requiring Lrrn2 and Lrrn3a for precise axonal targeting and connectivity within the optic tectum. Genetic targeting of these CAMs leads to circuit disorganization and impairments in contrast sensitivity, leading to deficits in visually guided behaviors. Additionally, ultrastructural circuit reconstruction and functional imaging analysis revealed their critical role in luminance processing. These studies define a fundamental visual processing pathway and establish Lrrn CAMs as essential molecular drivers of its assembly.

neuroscience↗

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

Two-photon voltage imaging with rhodopsin-based sensors

The recent advances in sophisticated optical techniques, coupled with two-photon sensitive genetic voltage indicators (GEVIs), have enabled in-depth voltage imaging in vivo at single spike and single-cell resolution. To date, these results have been only achieved using ASAP-type sensors, as the complex photocycle of rhodopsin-based voltage indicators posed challenges for their two-photon use, restricting their application to one-photon approaches. In this work, we demonstrate that rhodopsin-based GEVIs (FRET-opsin) can be used under two-photon illumination when their peculiar light intensity dependence of kinetics and sensitivity are considered. We rationally engineer a fully genetically-encoded, rhodopsin-based voltage indicator with the brightest known fluorophore AaFP1, Jarvis, and demonstrate its utility under both one- and two-photon illumination. We also showed two-photon usability of the similar FRET-opsin sensor pAce. Our comparison of 2P scanless with fast 2P scanning illumination revealed that the latter approach is less suitable for this class of indicators and, on the contrary, both sensors responded well when scanless approaches were used. Furthermore, utilising Jarvis, we demonstrated high-fidelity, high-SNR action potential detection at kilohertz-imaging rates both in mouse hippocampal slices and in zebrafish larvae. To the best of our knowledge, this study represents the first report of a fully genetically-encoded rhodopsin-based voltage indicator for high contrast action potential detection under two-photon illumination in vitro and in vivo.

neuroscience↗

bioRxiv2023

Meteorins regulate the formation of the left-right organizer and the establishment of vertebrate body asymmetry

While the exterior of vertebrate bodies appears bilaterally symmetrical, internal organ positioning and morphology frequently exhibit left-right (L-R) asymmetries. In several vertebrates, including human, mouse, frog and zebrafish, left-right symmetry-breaking during embryonic development is initiated by a ciliated organ called the Node or left-right organizer. Within the Node, a leftward flow of extraembryonic fluid named the Nodal flow mediates the asymmetric expressions of Nodal factors. Although downstream Nodal pathway components leading to the establishment of the embryonic left-right axis are well known, less is known about the development and formation of the embryonic Node itself. Here we reveal a novel role for the Meteorin protein family in the establishment of the left-right axis and in the formation of the Kupffers vesicle, the Node equivalent structure in zebrafish. We show that the genetic inactivation of each or all three members of the zebrafish Meteorin family (metrn, metrn-like a and metrn-like b) leads to defects in properties of the Kupffers vesicle, caused by impaired assembly and migration of the Kupffers vesicle forming dorsal forerunner cells. In addition, we demonstrate that Meteorins genetically interact with integrins ItgaV and Itg{beta}1b regulating the dorsal forerunner cell clustering and that meteorins loss-of-function results in disturbed Nodal factor expression and consequently in randomized or symmetric heart looping and jogging. These results identify a new role for the Meteorin protein family in the left-right asymmetry patterning during embryonic vertebrate development.

developmental biology↗

bioRxiv2023

Tunneling nanotubes enable intercellular transfer in zebrafish embryos

Tunneling nanotubes (TNTs) are thin intercellular connections facilitating the transport of diverse cargoes, ranging from ions to organelles. While TNT studies have predominantly been conducted in cell cultures, the existence of open-ended TNTs within live organisms remains unverified. Despite the observation of intercellular connections during embryonic development across various species, their functional role has not been confirmed. In this study, we performed mosaic labeling of gastrula cells in zebrafish embryos to demonstrate the coexistence of TNT-like structures alongside other cellular protrusions. These embryonic TNT-like connections exhibited similar morphology to TNTs described in cell culture, appeared to have similar formation mechanisms and could be induced by Eps8 overexpression and CK666 treatment. Most notably, to classify them as TNTs, we demonstrated their capability to transfer both soluble cargoes and organelles, which is a defining feature of open-ended TNTs. This study marks the first demonstration of functional TNTs in a living embryo.

cell biology↗

bioRxiv2022

In-vivo fast non-linear microscopy reveals impairment of fast axonal transport induced by molecular motor imbalances in the brain of zebrafish larvae

Cargo transport by molecular motors along microtubules is essential for the function of eucaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and of the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of {approx} 90 mx60 m size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole, and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having non-linear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate, and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated to disease models.

neuroscience↗

bioRxiv2022

The Adaptor Protein 2 (AP2) complex modulates habituation and behavioral selection across multiple pathways and time windows

Animals constantly perceive and integrate information across sensory modalities, and their nervous systems must select behavioral responses appropriate to the current situation and prior experience. Genetic factors supporting this behavioral flexibility are often disrupted in neuropsychiatric conditions, and our previous work revealed the disease-associated ap2s1 gene critically supports habituation learning in acoustically-evoked escape behavior of zebrafish. ap2s1 encodes a subunit of the AP2 endocytosis adaptor complex and has been linked to autism spectrum disorder, though its mechanism and direct behavioral importance have not been established. Here, we show that multiple subunits of the AP2 complex regulate acoustically-evoked behavior selection and habituation learning. Furthermore, ap2s1 biases the choice between distinct escape behaviors in sensory modality-specific manners, and more broadly regulates action selection across different sensory contexts. Using tissue-specific and inducible transgenic rescue, we demonstrate that the AP2 complex functions acutely and in the nervous system to modulate acoustically-evoked habituation learning, suggesting several spatially and/or temporally distinct mechanisms through which AP2 regulates different aspects of escape behavior selection and performance. Altogether, we demonstrate that the AP2 complex coordinates action selection across stimulus modalities and contexts, providing a new vertebrate model for the role of ap2s1 in human conditions including autism spectrum disorder. SIGNIFICANCE STATEMENTThe AP2S1 gene has been linked to learning disabilities and autism spectrum disorders (ASD), though the mechanisms underlying its impact on human behavior are unknown. We explored how, when, and where this gene regulates vertebrate behavior, developing a zebrafish model to identify the roles and mechanisms through which ap2s1 modulates behavior. We find that ap2s1 regulates simple acoustically-evoked learning, as well as how individuals bias behavioral choice in a wide variety of contexts. We show that ap2s1 acts at multiple distinct time periods and locations both within and outside of neuronal tissues, revealing the diverse mechanisms and pathways through which it modulates vertebrate behavior.

neuroscience↗

bioRxiv2021

Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

Vesicle trafficking and the establishment of apico-basal polarity are essential processes in epithelium morphogenesis. Myosin-1b, an actin-motor able to bind membranes, regulates membrane shaping and vesicle trafficking. Here, we investigate Myosin-1b function in gut morphogenesis and congenital disorders using cell line and zebrafish larvae as well as patient biopsies. In a 3D Caco-2 cyst model, lumen formation is impaired in absence of Myosin-1b. In zebrafish, both Morpholino knock-down and genetic mutation of myo1b result in intestinal bulb epithelium folding defects associated with vesicle accumulation, reminiscent of a villous atrophy phenotype. We show that Myosin-1b interacts with the chaperone UNC45A, genetic deletion of which also results in gut folding defects in zebrafish. Loss of function mutations in UNC45A have been reported in complex hereditary syndromes, notably exhibiting intestinal disorders associated with villous atrophy. In UNC45A-depleted cells and in patient biopsies, Myosin-1b protein level is strikingly decreased. The appearance of Myosin-1b aggregates upon proteasome inhibition in cells points at a degradation mechanism of misfolded Myosin-1b in the absence of its chaperone. In conclusion, Myosin-1b plays an unexpected role in the development of the intestinal epithelium folds or villi downstream UNC45A, establishing its role in the gut defects reported in UNC45A patients. Summary statementMyosin-1b is important for intestinal epithelium folding during zebrafish development and participates in the villous atrophy clinical manifestation downstream UNC45A loss of function.

developmental biology↗

bioRxiv2021

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo.

Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing the NRN PAM sequence, we could introduce point mutations for the first time in an animal model and achieved up to 100% efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we applied our method to create a new zebrafish model for melanoma predisposition based on the simultaneous editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

developmental biology↗

bioRxiv2021

Evolutionary divergence of locomotion in two related vertebrate species

Locomotion exists in diverse forms in nature and is adapted to the environmental constraints of each species1. However, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. We established a powerful approach in comparative neuroethology to investigate evolution of neuronal circuits in vertebrates by comparing divergent swimming pattern of two closely related larval fish species, Danionella translucida (DT) and Danio rerio or zebrafish (ZF)2,3. During swimming, we demonstrate that DT utilizes lower half tail-beat frequency and amplitude to generate a slower and continuous swimming pattern when compared to the burst-and-glide swimming pattern in ZF. We found a high degree of conservation in the brain anatomy between the two species. However, we revealed that the activity of a higher motor region, referred here as the Mesencephalic Locomotion Maintenance Neurons (MLMN) correlates with the duration of swim events and differs strikingly between DT and ZF. Using holographic stimulation, we show that the activation of the MLMN is sufficient to increase the frequency and duration of swim events in ZF. Moreover, we propose two characteristics, availability of dissolved oxygen and timing of swim bladder inflation, which drive the observed differences in the swim pattern. Our findings uncover the neuronal circuit substrate underlying the evolutionary divergence of navigational strategies and how they are adapted to their respective environmental constraints.

neuroscience↗

bioRxiv2020

Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes among which cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

developmental biology↗

bioRxiv2020

Expression of a Barhl1a reporter in subsets of retinal ganglion cells and commissural neurons of the developing zebrafish brain

Promoting the regeneration or survival of retinal ganglion cells (RGCs) is one focus of regenerative medicine. Homeobox Barhl transcription factors might be instrumental in these processes. In mammals, only barhl2 is expressed in the retina and is required for both subtype identity acquisition of amacrine cells and for the survival of RGCs downstream of Atoh7, a transcription factor necessary for RGC genesis. The underlying mechanisms of this dual role of Barhl2 in mammals have remained elusive. Whole genome duplication in the teleost lineage generated the barhl1a and barhl2 paralogues. In the Zebrafish retina, Barhl2 functions as determinant of subsets of amacrine cells lineally related to RGCs independently of Atoh7. In contrast, barhl1a expression depends on Atoh7 but its expression dynamics and function have not been studied. Here we describe for the first time a Barhl1a:GFP reporter line in vivo showing that Barhl1a turns on exclusively in subsets of RGCs and their post-mitotic precursors. We also show transient expression of Barhl1a:GFP in diencephalic neurons extending their axonal projections as part of the post-optic commissure, at the time of optic chiasm formation. This work sets the ground for future studies on RGC subtype identity, axonal projections and genetic specification of Barhl1a-positive RGCs and commissural neurons.

developmental biology↗

bioRxiv2019

Redox signaling via lipid peroxidation regulates retinal progenitor cell differentiation

Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However their regulation and mechanism of action remain poorly studied in vivo during normal development. Here we reveal that the fine regulation of hydrogen peroxide (H2O2) levels at the degradation step by its scavenger Catalase is crucial to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs). We further show that altering the levels of downstream products of the Redox signaling can also affect this switch. Indeed, we identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. In fact, RPCs exposed to higher amounts of 9-HSA failed to differentiate and remained proliferative. We found that 9-HSA exerts its biological function in vivo by inhibiting the activity of histone deacetylase 1. We finally show that the local and temporal manipulation of H2O2 levels by catalase overexpression in RPCs was sufficient to trigger their premature differentiation. Therefore the amount of H2O2 in RPCs is instructive of their ability to switch from proliferation to differentiation. We propose a mechanism that acts in RPC and linking H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation.

developmental biology↗

bioRxiv2018

Live tracking of inter-organ communication by endogenous exosomes in vivo

Extracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed an in vivo model to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVs in vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.\n\nHighlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin\n- In the YSL, syntenin regulates exosome release into the blood for their propagation\n- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells\n- Uptake is scavenger receptor and dynamin-dependent and provides trophic support\n\n\nBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

cell biology↗