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International audience; 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 ≈ 90 μ m×60 μ 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.

Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as ‘ciliopathies.’ Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.

In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods., This work was supported by Programme Investissements d’Avenir IHU FOReSIGHT (ANR-18-IAHU-01) (A.C. and F.D.B.), INSERM cross-cutting program HuDeCA 2018 (A.C.), NIH R01OD011116 (I.B.), and UQ Amplify Fellowship (R.S.)

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 appropriatein vivotools. 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 ≈ 90μm×60μm 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 transportin vivoin 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.

Danionella translucida (DT) are cyprinid fish with the smallest known vertebrate brain, a small transparent body, and a partially open cranium. They are also very closely related to the more popular genetic model zebrafish. This makes the adaptability of established genetic tools for transgenesis and mutagenesis easier. These features, along with a diverse behavioral repertoire, make them an interesting model organism in neuroscience to investigate complex behaviors. In addition to neuroscience, they can also be a valuable model organism in developmental and evolutionary biology, and for modeling adult-onset diseases. Here, we describe the biology and ecology of this fish with an emphasis on the research questions that are made more accessible with this new fish model. We also report the husbandry and breeding practices used in our laboratory, which can make it easier for other laboratories to house this fish species.

International audience; Dysregulated transforming growth factor TGF-beta signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-beta-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-beta signaling, ipo8(-/-) zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8(-/-) zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-beta signaling during development and reinforces the existing link between TGF-beta signaling and connective tissue defects.

International audience; Reelin, a glycoprotein of the extracellular matrix, has been the focus of several studies over the years, mostly for its role in cell migration. Here we report the role of this molecule and of its downstream pathways in post-mitotic neurons and how they contribute to neural circuit assembly, refinement and function. Accumulating evidence has pointed at a major role for Reelin in axonal guidance, synaptogenesis and dendritic spine formation. In particular new evidence points at a direct role in axonal targeting and refinement at the target site. In addition, recent advances highlight new functions of Reelin in the modulation of synaptic activity, plasticity and behavior and it directly regulates GABA receptors expression and stability. We discuss these findings in the context of neurodevelopmental disorders. Highlights  Reelin signaling is required for axonal targeting and refinement at the target site.  Reelin signaling regulates dendritogenesis and spine formation in mature neurons.  Reelin modulates GABA receptors expression and stability, pre-and-post-synaptically.  Reelin is important for hippocampal integrity, synaptic plasticity and behavior.

International audience; 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 by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H2O2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation.

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.

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 protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including 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.

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 a 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 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.

β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs. In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters. In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs. Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated by the increased RBC enucleation rate and the reduced apoptosis and oxidative stress. We then compared BE strategies that either disrupt the LRF BS or create a de novo KLF1 BS in single colonies derived from erythroid progenitors. Generation of the KLF1 BS was associated with higher levels of HbF compared to the LRF BS disruption. These results suggest that eviction of the LRF repressor is sufficient to reactivate HBG genes, but recruitment of an activator is more effective to achieve high levels of gene expression. HbF expression induced by both LRF BS disruption and KLF1 BS generation was sufficient to rescue the SCD cell phenotype, but higher HbF levels - achieved only through KLF1 BS generation - were necessary to fully correct the β-thalassemia phenotype. In the majority of cases, we detected no DSB-induced insertions, deletions, or large genomic rearrangements in base-edited samples. Accordingly, DSB-induced DNA damage response (DDR) was absent in base-edited HSPCs, as measured by evaluating the expression of p21, a readout of p53-induced DDR. DNA off-target activity was assessed by GUIDE-seq and targeted sequencing of the potential off-target sites in edited HSPCs, while RNA off-target activity was evaluated by RNA-seq in HSPCs. Finally, BE-treated HSPCs were transplanted in immunodeficient mice to evaluate the engraftment and differentiation capability of edited HSCs. We detected good frequencies of human cells with up to ~60% of edited promoters in the peripheral blood of transplanted mice. In conclusion, we developed a clinically-relevant strategy to perform efficient BE in the HBG promoters that led to therapeutically-relevant HbF levels and rescued both the SCD and β-thalassemia phenotypes, thus providing sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies. Disclosures El Nemer: Hemanext: Consultancy.

Atkins et al. identify a new role for Fidgetin-like 1 in motor axon navigation via its regulation of bidirectional axonal transport. They show that Fidgetin-like 1 binds Kif1bβ and the opposed polarity-directed motor dynein/dynactin in a molecular complex and controls circuit wiring by reducing dynein velocity in developing motor axons., Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are 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 biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes. ispartof: DEVELOPMENTAL CELL vol:48 issue:4 pages:573-+ ispartof: location:United States status: published

Throughout vertebrates, cerebrospinal fluid-contacting neurons (CSF-cNs) are ciliated cells surrounding the central canal in the ventral spinal cord. Their contribution to modulate locomotion remains undetermined. Recently, we have shown CSF-cNs modulate locomotion by directly projecting onto the locomotor central pattern generators (CPGs), but the sensory modality these cells convey to spinal circuits and their relevance to innate locomotion remain elusive. Here, we demonstrate in vivo that CSF-cNs form an intraspinal mechanosensory organ that detects spinal bending. By performing calcium imaging in moving animals, we show that CSF-cNs respond to both passive and active bending of the spinal cord. In mutants for the channel Pkd2l1, CSF-cNs lose their response to bending and animals show a selective reduction of tail beat frequency, confirming the central role of this feedback loop for optimizing locomotion. Altogether, our study reveals that CSF-cNs constitute a mechanosensory organ operating during locomotion to modulate spinal CPGs., CSF-contacting neurons are known to project to locomotor CPGs although their relevance to active locomotion is unclear. Here, the authors show that these cells constitute a mechanosensory organ relying on PKD2L1 channels to detect spinal cord curvature and modulate locomotor frequency in freely moving animals.

Escape behaviors deliver organisms away from imminent catastrophe. Here, we characterize behavioral responses of freely swimming larval zebrafish to looming visual stimuli simulating predators. We report that the visual system alone can recruit lateralized, rapid escape motor programs, similar to those elicited by mechanosensory modalities. Two-photon calcium imaging of retino-recipient midbrain regions isolated the optic tectum as an important center processing looming stimuli, with ensemble activity encoding the critical image size determining escape latency. Furthermore, we describe activity in retinal ganglion cell terminals and superficial inhibitory interneurons in the tectum during looming and propose a model for how temporal dynamics in tectal periventricular neurons might arise from computations between these two fundamental constituents. Finally, laser ablations of hindbrain circuitry confirmed that visual and mechanosensory modalities share the same premotor output network. Together, we establish a circuit for the processing of aversive stimuli in the context of an innate visual behavior.

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF). Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in >70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance. Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels. Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo. To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C>T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C>T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation. This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies. Disclosures Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure., ファイル公開：2020-11-15

SummaryReactive 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 studiedin vivoduring 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 identify9-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 of9-HSA failed to differentiate and remained proliferative. We found that9-HSA exerts its biological functionin vivoby inhibiting the activity of histone deacetylase 1. We finally show that the local and temporal manipulation of H2O2levels bycatalaseoverexpression in RPCs was sufficient to trigger their premature differentiation. Therefore the amount of H2O2in RPCs is instructive of their ability to switch from proliferation to differentiation. We propose a mechanism that acts in RPC and linking H2O2homeostasis and neuronal differentiationviathe modulation of lipid peroxidation.

Motor proteins are responsible for transport of vesicles and organelles within the cell cytoplasm. They interact with the actin cytoskeleton and with microtubules to ensure communication and supply throughout the cell. Much work has been done in vitro and in silico to unravel the key players, including the dynein motor complex, the kinesin and myosin superfamilies, and their interacting regulatory complexes, but there is a clear need for in vivo data as recent evidence suggests previous models might not recapitulate physiological conditions. The zebrafish embryo provides an excellent system to study these processes in intact animals due to the ease of genetic manipulation and the optical transparency allowing live imaging. We present here the advantages of the zebrafish embryo as a system to study live in vivo processive transport in neurons and provide technical recommendations for successful analysis.

SUMMARYExtracellular 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 anin vivomodel 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 EVsin 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.Highlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin- In the YSL, syntenin regulates exosome release into the blood for their propagation- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells- Uptake is scavenger receptor and dynamin-dependent and provides trophic supportBlurbWe 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.

Understanding how distributed neuronal circuits integrate sensory information and generate behavior is a central goal of neuroscience. However, it has been difficult to study neuronal networks at single-cell resolution across the entire adult brain in vertebrates because of their size and opacity. We address this challenge here by introducing the fish Danionella translucida to neuroscience as a potential model organism. This teleost remains small and transparent even in adulthood, when neural circuits and behavior have matured. Despite having the smallest known adult vertebrate brain, D. translucida displays a rich set of complex behaviors, including courtship, shoaling, schooling, and acoustic communication. In order to carry out optical measurements and perturbations of neural activity with genetically encoded tools, we established CRISPR-Cas9 genome editing and Tol2 transgenesis techniques. These features make D. translucida a promising model organism for the study of adult vertebrate brain function at single-cell resolution.

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

The zebrafish (Danio rerio) has emerged in recent years as a powerful vertebrate model to study neuronal circuit development and function, thanks to its relatively small size, rapid external development and translucency. These features allow the easy application of in vivo microscopy analysis and optical perturbation of neuronal function. So far, genetic manipulation in zebrafish has been limited to the generation of constitutive loss-of-function alleles and transgenic models. CRISPR/Cas9 offers unprecedented possibilities for genomic manipulation that can be exploited to study neuronal function. In the past few years, we have successfully used CRISPR/Cas9-based technology in zebrafish to achieve two goals crucial for neuronal circuit analysis by developing two CRISPR/Cas9-based approaches that overcome previous major limitations to the study of gene and neuron functions in zebrafish. The study of gene function via tissue- or cell-specific mutagenesis remains challenging in zebrafish when the study of the function of certain loci might require tight spatiotemporal control of gene inactivation, which is particularly true in studying the function of a particular gene in post mitotic neurons, when the same gene may have had an earlier developmental function. To circumvent this limitation, we developed a simple and versatile protocol to achieve tissue-specific and temporally controlled gene disruption based on Cas9 expression under the control of the Gal4/UAS binary system (Di Donato et al. 2016). This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells and to follow them in vivo via reporter gene expression. We have also been able to target endogenous genomic loci to specifically label the great variety of neuronal cell types with reporter genes such as the transcriptional activator Gal4 (Auer et al. 2014). As a result, we can specifically target the expression of fluorescent proteins, a genetically encoded calcium indicator or optogenetic actuators in defined neuronal subpopulations.