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Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis -regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals. The DANIO-CODE consortium leverages a large-scale multiomic dataset to improve zebrafish genome annotation. They identify ~140,000 cis -regulatory elements throughout development and perform a comparison with the mouse regulatory landscape.

Background Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. Results We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. Conclusions Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.

Here it is shown that epithelia extrude live but not dying cells at sites of high strain, elucidating a mechanism for maintaining homeostatic cell numbers. Crowd control in epithelia For an epithelial-cell layer to retain its structure and provide a protective barrier, it needs to maintain a balance between the number of cells dividing and the number dying. Buzz Baum and colleagues study this process in Drosophila tissues and demonstrate a direct link between physical forces in a tissue and the rates of cell loss. In regions of tissue that are overcrowded, some of the cells undergo a loss of cell-adhesive junctions and are squeezed out by neighbouring cells. This process of live-cell delamination buffers epithelial cells against variations in growth and contributes to normal tissue homeostasis. As a link between epithelial hyperplasia and cell invasion, it may have relevance to the early stages of cancer development. In a second paper, Jody Rosenblatt and colleagues study epithelial-cell monolayers and find that epithelia extrude live but not dying cells at sites of high strain. The extruded cells undergo cell death owing to loss of survival factors. Hence, extrusion could provide a tumour-suppressive mechanism that could be used to eliminate excess cells. In carcinomas with high levels of survival signalling pathways, extrusion may promote tumour-cell invasion. For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells 1 , 2 , 3 , it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier 4 . Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out 4 , 5 , 6 . However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro . Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

How fish larvae are protected from infection before the maturation of adaptive immunity, a process which may take up to several weeks in most species, has long been a matter of speculation. Using a germ-free model, we show that colonization by commensals in newly hatched zebrafish primes neutrophils and induces several genes encoding proinflammatory and antiviral mediators, increasing the resistance of larvae to viral infection. Commensal microbe recognition was found to be mediated mainly through a TLR/MyD88 signaling pathway, and professional phagocytes were identified as the source of these immune mediators. However, the induction of proinflammatory and antiviral genes, but not of antimicrobial effector genes, also required the covalent modification of histone H3 at gene promoters. Interestingly, chromatin modifications were not altered by commensal microbes or hatching. Taken together, our results demonstrate that gene-specific chromatin modifications are associated with the protection of zebrafish larvae against infectious agents before adaptive immunity has developed and prevent pathologies associated with excessive inflammation during development.

Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss-and gain-of-function experiments show that L-opsin expression requires trftë activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.

The transcriptional effector SMAD4 is a core component of the TGF-β family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-β family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-β family ligands, which has implications for diseases where Smad4 is mutated or deleted. The role of the transcriptional effector SMAD4 in vertebrate embryo development remains unresolved. Here the authors show that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling, while Nodal signaling is maintained, but insufficient for optimal endoderm specification.

The protein heterodimer calprotectin and its component proteins, S100A8 and S100A9, play important antibacterial and pro-inflammatory roles in the mammalian innate immune response. Gaining mechanistic insights into the regulation and biological function of calprotectin will help facilitate patient diagnostics and therapy for inflammation and further our understanding of the host-microbe interface. Recent literature has identified zebrafish s100a10b as zebrafish calprotectin based on sequence similarity, genomic context, and transcriptional upregulation during the immune response to bacterial infections. The field would benefit from expanding the breadth of calprotectin studies into a zebrafish innate immunity model. Here, we carefully evaluated the possibility that zebrafish possess a calprotectin ortholog or a paralog that convergently evolved similar function. Using careful bioinformatics approaches, we found that zebrafish do not have an ortholog of either mammalian S100A8 or S100A9. To look for paralogs with convergent function, we identified four zebrafish s100 proteins—including s100a10b—that are expressed in immune cells and upregulated during the immune response. We recombinantly expressed and purified these proteins and measured their antimicrobial activity. None of the zebrafish proteins exhibited activity comparable to mammalian calprotectin. We also generated structural models of all homodimers and heterodimers of all annotated zebrafish s100 genes. None of these complexes were predicted to have an antimicrobial transition metal binding site equivalent to calprotectin. Finally, we measured the ability of our four purified zebrafish s100 proteins to activate inflammation via Toll-like receptor 4, a key feature of human S100A9; none of the proteins activated the receptor. Our work demonstrates conclusively that zebrafish have no ortholog of calprotectin and suggests that similar proteins have not convergently evolved analogous functions.

Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent miRNA biogenesis pathway that uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in bhlago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2 catalytic activity in vivo. mutants showed delayed erythropoiesis that could be rescued by wild-type Ago2 or miR-451-duplex but not by catalytically dead Ago2. Changing the secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA function and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs independently of Dicer.

Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization.