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During development, oligodendrocytes contact and wrap neuronal axons with myelin. Similarly to neurons and synapses, excess myelin sheaths are produced and selectively eliminated, but how elimination occurs is unknown. Microglia, the resident immune cells of the central nervous system, engulf surplus neurons and synapses. To determine whether microglia also prune myelin sheaths, we used zebrafish to visualize and manipulate interactions between microglia, oligodendrocytes, and neurons during development. We found that microglia closely associate with oligodendrocytes and specifically phagocytose myelin sheaths. By using a combination of optical, genetic, chemogenetic, and behavioral approaches, we reveal that neuronal activity bidirectionally balances microglial association with neuronal cell bodies and myelin phagocytosis in the optic tectum. Furthermore, multiple strategies to deplete microglia resulted in oligodendrocytes maintaining excessive and ectopic myelin. Our work reveals a neuronal activity-regulated role for microglia in modifying developmental myelin targeting by oligodendrocytes.Microglia refine the developing CNS by engulfing excess neurons and synapses. Hughes and Appel here show that microglia also prune myelin sheaths in a neuronal activity-regulated manner to sculpt developmental myelination.

Migrasomes are recently identified vesicular organelles that form on retraction fibres behind migrating cells. Whether migrasomes are present in vivo and, if so, the function of migrasomes in living organisms is unknown. Here, we show that migrasomes are formed during zebrafish gastrulation and signalling molecules, such as chemokines, are enriched in migrasomes. We further demonstrate that Tspan4 and Tspan7 are required for migrasome formation. Organ morphogenesis is impaired in zebrafish MZ tspan4a and MZ tspan7 mutants. Mechanistically, migrasomes are enriched on a cavity underneath the embryonic shield where they serve as chemoattractants to ensure the correct positioning of dorsal forerunner cells vegetally next to the embryonic shield, thereby affecting organ morphogenesis. Our study shows that migrasomes are signalling organelles that provide specific biochemical information to coordinate organ morphogenesis. Yu and colleagues report the formation of migrasomes during zebrafish gastrulation. Migrasomes provide signalling molecules to guide the migration of dorsal forerunner cells, thus controlling organ morphogenesis.

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4 CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4 CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity. Zebrafish p63 isoforms were identified as thalidomide-dependent neosubstrates of the cereblon-containing E3 ligase complex. ∆Np63α and TAp63α are responsible for thalidomide-induced malformations of pectoral fins and otic vesicles, respectively.

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable “base editing” system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5′-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish. The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity.

Epithelial tissues require the removal and replacement of damaged cells to sustain a functional barrier. Dying cells provide instructive cues that can influence surrounding cells to proliferate, but how these signals are transmitted to their healthy neighbors to control cellular behaviors during tissue homeostasis remains poorly understood. Here we show that dying stem cells facilitate communication with adjacent stem cells by caspase-dependent production of Wnt8a-containing apoptotic bodies to drive cellular turnover in living epithelia. Basal stem cells engulf apoptotic bodies, activate Wnt signaling, and are stimulated to divide to maintain tissue-wide cell numbers. Inhibition of either cell death or Wnt signaling eliminated the apoptosis-induced cell division, while overexpression of Wnt8a signaling combined with induced cell death led to an expansion of the stem cell population. We conclude that ingestion of apoptotic bodies represents a regulatory mechanism linking death and division to maintain overall stem cell numbers and epithelial tissue homeostasis. Damaged epithelial tissues are known to compensate for cell death through compensatory cell divisions to maintain epithelial integrity. Here, the authors show in living epithelia that dying cells stimulate adjacent stem cells to divide through caspase-dependent production of Wnt8a-containing apoptotic bodies.

How diverse cell fates and complex forms emerge and feed back to each other to sculpt functional organs remains unclear. In the developing heart, the myocardium transitions from a simple epithelium to an intricate tissue that consists of distinct layers: the outer compact and inner trabecular layers. Defects in this process, which is known as cardiac trabeculation, cause cardiomyopathies and embryonic lethality, yet how tissue symmetry is broken to specify trabecular cardiomyocytes is unknown. Here we show that local tension heterogeneity drives organ-scale patterning and cell-fate decisions during cardiac trabeculation in zebrafish. Proliferation-induced cellular crowding at the tissue scale triggers tension heterogeneity among cardiomyocytes of the compact layer and drives those with higher contractility to delaminate and seed the trabecular layer. Experimentally, increasing crowding within the compact layer cardiomyocytes augments delamination, whereas decreasing it abrogates delamination. Using genetic mosaics in trabeculation-deficient zebrafish models—that is, in the absence of critical upstream signals such as Nrg–Erbb2 or blood flow—we find that inducing actomyosin contractility rescues cardiomyocyte delamination and is sufficient to drive cardiomyocyte fate specification, as assessed by Notch reporter expression in compact layer cardiomyocytes. Furthermore, Notch signalling perturbs the actomyosin machinery in cardiomyocytes to restrict excessive delamination, thereby preserving the architecture of the myocardial wall. Thus, tissue-scale forces converge on local cellular mechanics to generate complex forms and modulate cell-fate choices, and these multiscale regulatory interactions ensure robust self-organized organ patterning. Differences in the mechanical properties of individual cardiomyocytes drive their segregation into compact versus trabecular layer, thereby transforming the myocardium in a developing heart from a simple epithelium into an intricately patterned tissue with distinct cell fates.

The larval zebrafish ( Danio rerio ) is an excellent vertebrate model for in vivo imaging of biological phenomena at subcellular, cellular and systems levels. However, the optical accessibility of highly pigmented tissues, like the eyes, is limited even in this animal model. Typical strategies to improve the transparency of zebrafish larvae require the use of either highly toxic chemical compounds (e.g. 1-phenyl-2-thiourea, PTU) or pigmentation mutant strains (e.g. casper mutant). To date none of these strategies produce normally behaving larvae that are transparent in both the body and the eyes. Here we present crystal , an optically clear zebrafish mutant obtained by combining different viable mutations affecting skin pigmentation. Compared to the previously described combinatorial mutant casper , the crystal mutant lacks pigmentation also in the retinal pigment epithelium, therefore enabling optical access to the eyes. Unlike PTU-treated animals, crystal larvae are able to perform visually guided behaviours, such as the optomotor response, as efficiently as wild type larvae. To validate the in vivo application of crystal larvae, we performed whole-brain light-sheet imaging and two-photon calcium imaging of neural activity in the retina. In conclusion, this novel combinatorial pigmentation mutant represents an ideal vertebrate tool for completely unobstructed structural and functional in vivo investigations of biological processes, particularly when imaging tissues inside or between the eyes.

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase ( glul ) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis. Cox et al.  report that Yap induces the expression of glutamine synthetase, thereby elevating glutamine and nitrogen levels for de novo nucleotide synthesis. They show that this promotes hepatomegaly and growth of liver cancer cells in zebrafish.

Cardiac regeneration Zebrafish are able to efficiently regenerate lost cardiac muscle, and is used as a model to understand why natural heart regeneration is blocked in mammals. Two groups reporting in the issue of Nature used genetic fate-mapping approaches to identify which population of cardiomyocytes contribute prominently to cardiac muscle regeneration after an injury approximating myocardial infarction. They show that cardiac muscle regenerates through activation and expansion of existing cardiomyocytes, and does not involve activation of a stem cell population. Zebrafish are able to replace lost heart muscle efficiently, and are used as a model to understand why natural heart regeneration — after a heart attack, for instance — is blocked in mammals. Here, and in an accompanying paper, genetic fate-mapping approaches reveal which cell population contributes prominently to cardiac muscle regeneration after an injury approximating myocardial infarction. The results show that cardiac muscle regenerates through activation and expansion of existing cardiomyocytes, without involving a stem-cell population. Although mammalian hearts show almost no ability to regenerate, there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response. In contrast to mammals, several non-mammalian vertebrate species are able to regenerate their hearts 1 , 2 , 3 , including the zebrafish 4 , 5 , which can fully regenerate its heart after amputation of up to 20% of the ventricle. To address directly the source of newly formed cardiomyocytes during zebrafish heart regeneration, we first established a genetic strategy to trace the lineage of cardiomyocytes in the adult fish, on the basis of the Cre/ lox system widely used in the mouse 6 . Here we use this system to show that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes. Furthermore, we show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure, detachment from one another and the expression of regulators of cell-cycle progression. Specifically, we show that the gene product of polo-like kinase 1 ( plk1 ) is an essential component of cardiomyocyte proliferation during heart regeneration. Our data provide the first direct evidence for the source of proliferating cardiomyocytes during zebrafish heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process.

Cardiac regeneration Zebrafish are able to efficiently regenerate lost cardiac muscle, and is used as a model to understand why natural heart regeneration is blocked in mammals. Two groups reporting in the issue of Nature used genetic fate-mapping approaches to identify which population of cardiomyocytes contribute prominently to cardiac muscle regeneration after an injury approximating myocardial infarction. They show that cardiac muscle regenerates through activation and expansion of existing cardiomyocytes, and does not involve activation of a stem cell population. Zebrafish are able to replace lost heart muscle efficiently, and are used as a model to understand why natural heart regeneration — after a heart attack, for instance — is blocked in mammals. Here, and in an accompanying paper, genetic fate-mapping approaches reveal which cell population contributes prominently to cardiac muscle regeneration after an injury approximating myocardial infarction. The results show that cardiac muscle regenerates through activation and expansion of existing cardiomyocytes, without involving a stem-cell population. Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life 1 , 2 . Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction 3 . By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced 4 , 5 . In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart.