BackgroundStudies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening.ResultsHere we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.ConclusionsThis approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay.See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

SummaryWiskott–Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are immunodeficiencies in which the function of several haematopoietic cell lineages is perturbed as a result of mutations in the actin regulator WASp. From in vitro cell biology experiments, and biochemical and structural approaches, we know much about the functional domains of WASp and how WASp might regulate the dynamic actin cytoskeleton downstream of activators such as Cdc42, but in vivo experiments are much more challenging. In patients, there is a correlation between clinical disease and genotype, with severe reductions in WASp expression or function associating with complex multilineage immunodeficiency, whereas specific mutations that cause constitutive activation of WASp result in congenital neutropenia. Here, we take advantage of the genetic tractability and translucency of zebrafish larvae to first characterise how a null mutant in zfWASp influences the behaviour of neutrophils and macrophages in response to tissue damage and to clearance of infections. We then use this mutant background to study how leukocyte lineage-specific transgenic replacement with human WASp variants (including normal wild type and point mutations that either fail to bind Cdc42 or cannot be phosphorylated, and a constitutively active mutant equivalent to that seen in XLN patients) alter the capacity for generation of neutrophils, their chemotactic response to wounds and the phagocytic clearance capacity of macrophages. This model provides a unique insight into WASp-related immunodeficiency at both a cellular and whole organism level.

SUMMARYIn humans, skin is the largest organ and serves as a barrier between our body and the outside world. Skin protects our internal organs from external pathogens and other contaminants, and melanocytes within the skin protect the body from damage by ultraviolet light. These same pigment cells also determine our skin colour and complexion. Skin wounding triggers a repair response that includes a robust recruitment of inflammatory cells, which function to kill invading microbes and clear away cell and matrix debris. Once at the wound site, these innate immune cells release a barrage of cytokines that direct the activities of other cells during the repair process. Tissue damage and repair also frequently lead to alterations in skin pigmentation, in particular to wound hyperpigmentation. In this study, we describe a model of wound hyperpigmentation in the translucent zebrafish larva, where we can live-image the recruitment of melanocytes and their precursors, melanoblasts, to the wound site. We show that these pigment cells are drawn in after the initial recruitment of innate immune cells and that the inflammatory response is essential for wound hyperpigmentation. This new model will allow us to uncover the molecular link between immune and pigment cells during tissue repair and to screen for potential therapeutics to dampen wound hyperpigmentation.

The heart develops from two sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay in combination with genetic lineage tracing, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are pre-patterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function and disease.

The function of the FAM83F protein, like the functions of many members of the FAM83 family, is poorly understood. Here we show that injection of Fam83f mRNA into Xenopus embryos causes axis duplication, a phenotype indicative of enhanced Wnt signalling.Consistent with this, overexpression of FAM83F activates Wnt signalling, whilst ablation of FAM83F from human colorectal cancer (CRC) cells attenuates it. We demonstrate that FAM83F is farnesylated and interacts and co-localises with CK1α at the plasma membrane.This interaction with CK1α is essential for FAM83F to activate Wnt signalling, and FAM83F mutants that do not interact with CK1α fail to induce axis duplication in Xenopus embryos and to activate Wnt signalling in cells. FAM83F acts upstream of the β-catenin destruction complex, because the attenuation of Wnt signalling caused by loss of FAM83F can be rescued by GSK-3 inhibition. Introduction of a farnesyl-deficient mutant mis-localises the FAM83F-CK1α complex to the nucleus and significantly attenuates Wnt signalling, indicating that FAM83F exerts its effects on Wnt signalling at the plasma membrane.

Cadherin EGF LAG seven-pass G-type receptor (Celsr) proteins 1-3 comprise a subgroup of adhesion GPCRs whose functions range from planar cell polarity (PCP) signaling to axon pathfinding and ciliogenesis. Like its Drosophila ortholog, Flamingo, mammalian Celsr1 is a core component of the PCP pathway, which, among other roles, is responsible for the coordinated alignment of hair follicles across the skin surface. Although the role of Celsr1 in epidermal planar polarity is well established, the contribution of the other major epidermally expressed Celsr protein, Celsr2, has not been investigated. Here, using two new CRISPR/Cas9-targeted Celsr1 and Celsr2 knockout mouse lines, we define the relative contributions of Celsr1 and Celsr2 to PCP establishment in the skin. We find that Celsr1 is the major Celsr family member involved in epidermal PCP. Removal of Celsr1 function alone abolishes PCP protein asymmetry and hair follicle polarization, whereas epidermal PCP is unaffected by loss of Celsr2. Further, elimination of both Celsr proteins only minimally enhances the Celsr1−/− phenotype. Using FRAP and junctional enrichment assays to measure differences in Celsr1 and Celsr2 adhesive interactions, we find that compared to Celsr1, which stably enriches at junctional interfaces, Celsr2 is much less efficiently recruited to and immobilized at junctions. As the two proteins seem equivalent in their ability to interact with core PCP proteins Vangl2 and Fz6, we suggest that perhaps differences in homophilic adhesion contribute to the differential involvement of Celsr1 and Celsr2 in epidermal PCP.

The function of the FAM83F protein, like the functions of many members of the FAM83 family, is poorly understood. Here, we show that injection of Fam83f mRNA into Xenopus embryos causes axis duplication, a phenotype indicative of enhanced Wnt signalling. Consistent with this, overexpression of FAM83F activates Wnt signalling, whereas ablation of FAM83F from human colorectal cancer (CRC) cells attenuates it. We demonstrate that FAM83F is farnesylated and interacts and co-localises with CK1α at the plasma membrane. This interaction with CK1α is essential for FAM83F to activate Wnt signalling, and FAM83F mutants that do not interact with CK1α fail to induce axis duplication in Xenopus embryos and to activate Wnt signalling in cells. FAM83F acts upstream of GSK-3β because the attenuation of Wnt signalling caused by loss of FAM83F can be rescued by GSK-3 inhibition. Introduction of a farnesyl-deficient mutant of FAM83F in cells through CRISPR/Cas9 genome editing redirects the FAM83F–CK1α complex away from the plasma membrane and significantly attenuates Wnt signalling, indicating that FAM83F exerts its effects on Wnt signalling at the plasma membrane.