Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

Unexplained mismatch repair deficiency: Case closed.

To identify Lynch syndrome (LS) carriers, DNA mismatch repair (MMR) immunohistochemistry (IHC) is performed on colorectal cancers (CRCs). Upon subsequent LS diagnostics, MMR deficiency (MMRd) sometimes remains unexplained (UMMRd). Recently, the importance of complete LS diagnostics to explain UMMRd, involving MMR methylation, germline, and somatic analyses, was stressed. To explore why some MMRd CRCs remain unsolved, we performed a systematic review of the literature and mapped patients with UMMRd diagnosed in our center. A systematic literature search was performed in Ovid Medline, Embase, Web of Science, Cochrane CENTRAL, and Google Scholar for articles on UMMRd CRCs after complete LS diagnostics published until December 15, 2021. Additionally, UMMRd CRCs diagnosed in our center since 1993 were mapped. Of 754 identified articles, 17 were included, covering 74 patients with UMMRd. Five CRCs were microsatellite stable. Upon complete diagnostics, 39 patients had single somatic MMR hits, and six an MMR germline variant of unknown significance (VUS). Ten had somatic pathogenic variants (PVs) in POLD1, MLH3, MSH3, and APC. The remaining 14 patients were the only identifiable cases in the literature without a plausible identified cause of the UMMRd. Of those, nine were suspected to have LS. In our center, complete LS diagnostics in approximately 5,000 CRCs left seven MMRd CRCs unexplained. All had a somatic MMR hit or MMR germline VUS, indicative of a missed second MMR hit. In vitually all patients with UMMRd, complete LS diagnostics suggest MMR gene involvement. Optimizing detection of currently undetectable PVs and VUS interpretation might explain all UMMRd CRCs, considering UMMRd a case closed.

High-yield identification of pathogenic NF1 variants by skin fibroblast transcriptome screening after apparently normal diagnostic DNA testing.

Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.

Dominant-acting CSF1R variants cause microglial depletion and altered astrocytic phenotype in zebrafish and adult-onset leukodystrophy.

Tissue-resident macrophages of the brain, including microglia, are implicated in the pathogenesis of various CNS disorders and are possible therapeutic targets by their chemical depletion or replenishment by hematopoietic stem cell therapy. Nevertheless, a comprehensive understanding of microglial function and the consequences of microglial depletion in the human brain is lacking. In human disease, heterozygous variants in CSF1R, encoding the Colony-stimulating factor 1 receptor, can lead to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) possibly caused by microglial depletion. Here, we investigate the effects of ALSP-causing CSF1R variants on microglia and explore the consequences of microglial depletion in the brain. In intermediate- and late-stage ALSP post-mortem brain, we establish that there is an overall loss of homeostatic microglia and that this is predominantly seen in the white matter. By introducing ALSP-causing missense variants into the zebrafish genomic csf1ra locus, we show that these variants act dominant negatively on the number of microglia in vertebrate brain development. Transcriptomics and proteomics on relatively spared ALSP brain tissue validated a downregulation of microglia-associated genes and revealed elevated astrocytic proteins, possibly suggesting involvement of astrocytes in early pathogenesis. Indeed, neuropathological analysis and in vivo imaging of csf1r zebrafish models showed an astrocytic phenotype associated with enhanced, possibly compensatory, endocytosis. Together, our findings indicate that microglial depletion in zebrafish and human disease, likely as a consequence of dominant-acting pathogenic CSF1R variants, correlates with altered astrocytes. These findings underscore the unique opportunity CSF1R variants provide to gain insight into the roles of microglia in the human brain, and the need to further investigate how microglia, astrocytes, and their interactions contribute to white matter homeostasis.

M-CSFR/CSF1R signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands.

Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. The CSF1R pathway is a promising therapeutic target in many human diseases, including neurological disorders and cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands (Csf1a, Csf1b, and Il34) in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors, Csf1ra and Csf1rb, however there is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Furthermore, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation, resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rbΔ4bp-deficient and il34Δ5bp-deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors, and the expression of csf1ra and csf1rb is nonoverlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.

Müller glia-myeloid cell crosstalk accelerates optic nerve regeneration in the adult zebrafish.

Neurodegenerative disorders, characterized by progressive neuronal loss, eventually lead to functional impairment in the adult mammalian central nervous system (CNS). Importantly, these deteriorations are irreversible, due to the very limited regenerative potential of these CNS neurons. Stimulating and redirecting neuroinflammation was recently put forward as an important approach to induce axonal regeneration, but it remains elusive how inflammatory processes and CNS repair are intertwined. To gain more insight into these interactions, we investigated how immunomodulation affects the regenerative outcome after optic nerve crush (ONC) in the spontaneously regenerating zebrafish. First, inducing intraocular inflammation using zymosan resulted in an acute inflammatory response, characterized by an increased infiltration and proliferation of innate blood-borne immune cells, reactivation of Müller glia, and altered retinal cytokine expression. Strikingly, inflammatory stimulation also accelerated axonal regrowth after optic nerve injury. Second, we demonstrated that acute depletion of both microglia and macrophages in the retina, using pharmacological treatments with both the CSF1R inhibitor PLX3397 and clodronate liposomes, compromised optic nerve regeneration. Moreover, we observed that csf1ra/b double mutant fish, lacking microglia in both retina and brain, displayed accelerated RGC axonal regrowth after ONC, which was accompanied with unusual Müller glia proliferative gliosis. Altogether, our results highlight the importance of altered glial cell interactions in the axonal regeneration process after ONC in adult zebrafish. Unraveling the relative contribution of the different cell types, as well as the signaling pathways involved, may pinpoint new targets to stimulate repair in the vertebrate CNS.

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes.

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, csf1r deficiency disrupts embryonic to adult macrophage development. Zebrafish deficient for csf1r are viable and permit analyzing the consequences of macrophage loss throughout life.

Immune cells called macrophages are found in all organs in the body. These cells are highly effective at eating and digesting large particles including dead cells and debris, and microorganisms such as bacteria. Macrophages are also instrumental in shaping developing organs and repairing tissues during life. Macrophages were, until recently, thought to be constantly replenished from cells circulating in the bloodstream. However, it turns out that separate populations of macrophages become established in most tissues during embryonic development and are maintained throughout life without further input. Previous studies of zebrafish, rodents and humans have shown that, when a gene called CSF1R is non-functional, macrophages are absent from many organs including the brain. However, some tissue-specific macrophages still persist, and it was not clear why these cells do not rely on the CSF1R gene while others do. Kuil et al. set out to decipher the precise requirement for the CSF1R gene in macrophage development in living zebrafish. The experiments used zebrafish that make a green fluorescent protein in their macrophages. As these fish are transparent, this meant that Kuil et al. could observe the cells within the living fish and isolate them to determine which genes are switched on and off. This approach revealed that zebrafish with a mutated version of the CSF1R gene make macrophages as embryos but that these cells then fail to multiply and migrate into the developing organs. This results in fewer macrophages in the zebrafish's tissues, and an absence of these cells in the brain. Kuil et al. went on to show that new macrophages did emerge in zebrafish that were about two to three weeks old. However, unexpectedly, these new cells were not regular macrophages. Instead, they were a new recently identified cell-type called metaphocytes, which share similarities with macrophages but have a completely different origin, move faster and do not eat particles. Zebrafish lacking the CSF1R gene thus lose nearly all their macrophages but retain metaphocytes. These macrophage-free mutant zebrafish constitute an unprecedented tool for further studies looking to discriminate the different roles of macrophages and metaphocytes.

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis.

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes.

Gene expression profiling reveals a conserved microglia signature in larval zebrafish.

Microglia are the resident macrophages of the brain. Over the past decade, our understanding of the function of these cells has significantly improved. Microglia do not only play important roles in the healthy brain but are involved in almost every brain pathology. Gene expression profiling allowed to distinguish microglia from other macrophages and revealed that the full microglia signature can only be observed in vivo. Thus, animal models are irreplaceable to understand the function of these cells. One of the popular models to study microglia is the zebrafish larva. Due to their optical transparency and genetic accessibility, zebrafish larvae have been employed to understand a variety of microglia functions in the living brain. Here, we performed RNA sequencing of larval zebrafish microglia at different developmental time points: 3, 5, and 7 days post fertilization (dpf). Our analysis reveals that larval zebrafish microglia rapidly acquire the core microglia signature and many typical microglia genes are expressed from 3 dpf onwards. The majority of changes in gene expression happened between 3 and 5 dpf, suggesting that differentiation mainly takes place during these days. Furthermore, we compared the larval microglia transcriptome to published data sets of adult zebrafish microglia, mouse microglia, and human microglia. Larval microglia shared a significant number of expressed genes with their adult counterparts in zebrafish as well as with mouse and human microglia. In conclusion, our results show that larval zebrafish microglia mature rapidly and express the core microglia gene signature that seems to be conserved across species.

Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain.

Microglia are brain-resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, by Cas9/gRNA complex injections, followed by Neutral-Red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that, in zebrafish, il34 is required during the earliest seeding of the brain by microglia. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.

Dynamic control of proinflammatory cytokines Il-1ß and Tnf-α by macrophages in zebrafish spinal cord regeneration.

Spinal cord injury leads to a massive response of innate immune cells in non-regenerating mammals, but also in successfully regenerating zebrafish. However, the role of the immune response in successful regeneration is poorly defined. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in spinal-lesioned zebrafish larvae. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary for repair. Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Tnf-α and Il-1ß. Inhibiting Tnf-α does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. In contrast, decreasing Il-1ß levels or number of Il-1ß+ neutrophils rescue functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1ß function impairs regeneration in irf8 and wildtype animals. Hence, inflammation is dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

Identification of a conserved and acute neurodegeneration-specific microglial transcriptome in the zebrafish.

Microglia are brain resident macrophages important for brain development, connectivity, homeostasis and disease. However, it is still largely unclear how microglia functions and their identity are regulated at the molecular level. Although recent transcriptomic studies have identified genes specifically expressed in microglia, the function of most of these genes in microglia is still unknown. Here, we performed RNA sequencing on microglia acutely isolated from healthy and neurodegenerative zebrafish brains. We found that a large fraction of the mouse microglial signature is conserved in the zebrafish, corroborating the use of zebrafish to help understand microglial genetics in mammals in addition to studying basic microglia biology. Second, our transcriptome analysis of microglia following neuronal ablation suggested primarily a proliferative response of microglia, which we confirmed by immunohistochemistry and in vivo imaging. Together with the recent improvements in genome editing technology in zebrafish, these data offer opportunities to facilitate functional genetic research on microglia in vivo in the healthy as well as in the diseased brain. GLIA 2016;65:138-149.

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed.

Apoptosis is generally believed to be a process that requires several hours, in contrast to non-programmed forms of cell death that can occur in minutes. Our findings challenge the time-consuming nature of apoptosis as we describe the discovery and characterization of a small molecule, named Raptinal, which initiates intrinsic pathway caspase-dependent apoptosis within minutes in multiple cell lines. Comparison to a mechanistically diverse panel of apoptotic stimuli reveals that Raptinal-induced apoptosis proceeds with unparalleled speed. The rapid phenotype enabled identification of the critical roles of mitochondrial voltage-dependent anion channel function, mitochondrial membrane potential/coupled respiration, and mitochondrial complex I, III, and IV function for apoptosis induction. Use of Raptinal in whole organisms demonstrates its utility for studying apoptosis in vivo for a variety of applications. Overall, rapid inducers of apoptosis are powerful tools that will be used in a variety of settings to generate further insight into the apoptotic machinery.

Immune cell dynamics in the CNS: Learning from the zebrafish.

A major question in research on immune responses in the brain is how the timing and nature of these responses influence physiology, pathogenesis or recovery from pathogenic processes. Proper understanding of the immune regulation of the human brain requires a detailed description of the function and activities of the immune cells in the brain. Zebrafish larvae allow long-term, noninvasive imaging inside the brain at high-spatiotemporal resolution using fluorescent transgenic reporters labeling specific cell populations. Together with recent additional technical advances this allows an unprecedented versatility and scope of future studies. Modeling of human physiology and pathology in zebrafish has already yielded relevant insights into cellular dynamics and function that can be translated to the human clinical situation. For instance, in vivo studies in the zebrafish have provided new insight into immune cell dynamics in granuloma formation in tuberculosis and the mechanisms involving treatment resistance. In this review, we highlight recent findings and novel tools paving the way for basic neuroimmunology research in the zebrafish. GLIA 2015;63:719-735.

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.

An in vivo zebrafish screen identifies organophosphate antidotes with diverse mechanisms of action.

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry-based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

Apoptotic cells are cleared by directional migration and elmo1- dependent macrophage engulfment.

Apoptotic cell death is essential for development and tissue homeostasis. Failure to clear apoptotic cells can ultimately cause inflammation and autoimmunity. Apoptosis has primarily been studied by staining of fixed tissue sections, and a clear understanding of the behavior of apoptotic cells in living tissue has been elusive. Here, we use a newly developed technique to track apoptotic cells in real time as they emerge and are cleared from the zebrafish brain. We find that apoptotic cells are remarkably motile, frequently migrating several cell diameters to the periphery of living tissues. F-actin remodeling occurs in surrounding cells, but also within the apoptotic cells themselves, suggesting a cell-autonomous component of motility. During the first 2 days of development, engulfment is rare, and most apoptotic cells lyse at the brain periphery. By 3 days postfertilization, most cell corpses are rapidly engulfed by macrophages. This engulfment requires the guanine nucleotide exchange factor elmo1. In elmo1-deficient macrophages, engulfment is rare and may occur through macropinocytosis rather than directed engulfment. These findings suggest that clearance of apoptotic cells in living vertebrates is accomplished by the combined actions of apoptotic cell migration and elmo1-dependent macrophage engulfment.

A FRET sensor for non-invasive imaging of amyloid formation in vivo.

Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.