Loss of function of FAM177A1, a Golgi complex localized protein, causes a novel neurodevelopmental disorder.

PURPOSE: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in 4 siblings. METHODS: We identified 5 individuals from 3 unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort. RESULTS: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA sequencing and metabolomic data sets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation. CONCLUSION: Our data shed light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis.

Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.

Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases.

Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome.

CNS myelination requires cytoplasmic dynein function.

BACKGROUND: Cytoplasmic dynein provides the main motor force for minus-end-directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration, and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known. RESULTS: We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression. CONCLUSIONS: In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination.

Chemokine GPCR signaling inhibits ß-catenin during zebrafish axis formation.

Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal ß-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect ß-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca(2+) stores generates Ca(2+) transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal ß-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca(2+) downstream of non-canonical Wnt ligands was proposed to inhibit ß-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of ß-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of ß-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal ß-catenin and its axis-inducing activity and can also inhibit the Gsk3ß -insensitive form of ß-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit ß-catenin, likely by promoting Ca(2+) transients throughout the blastula. Our study delineates a novel negative, Gsk3ß-independent control mechanism of ß-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation.

Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.

Zinc-finger nucleases (ZFNs) allow targeted gene inactivation in a wide range of model organisms. However, construction of target-specific ZFNs is technically challenging. Here, we evaluate a straightforward modular assembly-based approach for ZFN construction and gene inactivation in zebrafish. From an archive of 27 different zinc-finger modules, we assembled more than 70 different zinc-finger cassettes and evaluated their specificity using a bacterial one-hybrid assay. In parallel, we constructed ZFNs from these cassettes and tested their ability to induce lesions in zebrafish embryos. We found that the majority of zinc-finger proteins assembled from these modules have favorable specificities and nearly one-third of modular ZFNs generated lesions at their targets in the zebrafish genome. To facilitate the application of ZFNs within the zebrafish community we constructed a public database of sites in the zebrafish genome that can be targeted using this archive. Importantly, we generated new germline mutations in eight different genes, confirming that this is a viable platform for heritable gene inactivation in vertebrates. Characterization of one of these mutants, gata2a, revealed an unexpected role for this transcription factor in vascular development. This work provides a resource to allow targeted germline gene inactivation in zebrafish and highlights the benefit of a definitive reverse genetic strategy to reveal gene function.

VPS13B is localized at the cis-trans Golgi complex interface and is a functional partner of FAM177A1.

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

Oligodendrocyte progenitor cell numbers and migration are regulated by the zebrafish orthologs of the NF1 tumor suppressor gene.

Neurofibromatosis type 1 is the most commonly inherited human cancer predisposition syndrome. Neurofibromin (NF1) gene mutations lead to increased risk of neurofibromas, schwannomas, low grade, pilocytic optic pathway gliomas, as well as malignant peripheral nerve sheath tumors and glioblastomas. Despite the evidence for NF1 tumor suppressor function in glial cell tumors, the mechanisms underlying transformation remain poorly understood. In this report, we used morpholinos to knockdown the two nf1 orthologs in zebrafish and show that oligodendrocyte progenitor cell (OPC) numbers are increased in the developing spinal cord, whereas neurons are unaffected. The increased OPC numbers in nf1 morphants resulted from increased proliferation, as detected by increased BrdU labeling, whereas TUNEL staining for apoptotic cells was unaffected. This phenotype could be rescued by the forced expression of the GTPase-activating protein (GAP)-related domain of human NF1. In addition, the in vivo analysis of OPC migration following nf1 loss using time-lapse microscopy demonstrated that olig2-EGFP(+) OPCs exhibit enhanced cell migration within the developing spinal cord. OPCs pause intermittently as they migrate, and in nf1 knockdown animals, they covered greater distances due to a decrease in average pause duration, rather than an increase in velocity while in motion. Interestingly, nf1 knockdown also leads to an increase in ERK signaling, principally in the neurons of the spinal cord. Together, these results show that negative regulation of the Ras pathway through the GAP activity of NF1 limits OPC proliferation and motility during development, providing insight into the oncogenic mechanisms through which NF1 loss contributes to human glial tumors.

Six3 represses nodal activity to establish early brain asymmetry in zebrafish.

The vertebrate brain is anatomically and functionally asymmetric; however, the molecular mechanisms that establish left-right brain patterning are largely unknown. In zebrafish, asymmetric left-sided Nodal signaling within the developing dorsal diencephalon is required for determining the direction of epithalamic asymmetries. Here, we show that Six3, a transcription factor essential for forebrain formation and associated with holoprosencephaly in humans, regulates diencephalic Nodal activity during initial establishment of brain asymmetry. Reduction of Six3 function causes brain-specific deregulation of Nodal pathway activity, resulting in epithalamic laterality defects. Based on misexpression and genetic epistasis experiments, we propose that Six3 acts in the neuroectoderm to establish a prepattern of bilateral repression of Nodal activity. Subsequently, Nodal signaling from the left lateral plate mesoderm alleviates this repression ipsilaterally. Our data reveal a Six3-dependent mechanism for establishment of correct brain laterality and provide an entry point to understanding the genetic regulation of Nodal signaling in the brain.

Model organisms contribute to diagnosis and discovery in the undiagnosed diseases network: current state and a future vision.

Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000-13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support.

In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development.

Myelinating oligodendrocytes arise from migratory and proliferative oligodendrocyte progenitor cells (OPCs). Complete myelination requires that oligodendrocytes be uniformly distributed and form numerous, periodically spaced membrane sheaths along the entire length of target axons. Mechanisms that determine spacing of oligodendrocytes and their myelinating processes are not known. Using in vivo time-lapse confocal microscopy, we show that zebrafish OPCs continuously extend and retract numerous filopodium-like processes as they migrate and settle into their final positions. Process remodeling and migration paths are highly variable and seem to be influenced by contact with neighboring OPCs. After laser ablation of oligodendrocyte-lineage cells, nearby OPCs divide more frequently, orient processes toward the ablated cells and migrate to fill the unoccupied space. Thus, process activity before axon wrapping might serve as a surveillance mechanism by which OPCs determine the presence or absence of nearby oligodendrocyte-lineage cells, facilitating uniform spacing of oligodendrocytes and complete myelination.

Normal forebrain development may require continual Wnt antagonism until mid-somitogenesis in zebrafish.

During normal forebrain development in vertebrates, rostral neural tissue must be protected from Wnt signals via the actions of locally expressed Wnt antagonistic factors. In zebrafish zygotic oep (Zoep) mutants, forebrain structure is severely disrupted with reduced expression of the Wnt antagonists secreted frizzled related protein1 and dickkopf1. To analyze the temporal effects of Wnt antagonism on forebrain development, we generated transgenic zebrafish that overexpressed the dominant negative form of frizzled8a (DNfz8a) in wild-type and Zoep mutants under the control of a heat-inducible promoter. This model allowed for assessment of the dynamics of Wnt antagonistic signaling during forebrain development. Our results demonstrated that overexpression of DNfz8a in Zoep embryos between 7 and 16hpf increased putative forebrain region demarcated by anf and distal-less2 expressions. These results suggest that normal forebrain development requires continual Wnt antagonism from the early gastrula to the mid-somitogenesis stage.

Spatio-temporal regulation of concurrent developmental processes by generic signaling downstream of chemokine receptors.

Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration.

A missense variant in SLC39A8 is associated with severe idiopathic scoliosis.

Genetic factors predictive of severe adolescent idiopathic scoliosis (AIS) are largely unknown. To identify genetic variation associated with severe AIS, we performed an exome-wide association study of 457 severe AIS cases and 987 controls. We find a missense SNP in SLC39A8 (p.Ala391Thr, rs13107325) associated with severe AIS (P = 1.60 × 10-7, OR = 2.01, CI = 1.54-2.62). This pleiotropic SNP was previously associated with BMI, blood pressure, cholesterol, and blood manganese level. We replicate the association in a second cohort (841 cases and 1095 controls) resulting in a combined P = 7.02 × 10-14, OR = 1.94, CI = 1.63-2.34. Clinically, the minor allele of rs13107325 is associated with greater spinal curvature, decreased height, increased BMI and lower plasma manganese in our AIS cohort. Functional studies demonstrate reduced manganese influx mediated by the SLC39A8 p.Ala391Thr variant and vertebral abnormalities, impaired growth, and decreased motor activity in slc39a8 mutant zebrafish. Our results suggest the possibility that scoliosis may be amenable to dietary intervention.

Eye field requires the function of Sfrp1 as a Wnt antagonist.

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the specification of vertebrate forebrain region. Previous studies have shown that Wnt activation by LiCl treatment causes an expansion of optic stalk and mid-hindbrain boundary, whereas eye and ventral diencephalon in the forebrain region were reduced. However, the molecular mechanism, by which inhibits Wnt activity in the forebrain remains poorly defined. To investigate relationship between forebrain specification and Wnt signaling, the zebrafish homologue of secreted frizzled related protein1 (sfrp1) has been characterized. The transcripts of sfrp1 are detected in the presumptive forebrain at gastrula and in the ventral telencephalon, ventral diencephalon, midbrain and optic vesicles at 24h after postfertilization (hpf). Overexpression of sfrp1 causes an anteriorization of embryo, with enlarged head and reduced posterior structure as in the embryo overexpressing dominant-negative form of Frizzled8a or Dkk1. Its overexpression restored the eye defects in the Wnt8b-overexpressing embryos, but not in the LiCl-treated embryos. These results suggest that Sfrp1 expressed in the forebrain and eye field plays a critical role in the extracellular events of antagonizing Wnt activity for the forebrain specification.

Oligodendrocyte specification in zebrafish requires notch-regulated cyclin-dependent kinase inhibitor function.

Cyclin-dependent kinase inhibitors (Cdkis) influence both cell-cycle progression and differentiation of neural cells. However, the precise roles of Cdkis in coordinating formation of neurons and glia and the mechanisms that regulate expression of genes that encode Cdkis in the vertebrate CNS remain unknown. Here, we report that, in zebrafish, expression of the Cdki gene cyclin-dependent kinase inhibitor 1c (cdkn1c), a p57 homolog, is negatively regulated by Delta-Notch signaling and that Cdkn1c function is required for neural plate cells to stop dividing and differentiate as neurons on schedule, even in the absence of Notch signaling activity. Furthermore, Cdkn1c function is required for specification of oligodendrocytes from ventral spinal cord precursors. We propose that levels of cdkn1c expression are an important factor in regulating neural development: high levels of Cdkn1c promote cell-cycle exit and neuronal development, whereas, during late embryogenesis, neural cells that have low but functional levels of Cdkn1c, regulated by Notch activity, are specified for oligodendrocyte fate.

Activated ALK collaborates with MYCN in neuroblastoma pathogenesis.

Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analog following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma.

Zebrafish neurofibromatosis type 1 genes have redundant functions in tumorigenesis and embryonic development.

Neurofibromatosis type 1 (NF1) is a common, dominantly inherited genetic disorder that results from mutations in the neurofibromin 1 (NF1) gene. Affected individuals demonstrate abnormalities in neural-crest-derived tissues that include hyperpigmented skin lesions and benign peripheral nerve sheath tumors. NF1 patients also have a predisposition to malignancies including juvenile myelomonocytic leukemia (JMML), optic glioma, glioblastoma, schwannoma and malignant peripheral nerve sheath tumors (MPNSTs). In an effort to better define the molecular and cellular determinants of NF1 disease pathogenesis in vivo, we employed targeted mutagenesis strategies to generate zebrafish harboring stable germline mutations in nf1a and nf1b, orthologues of NF1. Animals homozygous for loss-of-function alleles of nf1a or nf1b alone are phenotypically normal and viable. Homozygous loss of both alleles in combination generates larval phenotypes that resemble aspects of the human disease and results in larval lethality between 7 and 10 days post fertilization. nf1-null larvae demonstrate significant central and peripheral nervous system defects. These include aberrant proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), dysmorphic myelin sheaths and hyperplasia of Schwann cells. Loss of nf1 contributes to tumorigenesis as demonstrated by an accelerated onset and increased penetrance of high-grade gliomas and MPNSTs in adult nf1a(+/-); nf1b(-/-); p53(e7/e7) animals. nf1-null larvae also demonstrate significant motor and learning defects. Importantly, we identify and quantitatively analyze a novel melanophore phenotype in nf1-null larvae, providing the first animal model of the pathognomonic pigmentation lesions of NF1. Together, these findings support a role for nf1a and nf1b as potent tumor suppressor genes that also function in the development of both central and peripheral glial cells as well as melanophores in zebrafish.