The role of Niemann-Pick type C2 in zebrafish embryonic development.

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

Gastrointestinal Tract Pathology in a BALB/c Niemann-Pick Disease Type C1 Null Mouse Model.

BACKGROUND: Niemann-Pick disease, type C (NPC) is a rare lysosomal storage disorder characterized by progressive neurodegeneration, splenomegaly, hepatomegaly, and early death. NPC is caused by mutations in either the NPC1 or NPC2 gene. Impaired NPC function leads to defective intracellular transport of unesterified cholesterol and its accumulation in late endosomes and lysosomes. A high frequency of Crohn disease has been reported in NPC1 patients, suggesting that gastrointestinal tract pathology may become a more prominent clinical issue if effective therapies are developed to slow the neurodegeneration. The Npc1 nih mouse model on a BALB/c background replicates the hepatic and neurological disease observed in NPC1 patients. Thus, we sought to characterize the gastrointestinal tract pathology in this model to determine whether it can serve as a model of Crohn disease in NPC1. METHODS: We analyzed the gastrointestinal tract and isolated macrophages of BALB/cJ cNctr-Npc1m1N/J (Npc1-/-) mouse model to determine whether there was any Crohn-like pathology or inflammatory cell activation. We also evaluated temporal changes in the microbiota by 16S rRNA sequencing of fecal samples to determine whether there were changes consistent with Crohn disease. RESULTS: Relative to controls, Npc1 mutant mice demonstrate increased inflammation and crypt abscesses in the gastrointestinal tract; however, the observed pathological changes are significantly less than those observed in other Crohn disease mouse models. Analysis of Npc1 mutant macrophages demonstrated an increased response to lipopolysaccharides and delayed bactericidal activity; both of which are pathological features of Crohn disease. Analysis of the bacterial microbiota does not mimic what is reported in Crohn disease in either human or mouse models. We did observe significant increases in cyanobacteria and epsilon-proteobacteria. The increase in epsilon-proteobacteria may be related to altered cholesterol homeostasis since cholesterol is known to promote growth of this bacterial subgroup. CONCLUSIONS: Macrophage dysfunction in the BALB/c Npc1-/- mouse is similar to that observed in other Crohn disease models. However, neither the degree of pathology nor the microbiota changes are typical of Crohn disease. Thus, this mouse model is not a good model system for Crohn disease pathology reported in NPC1 patients.

Establishment of the Vertebrate Germ Layers.

The process of germ layer formation is a universal feature of animal development. The germ layers separate the cells that produce the internal organs and tissues from those that produce the nervous system and outer tissues. Their discovery in the early nineteenth century transformed embryology from a purely descriptive field into a rigorous scientific discipline, in which hypotheses could be tested by observation and experimentation. By systematically addressing the questions of how the germ layers are formed and how they generate overall body plan, scientists have made fundamental contributions to the fields of evolution, cell signaling, morphogenesis, and stem cell biology. At each step, this work was advanced by the development of innovative methods of observing cell behavior in vivo and in culture. Here, we take an historical approach to describe our current understanding of vertebrate germ layer formation as it relates to the long-standing questions of developmental biology. By comparing how germ layers form in distantly related vertebrate species, we find that highly conserved molecular pathways can be adapted to perform the same function in dramatically different embryonic environments.

From multiallele fish to nonstandard environments, how ZFIN assigns phenotypes, human disease models, and gene expression annotations to genes.

Danio rerio is a model organism used to investigate vertebrate development. Manipulation of the zebrafish genome and resultant gene products by mutation or targeted knockdown has made the zebrafish a good system for investigating gene function, providing a resource to investigate genetic contributors to phenotype and human disease. Phenotypic outcomes can be the result of gene mutation, targeted knockdown of gene products, manipulation of experimental conditions, or any combination thereof. Zebrafish have been used in various genetic and chemical screens to identify genetic and environmental contributors to phenotype and disease outcomes. The Zebrafish Information Network (ZFIN, zfin.org) is the central repository for genetic, genomic, and phenotypic data that result from research using D. rerio. Here we describe how ZFIN annotates phenotype, expression, and disease model data across various experimental designs, how we computationally determine wild-type gene expression, the phenotypic gene, and how these results allow us to propagate gene expression, phenotype, and disease model data to the correct gene, or gene related entity.

Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds.

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1 NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-ß-cyclodextrin (2HPßCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPßCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds.This article has an associated First Person interview with the first author of the paper.

Extraembryonic signals under the control of MGA, Max, and Smad4 are required for dorsoventral patterning.

In vertebrates, extraembryonic tissues can act as signaling centers that impose a reproducible pattern of cell types upon the embryo. Here, we show that the zebrafish yolk syncytial layer (YSL) secretes a ventralizing signal during gastrulation. This activity is mediated by Bmp2b/Swirl (Swr) expressed under the control of Max's giant associated protein (MGA) and its binding partners, Max and Smad4. MGA coimmunoprecipitates with both Max and Smad4 in embryo extracts, and the three proteins form a complex in vitro. Furthermore, all three proteins bind to a DNA fragment upstream of the bmp2b transcription start site. Targeted depletion of MGA, its binding partners, or Bmp2b/Swr from the YSL reduces BMP signaling throughout the embryo, resulting in a mildly dorsalized phenotype. We conclude that MGA, Max, and Smad4 act in the extraembryonic YSL to initiate a positive feedback loop of Bmp signaling within the embryo.

Redistribution of GFAP and alphaB-crystallin after thermal stress in C6 glioma cell line.

Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein alphaB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, alphaB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein alphaB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.