Identification of a Mutation in the Clock1 Gene Affecting Zebrafish Circadian Rhythms

As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1^dg3, revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.     

Cell Proliferation Patterns in Early Zebrafish Development

Although cell proliferation is an essential cell behavior for animal development, a detailed analysis of spatial and temporal patterns of proliferation in whole embryos are still lacking for most model organisms. Zebrafish embryos are particularly suitable for this type of analysis due to their transparency and size. Therefore, the main objective of the present work was to analyze the spatial and temporal patterns of proliferation during the first day of zebrafish embryo development by indirect immunofluorescence against phosphorylated histone H3, a commonly used mitotic marker. Several interesting findings were established. First, we found that mitosis metasynchrony among blastomeres could begin at the 2‐ to 4‐cell stage embryos. Second, mitosis synchrony was lost before the midblastula transition (MBT). Third, we observed a novel pattern of mitotic clusters that coincided in time with the mitotic pseudo “waves” described to occur before the MBT. Altogether, our findings indicate that early development is less synchronic than anticipated and that synchrony is not a requirement for proper development in zebrafish. Anat Rec, 296:759–773, 2013. © 2013 Wiley Periodicals, Inc.     

hace1 Influences zebrafish cardiac development via ROS‐dependent mechanisms

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Rodlet cells and the sensory systems in zebrafish (Danio rerio)

This preliminary work was designed to study, using routine procedures for light and transmission electron microscopy, the presence of rodlet cells (RCs) in or near the sensory systems of 12 adult specimens (4.0 ± 1.2 cm, LT ± SD) of zebrafish, Danio rerio Hamilton, 1822. Rodlet cells, characterized by a distinctive cell cortex (range, 0.4–1.5 μm in thickness) and conspicuous inclusions named “rodlets,” have a round to ovoid nucleus with irregular outline. Mature RCs are 11.5 ± 1.2 μm (mean ± SD) long and 7.8 ± 1.1 μm (mean ± SD) wide. These cells are more numerous near neuromasts enclosed by an epithelial roof and/or ossified canal wall. In contrast, very few RCs were noticed near superficial neuromasts. Based on the presence of RCs around the two cranial neuromasts of each fish, a variable number from 1 to 15 rodlet cells was found (10.4 ± 3.6, mean ± SD). The RCs were located 1.5 μm (minimal) to 73.3 μm (maximal) from the neuromast (27.9 ± 17.2, mean ± SD). Moreover, RCs were found in olfactory epithelium and in proximity to some taste buds. Interestingly, RCs were absent in the inner ear, eye, and brain. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Cellular Source of the Poxviral N1R/p28 Gene Family

Full-length poxvirus N1R/p28 orthologous proteins feature a prominent C-terminal RING zinc-finger motif. The RING moiety is conspicuously mutated in a number of vaccinia virus strains relative to variola virus. This, together with empirical data, suggests that N1R/p28 proteins promote virulence by suppressing apoptosis. Poxvirus N1R/p28 orthologues are strikingly similar to the RING motif of the cellular Makorin family of zinc-finger proteins, suggesting a homologous relationship connecting the viral and cellular genes. Recently identified avipox N1R/p28 orthologues further encode additional Makorin-like zinc-finger motifs, consistent with this suggestion. Phylogenetic analysis supports a model of poxviral capture of a MKRNcDNA and fusion with an existing viral gene. Establishing an evolutionary link between the viral and cellular genes will facilitate the elucidation of their respective cellular functions, and of how they interact in modulating virulence.     

Identification and Expression Analysis of the Zebrafish Homologs of the ceramide synthase Gene Family

Background: Sphingolipids represent a major class of lipids which both serve as structural components of membranes and as bioactive molecules involved in lipid signaling. Ceramide synthases (cers) reside in the center of sphingolipid metabolism by producing ceramide through de novo synthesis or degradative pathways. While the six mammalian cers family members have been extensively studied in cell culture and in adult tissues, a systematic analysis of cers expression and function during embryogenesis is still lacking. Results: Using bioinformatic and phylogenetic analysis, we identified nine highly conserved homologs of the vertebrate cers gene family in the zebrafish genome. A systematic expression analysis throughout five developmental stages indicates that, whereas until 48 hours post fertilization most zebrafish cers homologs are expressed in distinct patterns, e.g., in the intermediate cell mass and the pronephric duct, they show a highly overlapping expression during later stages of embryonic development, mostprominently in the developing brain. Conclusions: In this study, the expression of the cers gene homologs is comprehensively analyzed for the first time during vertebrate embryogenesis. Our data indicate that each embryonic tissue has a unique profile of cers expression during zebrafish embryogenesis suggesting tissue‐specific profiles of ceramides and their derivatives. Developmental Dynamics 242:189–200, 2013. © 2012 Wiley Periodicals, Inc.     

Effects of Different Variants in the ENPP1 Gene on the Functional Properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E‐NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PP_i), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal‐recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PP_i generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild‐type E‐NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E‐NPP1 enzyme activity were still able to generate PP_i and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E‐NPP1 function, which would be potential therapeutical targets for conformational‐stabilizing agents.     

Functional bone histology of zebrafish reveals two types of endochondral ossification,different types of osteoblast clusters and a new bone type

The zebrafish is as an important vertebrate animal model system for studying developmental processes, gene functions and signalling pathways. It is also used as a model system for the understanding of human developmental diseases including those related to the skeleton. However, surprisingly little is known about normal zebrafish skeletogenesis and osteogenesis. As in most vertebrates, it is commonly known that the bones of adult zebrafish are cellular unlike that of some other teleosts. After careful histological analyses of each zebrafish adult bone, we identified several acellular bones, with no entrapped osteocytes in addition to several cellular bones. We show that both cellular and acellular bones can even occur within the same skeletal element and transitions between these two cell types can be found. Furthermore, we describe two types of osteoblast clusters during skeletogenesis and two different types of endochondral ossification. The epiphyseal plate, for example, lacks a zone of calcification and a degradation zone with osteoblasts. A new bone type that we term tubular bone was also identified. This bone is completely filled with adipose tissue, unlike spongy bones. This study provides important insight on how osteogenesis takes place in zebrafish, and especially on the transition from cellular to acellular bones. Overall, this study leads to a deeper understanding of the functional histological composition of adult zebrafish bones.     

FXR基因家族

FXR基因家族是与脆性X综合征发病相关的一个基因家族。有三个家族成员。在进化上高度保守,氨基酸序列高度相似,有共同的功能结构域：两个KH结构域,RGG盒,NES和NLS。能够与RNA和多聚核蛋白体结合。通过NES和NLS携带其mRNA在细胞核和细胞质之间穿梭。不同的蛋白亚型其组织分布各异。该家族成员在大脑和神经发育过程中发挥重要作用,影响认知过程和智力发育。     

Mapping And Characterization of the Eukaryotic Early Pregnancy Factor/Chaperonin 10 Gene Family

Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.     

Unexpanded and intermediate CAG polymorphisms at the SCA2 locus (ATXN2) in the Cuban population: evidence about the origin of expanded SCA2 alleles

The role of short, large or intermediate normal alleles (ANs) of the ataxin-2 gene in generating expanded alleles (EAs) causing spinocerebellar ataxia type 2 (SCA2) is poorly understood. It has been postulated that SCA2 prevalence is related to the frequency of large ANs. SCA2 shows the highest worldwide prevalence in Cuban population, which is therefore a unique source for studying the relationship between the frequency of large and intermediate alleles and the frequency of SCA2 mutation. Through genetic polymorphism analyses in a comprehensive sample (~3000 chromosomes), we show that the frequency of large ANs in the ataxin-2 gene is the highest worldwide, although short ANs are also frequent. This highly polymorphic population displayed also high variability in the CAG sequence, featured by loss of the anchor CAA interruption(s). In addition, large ANs showed germinal and somatic instability. Our study also includes related genotypic, genealogical and haplotypic data and provides substantial evidence with regard to the role of large and intermediate alleles in the generation of pathological EAs.     

ING基因家族的研究进展

ING基因最早是通过消减杂交寻找抑癌基因的方法得到的,这个基因家族在进化中十分保守,功能上与染色质结构的调节、基因转录、细胞周期调控、细胞增殖以及细胞凋亡有关.目前发现ING基因家族至少包括6个基因.本文重点回顾了ING基因家族中的ING1的研究过程及其进展,并试图探讨ING1的不同转录本所编码的蛋白在功能上的相互拮抗(antagonistic)现象.     

ISOLATION AND PHENOGENETICS OF A NOVEL CIRCADIAN RHYTHM MUTANT IN ZEBRAFISH

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag^dg2) mutant is shortened by 0.7 h in heterozygotes, and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1 (per1) is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag^dg2 mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.