Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

Impaired light detection of the circadian clock in a zebrafish melanoma model

The circadian clock controls the timing of the cell cycle in healthy tissues and clock disruption is known to increase tumourigenesis. Melanoma is one of the most rapidly increasing forms of cancer and the precise molecular circadian changes that occur in a melanoma tumor are unknown. Using a melanoma zebrafish model, we have explored the molecular changes that occur to the circadian clock within tumors. We have found disruptions in melanoma clock gene expression due to a major impairment to the light input pathway, with a parallel loss of light-dependent activation of DNA repair genes. Furthermore, the timing of mitosis in tumors is perturbed, as well as the regulation of certain key cell cycle regulators, such that cells divide arhythmically. The inability to co-ordinate DNA damage repair and cell division is likely to promote further tumourigenesis and accelerate melanoma development.     

Interaction of MAGED1 with nuclear receptors affects circadian clock function

The circadian clock has a central role in physiological adaption and anticipation of day/night changes. In a genetic screen for novel regulators of circadian rhythms, we found that mice lacking MAGED1 (Melanoma Antigen Family D1) exhibit a shortened period and altered rest–activity bouts. These circadian phenotypes are proposed to be caused by a direct effect on the core molecular clock network that reduces the robustness of the circadian clock. We provide in vitro and in vivo evidence indicating that MAGED1 binds to RORα to bring about positive and negative effects on core clock genes of Bmal1, Rev‐erbα and E4bp4 expression through the Rev‐Erbα/ROR responsive elements (RORE). Maged1 is a non‐rhythmic gene that, by binding RORα in non‐circadian way, enhances rhythmic input and buffers the circadian system from irrelevant, perturbing stimuli or noise. We have thus identified and defined a novel circadian regulator, Maged1, which is indispensable for the robustness of the circadian clock to better serve the organism.     

A zebrafish gene trap line expresses GFP recapturing expression pattern of foxj1b

Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZIO by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxj1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxj1b. Although normal expression offoxj1b is dramatically reduced, T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxjlb expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.     

Daily variation of visual sensitivity to luminance contrast: Effects of time of measurement and circadian typology

This study analyzed the fluctuation of the achromatic visual contrast sensitivity (CS) of adult males (M = 23.42 ± 2.6 years) during a daily period. Twenty-eight volunteers were divided into three groups according to circadian typology (CT): moderate morning (MM; n = 8); intermediate (I; n = 10) and moderate evening (ME; n = 10). The Pittsburgh Sleep Quality Index was used to evaluate sleep quality, and the Horne and Ostberg Morningness-Eveningness Questionnaire was used to measure CT. To measure CS, we used Metropsis software version 11.0 with vertical sinusoidal grids of 0.2, 0.6, 1, 3.1, 6.1, 8.8, 13.2 and 15.6 cycles per degree of visual angle (cpd). The stimuli were presented on a cathode ray tube (CRT) color video monitor with a 19-inch flat screen, a 1024 × 786 pixel resolution, a 100 Hz refresh rate and a photopic luminance of 39.6 cd/m². It was inferred that there is a tendency for visual contrast to vary according to daily rhythmicity and CT, mainly for the median spatial frequencies (1.0 cpd, χ² = 9.93, p < 0.05 and 3.1 cpd, χ² = 10.33, p < 0.05) and high spatial frequencies (13.2 cpd, χ² = 11.54, p < 0.05) of ME participants. ME participants had minimal visual contrast sensitivity during the morning shift and a progressive increase from afternoon to night.     

CUL1 regulates TOC1 protein stability in the Arabidopsis circadian clock

The circadian clock is the endogenous timer that coordinates physiological processes with daily and seasonal environmental changes. In Arabidopsis thaliana , establishment of the circadian period relies on targeted degradation of TIMING OF CAB EXPRESSION 1 (TOC1) by the 26S proteasome. ZEITLUPE (ZTL) is the F-box protein that associates with the SCF (Skp/Cullin/F-box) E3 ubiquitin ligase that is responsible for marking TOC1 for turnover. CULLIN1 (CUL1) is a core component of SCF complexes and is involved in multiple signaling pathways. To assess the contribution of CUL1-containing SCF complexes to signaling within the plant oscillator, circadian rhythms were examined in the recessive, temperature-sensitive CUL1 allele axr6-3 . The activity of CUL1 in this mutant declines progressively with increasing ambient temperature, resulting in more severe defects in CUL1-dependent activities at elevated temperature. Examination of circadian rhythms in axr6-3 revealed circadian phenotypes comparable to those observed in ztl null mutants; namely, lengthened circadian period, altered expression of core oscillator genes, and limited degradation of TOC1. In addition, treatment of seedlings with exogenous auxin did not alter TOC1 stability. These results demonstrate that CUL1 is required for TOC1 degradation and further suggest that this protein is the functional cullin for the SCF^ZTL complex.     

The zebrafish dyrk1b gene is important for endoderm formation

Nodal‐signaling is required for specification of mesoderm, endoderm, establishing left–right asymmetry, and craniofacial development. Wdr68 is a WD40‐repeat domain‐containing protein recently shown to be required for endothelin‐1 (edn1) expression and subsequent lower jaw development. Previous reports detected the Wdr68 protein in multiprotein complexes containing mammalian members of the dual‐specificity tyrosine‐regulated kinase (dyrk) family. Here we describe the characterization of the zebrafish dyrk1b homolog. We report the detection of a physical interaction between Dyrk1b and Wdr68. We also found perturbations of nodal signaling in dyrk1b antisense morpholino knockdown (dyrk1b‐MO) animals. Specifically, we found reduced expression of lft1 and lft2 (lft1/2) during gastrulation and a near complete loss of the later asymmetric lft1/2 expression domains. Although wdr68‐MO animals did not display lft1/2 expression defects during gastrulation, they displayed a near complete loss of the later asymmetric lft1/2 expression domains. While expression of ndr1 was not substantially effected during gastrulation, ndr2 expression was moderately reduced in dyrk1b‐MO animals. Analysis of additional downstream components of the nodal signaling pathway in dyrk1b‐MO animals revealed modestly expanded expression of the dorsal axial mesoderm marker gsc while the pan‐mesodermal marker bik was largely unaffected. The endodermal markers cas and sox17 were also moderately reduced in dyrk1b‐MO animals. Notably, and similar to defects previously reported for wdr68 mutant animals, we also found reduced expression of the pharyngeal pouch marker edn1 in dyrk1b‐MO animals. Taken together, these data reveal a role for dyrk1b in endoderm formation and craniofacial patterning in the zebrafish. genesis 48:20–30, 2010. © 2009 Wiley‐Liss, Inc.     

Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

The olfm1a and olfm1b genes in zebrafish encode conserved secreted glycoproteins. These genes are preferentially expressed in the brain and retina starting from 16 h post‐fertilization until adulthood. Functions of the Olfm1 gene is still unclear. Here, we produced and analyzed a null zebrafish mutant of both olfm1a and olfm1b genes (olfm1 null). olfm1 null fish were born at a normal Mendelian ratio and showed normal body shape and fertility as well as no visible defects from larval stages to adult. Olfm1 proteins were preferentially localized in the synaptosomes of the adult brain. Olfm1 co‐immunoprecipitated with GluR2 and soluble NSF attachment protein receptor complexes indicating participation of Olfm1 in both pre‐ and post‐synaptic events. Phosphorylation of GluR2 was not changed while palmitoylation of GluR2 was decreased in the brain synaptosomal membrane fraction of olfm1 null compared with wt fish. The levels of GluR2, SNAP25, flotillin1, and VAMP2 were markedly reduced in the synaptic microdomain of olfm1 null brain compared with wt. The internalization of GluR2 in retinal cells and the localization of VAMP2 in brain synaptosome were modified by olfm1 null mutation. This indicates that Olfm1 may regulate receptor trafficking from the intracellular compartments to the synaptic membrane microdomain, partly through the alteration of post‐translational GluR2 modifications such as palmitoylation. Olfm1 may be considered a novel regulator of the composition and function of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor complex.

     

Treatment with beta2-adrenoceptor agonist in vivo induces human clock gene, Per1, mRNA expression in peripheral blood

This study examined whether in vivo exposure to a β₂-adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real-time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

整体原位杂交法初步研究MMP11b在斑马鱼胚胎发育过程中的表达

克隆斑马鱼基质金属蛋白酶11b（MMP11b）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP11b RNA探针,采用全胚胎原位杂交方法研究MMP11b基因在斑马鱼胚胎的表达。MMP11b基因在胚胎受精后一个细胞时期就开始表达,并且一直持续到96h,从受精后24h起,在耳囊处表达明显,在受精后48h时期在胸鳍和肛门处也有特异性表达。MMP11b在斑马鱼胚胎发育不同时期表达明显,且在耳囊处有持续表达。     

Crystal structure of zebrafish complement 1qA globular domain

C1q contains three globular domains (C1qgD) that are the key functional component of the classical complement system. C1qgD can interact with important immune molecules, including IgG and C‐reactive protein (CRP) to form defense systems to protect animals. Here, the first non‐mammalian structure, zebrafish C1qA globular domain (Dare‐C1qAgD) was solved. Although the overall architecture of Dare‐C1qAgD is similar to human C1qA, residues involved in C1qBgD, C1qCgD, and CRP binding are somewhat different while residues involved in IgG binding are not present in zebrafish. The structure gives insight into how human and fish C1qA evolved from an ancestral protein.     

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation

Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.