Genome-wide profiling of gene expression in 29 normal human tissues with a cDNA microarray.

We have performed a comprehensive analysis of the expression profiles in 25 adult and 4 fetal human tissues by means of a cDNA microarray consisting of 23,040 human genes. This study revealed a number of genes that were expressed specifically in each of those tissues. Among the 29 tissues examined, 4,080 genes were highly expressed (at least a five-fold expression ratio) in one or only a few tissues and 1,163 of those were expressed exclusively (more than a ten-fold higher expression ratio) in a particular tissue. Expression of some of the genes in the latter category was confirmed by northern analysis. A hierarchical clustering analysis of gene-expression profiles in nerve tissues (adult brain, fetal brain, and spinal cord), lymphoid tissues (bone marrow, thymus, spleen, and lymph node), muscle tissues (heart and skeletal muscle), or adipose tissues (mesenteric adipose and mammary gland) identified a set of genes that were commonly expressed among related tissues. These data should provide useful information for medical research, especially for efforts to identify tissue-specific molecules as potential targets of novel drugs to treat human diseases.     

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0 °C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.     

Unlike mammalian GRIFIN, the zebrafish homologue (DrGRIFIN) represents a functional carbohydrate-binding galectin

Galectins, a family of β-galactoside-binding proteins, participate in a variety of biological processes, such as early development, tissue organization, immune regulation, and tumor evasion and metastasis. Although as many as fifteen bona fide galectins have been identified in mammals, but the detailed mechanisms of their biological roles still remain unclear for most. This fragmentary knowledge extends to galectin-like proteins such as the rat lens crystallin protein GRIFIN (Galectin-related inter fiber protein) and the galectin-related protein GRP (previously HSPC159; hematopoietic stem cell precursor) that lack carbohydrate-binding activity. Their inclusion in the galectin family has been debated, as they are considered products of evolutionary co-option. We have identified a homologue of the GRIFIN in zebrafish (Danio rerio) (designated DrGRIFIN), which like the mammalian equivalent is expressed in the lens, particularly in the fiber cells, as revealed by whole mount in situ hybridization and immunostaining of 2 dpf (days post fertilization) embryos. As evidenced by RT-PCR, it is weakly expressed in the embryos as early as 21 hpf (hour post fertilization) but strongly at all later stages tested (30 hpf and 3, 4, 6, and 7 dpf). In adult zebrafish tissues, however, DrGRIFIN is also expressed in oocytes, brain, and intestine. Unlike the mammalian homologue, DrGRIFIN contains all amino acids critical for binding to carbohydrate ligands and its activity was confirmed as the recombinant DrGRIFIN could be purified to homogeneity by affinity chromatography on a lactosyl-Sepharose column. Therefore, DrGRIFIN is a bona fide galectin family member that in addition to its carbohydrate-binding properties, may also function as a crystallin.     

The zebrafish genome contains two inducible, functional cyclooxygenase-2 genes

Cyclooxygenase is a key enzyme in prostanoid biosynthesis. Mammalian species have two cyclooxygenases, constitutively expressed cyclooxygenase-1 (Cox-1) and inducible cyclooxygenase-2 (Cox-2). Cox-1 and/or Cox-2 have been also identified in other vertebrates, including fish. We identified a second zebrafish Cox-2 gene orthologue, Cox-2b. All of the functionally important amino acids for cyclooxygenase enzymes are conserved in Cox-2b. The 3' untranslated region of the Cox-2b message contains AU rich elements characteristic of regulation at the level of mRNA stability. Constitutive tissue expression patterns for Cox-2a and Cox-2b are distinct, but overlap. Both Cox-2a and Cox-2b expression are inducible in the kidney when fish are exposed to tetradecanoylphorbol acetate. Like Cox-2a, Cox-2b protein, expressed in COS cells is functionally active. Thus, the zebrafish genome contains two functional, inducible Cox-2 genes. Database searching demonstrates that some fish genomes contain multiple Cox-1 or Cox-2 cyclooxygenase genes, suggesting alternate duplication and retention of this gene.     

Trypanosoma brucei brucei: A comparison of gene expression in the liver and spleen of infected mice utilizing cDNA microarray technology

Trypanosoma brucei brucei, the infectious agent of the disease known as Nagana, is a pathogenic trypanosome occurring in Africa, where it causes significant economic loss to domesticated livestock. Although many studies on the histopathology of organs of mice infected with T. b. brucei have been reported, little work has been done regarding gene expression in these organs in infected mice. In this paper, we describe the use of cDNA microarray to determine gene expression profiles in the liver and spleen of mice infected with T. b. brucei (STIB 920) at peak parasitaemia (12 days after infection). Our results showed that a total of 123 genes in the liver and 389 genes in the spleen were expressed differentially in T. b. brucei infected mice. In contrast, however, in an acute infection in mice caused by Trypanosoma brucei evansi, a species genetically related to T. b. brucei, 336 genes in the liver and 190 genes in the spleen were expressed, differentially, indicating that the liver of mice was more affected by the acute T. b. evansi infection whilst the spleen was more affected by the subacute T. b. brucei infection. Our results provide a number of possible reasons why mice infected with T. b. evansi die sooner than those infected with T. b. brucei: (1) mice infected with T. b. evansi may need more stress response proteins to help them pass through the infection and these are probably excessively consumed; (2) proliferating cell nuclear antigen was more down-regulated in the liver of mice infected with T. b. evansi, which indicated that the inhibition of proliferation of hepatocytes in mice infected with T. b. evansi might be more severe than that in T. b. brucei infection; and (3) more hepatocyte apoptosis occurred in the mice infected with T. b. evansi and this might be probably the most important reason why mice died sooner than those infected with T. b. brucei. Studies of the changes in the gene expression profile in the liver and spleen of mice infected with T. b. brucei may be helpful in understanding the mechanisms of pathogenesis in Nagana disease at the molecular level. By comparing the gene profiles of the liver and spleen of mice infected with T. b. brucei with T. b. evansi, we have identified a number of factors that could explain the differences in pathogenesis in mice infected with these two African trypanosomes.     

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.     

Timing and plasticity of shoaling behaviour in the zebrafish, Danio rerio

The zebrafish has become a major model system for biomedical research and is an emerging model for the study of behaviour. While adult zebrafish express a visually mediated shoaling preference, the onset of shoaling behaviour and of this preference is unknown. To assess the onset of these behaviours, we first manipulated the early social environment of larval zebrafish subjects, giving them three model shoaling partners of the same pigment phenotype. We then assayed the subjects' preferences using binary preference tests in which we presented subjects with two shoals, one shoal of fish exhibiting the same pigment pattern phenotype as their models and another shoal with a radically different pigment pattern. To determine whether or not the visually mediated preference could be altered once it was established, we further manipulated the social environment of a number of subjects, rearing them with one model shoal and testing them, then changing their social consorts and retesting them. Our results demonstrate that larval zebrafish shoal early in their development, but do not exhibit a shoaling preference until they are juveniles. Moreover, we find that the shoaling preference is stable, as changing the social environment of fish after they had acquired a preference did not change their preference. These data will facilitate investigations into the mechanisms underlying social behaviour in this vertebrate model system.     

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length

Females often choose to associate with males that have exaggerated traits. In fishes, this may reflect an overall preference for larger size in a potential mate. Female zebrafish (Danio rerio) prefer males with larger bodies but not longer fins. The availability of mutant and transgenic strains of zebrafish make this a unique model system in which to study the role of phenotypic variation in social and sexual behavior. We used mutant strains of zebrafish with truncated (short fin) and exaggerated (long fin) fins to further examine female preferences for fin length in dichotomous association tests. Wild type females showed no preferences between wild type males and short fin mutant males or between wild type males and long fin mutant males. short fin females also showed no preference for short fin males or wild type males while long fin females preferred to associate with long fin males over wild type males. These results suggest that the single gene long fin mutation that results in altered fin morphological may also be involved in a related female association preference.     

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

Hierarchical Bayes models for cDNA microarray gene expression

cDNA microarrays are used in many contexts to compare mRNA levels between samples of cells. Microarray experiments typically give us expression measurements on 1000-20 000 genes, but with few replicates for each gene. Traditional methods using means and standard deviations to detect differential expression are not satisfactory in this context. A handful of alternative statistics have been developed, including several empirical Bayes methods. In the present paper we present two full hierarchical Bayes models for detecting gene expression, of which one (D) describes our microarray data very well. We also compare the full Bayes and empirical Bayes approaches with respect to model assumptions, false discovery rates and computer running time. The proposed models are compared to existing empirical Bayes models in a simulation study and for a set of data (Yuen et al., 2002), where 27 genes have been categorized by quantitative real-time PCR. It turns out that the existing empirical Bayes methods have at least as good performance as the full Bayes ones.     

Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres

Mutations affecting endocytosis, such as those in clathrin and dynamin, unexpectedly cause defects in cytokinesis in a number of organisms. To explore the relationship between endocytosis and cytokinesis, we used the relatively large cells of the transparent zebrafish embryo. Using fluorescent markers for fluid-phase as well as plasma membrane uptake, we demonstrate that cytokinesis involves furrow-specific endocytosis. Clathrin-coated pits are visible near the furrow in ultrathin sections, while immunolabeling demonstrates that clathrin and caveolin are localized to the cleavage furrow. Hence, it is likely that both clathrin- and caveolae-mediated endocytosis occurs at the furrow during cytokinesis. Dynamin II is also localized to the furrow and may mediate furrow-specific endocytosis. Treatment of embryos with chlorpromazine or with methyl-beta-cyclodextrin, both of which inhibit endocytosis, prevents the normal completion of cytokinesis. These data suggest that furrow-specific endocytosis is an integral part of cytokinesis.     

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the <Emphasis Type="Italic">fmr1</Emphasis> gene

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.Edited by D. Tautz