Cardiac function-related gene expression profiles in human atrial myocytes

To obtain insights into the molecular pathogenesis of heart failure in humans, we have analyzed the expression profiles of>12,000 genes in a total of 17 human specimens of right atrial myocytes. From this large data set, we here tried to identify gene clusters, expression level of which is correlated precisely with clinical parameter values of cardiac function. We could reveal that cardiac myocytes with normal sinus rhythm were clearly differentiated, in the point of view of gene expression, from those with atrial fibrillation. Further, an expression profile-based prediction of arrhythmia by a newly developed "weighted-distance method" could efficiently diagnose our samples. We could even construct calculation formulae for the values of left ventricular ejection fraction based on the expression level of selected genes. To our best knowledge, this is the first report to indicate that pumping ability of heart can be predicted by any measures of atrium.     

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.     

Gene expression in placentation of farm animals: an overview of gene function during development

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish — a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] — for humans, and tissue-specific genes database (TiSGeD) — for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta.     

Genome-wide analysis of gene expression in human embryonic tooth germ

While numerous genes that play important regulatory roles during tooth development in mice have been identified, little is known about gene expression profile and their function during human odontogenesis. To unveil expression profile of odontogenic genes in humans, we conducted genome-wide gene expression analysis by microarray assays to analyze differential gene expression between tooth germ and lip tissue from 11-week old human fetuses. We identified 167 genes that are strongly expressed in the cap stage tooth germ as compared to the lip tissue. Among them, 145 genes were further identified by gene ontology enrichment analysis that are highly represented in multiple gene ontology classes, include extracellular components, sequence-specific DNA binding proteins, Wnt-protein binding molecules, system development, organogenesis, and cell differentiation. Sixty-seven genes that are known to be associated with mammalian tooth development and tooth abnormalities were identified. Real-time PCR was further employed to validate microarray data. Moreover, in situ hybridization assay demonstrated tooth type specific expression of ISL1 and BARX1 in the incisor, canine, and molar respectively, consistent with microarray results. Our results represent a set of reliable data that could provide a solid base for future elaboration of molecular mechanisms underlying human tooth development.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

Isolation of a ubiquitin-like (UBL5) gene from a screen identifying highly expressed and conserved iris genes

We have screened a human adult iris cDNA library to identify genes that are highly expressed and conserved between humans and pigs. We identified human iris cDNAs that hybridized at high stringency to a porcine choroidal ring cDNA probe. Of 1568 human iris cDNAs examined, 176 were found to have high expression in porcine choroidal rings. One of the 176 clones was identified as a previously uncharacterized cDNA that we have named the Ubiquitin-like 5 gene (UBL5). The UBL5 gene is located on chromosome 19p13.2, and its genomic structure has been examined. There is a UBL5 pseudogene on chromosome 17p11.2. We have also found homologues to the UBL5 gene in Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Northern blot analysis of the Ubiquitin-like gene 5 revealed expression in every tissue tested, with the highest levels of RNA expression in heart, skeletal muscle, kidney, liver, iris, and lymphoblasts. Intracellular localization experiments in COS-7 cells showed that the recombinant UBL5 protein is cytoplasmic. Western analysis demonstrated that the recombinant UBL5 protein is approximately 9 kDa, as predicted from the cDNA. A comparison between UBL5 and its homologues with other Ubiquitin-like proteins and Ubiquitin, using the PROTDIST program, suggests that the UBL5 genes are a separate class of Ubiquitin-like genes. Further characterization of the UBL5 gene will determine the function of the encoded protein and whether it is a candidate for ocular disease.     

Genetic and developmental regulation of the lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structural gene control enzyme expression

We report here a study of the developmental and genetic control of tissue-specific expression of lipoprotein lipase, the enzyme responsible for hydrolysis of triglycerides in chylomicrons and very low density lipoproteins. Lipoprotein lipase (LPL) mRNA is present in a wide variety of adult rat and mouse tissues examined, albeit at very different levels. A remarkable increase in the levels of LPL mRNA occurs in heart over a period of several weeks following birth, closely paralleling developmental changes in lipase activity and myocardial beta-oxidation capacity. Large increases in LPL mRNA also occur during differentiation of 3T3L1 cells to adipocytes. As previously reported, at least two separate genetic loci control the tissue-specific expression of LPL activity in mice. One of the loci, controlling LPL activity in heart, is associated with an alteration in LPL mRNA size, while the other, controlling LPL activity in adipose tissue, appears to affect the translation or post-translational expression of LPL. To examine whether these genetic variations are due to mutations of the LPL structural locus, we mapped the LPL gene to a region of mouse chromosome 8 using restriction fragment-length polymorphisms and analysis of hamster-mouse somatic cell hybrids. This region is homologous to the region of human chromosome 8 which contains the human LPL gene as judged by the conservation of linked genetic markers. Genetic variations affecting LPL expression in heart cosegregated with the LPL gene, while variations affecting LPL expression in adipose tissue did not. Furthermore, Southern blotting analysis indicates that LPL is encoded by a single gene and, thus, the genetic differences are not a consequence of independent regulation of two separate genes in the two tissues. These results suggest the existence of cis-acting elements for LPL gene expression that operate in heart but not adipose tissue. Our results also indicate that two genetic mutations resulting in deficiencies of LPL in mice, the W mutation on chromosome 5 and the cld mutation on mouse chromosome 17, do not involve the LPL structural gene locus. Finally, we show that the gene for hepatic lipase, a member of a gene family with LPL, is unlinked to the gene for LPL. This indicates that combined deficiencies of LPL and hepatic lipase, observed in humans as well as in certain mutant strains of mice, do not result from focal disruptions of a cluster of lipase genes.