The isolation and characterization of tescalcin, a novel gene differentially expressed in the mouse testis during the early stages of gonadal differentiation

Gonadal development is an ideal model to study organogenesis because a variety of developmental processes can be studied during the differentiation of the bipotential primordium into testis or ovary. To better understand this process, Representational Difference Analysis of cDNA was used to identify genes that are differentially expressed in mouse gonads at 13.5 days post-coitus. The analysis led to the identification of three testis specific genes and a sequence that was only expressed in the ovary. The male genes identified: renin, Col9a3, and a novel gene termed tescalcin had patterns of expression that suggested a role in testis determination. ^ Studies of the tescalcin gene revealed that it is organized into eight exons and seven introns. The gene was located at 64 cM in mouse chromosome 5, where it spans approximately 35 Kb. Three mRNA variants resulting from alternative splicing of intron 5 were identified in mouse tissues. Gel mobility shift assays demonstrated that Sp1 and Sp3 from Y-1, msc-1, and MIN-6 cells nuclear extracts bind the GC-boxes within the tescalcin proximal promoter. Bisulfite sequencing analysis of tescalcin CpG island revealed that it is differentially methylated in male and female mouse embryonic gonads, and that hypermethylation of this region represses expression of tescalcin in the β-TC3 cell line. ^ The major tescalcin mRNA encodes a protein with 214 amino acids that contains a consensus EF-hand Ca2+-binding domain and an N-myristoylation motif. The amino acid sequence of tescalcin is highly conserved among various species, and it showed the highest homology with calcineurin B homologous proteins 1 and 2, and calcineurin B. Western blot analysis using antibodies generated against the tescalcin protein confirmed its presence in specific mouse tissues and cell lines. Immunohistochemical analysis of mouse embryos confirmed the pattern of expression of tescalcin mRNA in fetal testis. Using pull-down assays, glyceraidehydes-3-phosphate dehydrogenase was identified as an interacting and potential functional partner of tescalcin. ^ The identification and characterization of tescalcin as a novel embryonic testicular marker will contribute to the elucidation of the genetic pathways involved in testis development and likely to the understanding of pathological conditions such as sex reversal and infertility. ^

Evaluation of Some Statistical Methods for the Identification of Differentially Expressed Genes

Microarray platforms have been around for many years and while there is a rise of new technologies in laboratories, microarrays are still prevalent. When it comes to the analysis of microarray data to identify differentially expressed (DE) genes, many methods have been proposed and modified for improvement. However, the most popular methods such as Significance Analysis of Microarrays (SAM), samroc, fold change, and rank product are far from perfect. When it comes down to choosing which method is most powerful, it comes down to the characteristics of the sample and distribution of the gene expressions. The most practiced method is usually SAM or samroc but when the data tends to be skewed, the power of these methods decrease. With the concept that the median becomes a better measure of central tendency than the mean when the data is skewed, the tests statistics of the SAM and fold change methods are modified in this thesis. This study shows that the median modified fold change method improves the power for many cases when identifying DE genes if the data follows a lognormal distribution.

The potential role of environmental exposures and genomic signaling in development of central nervous system tumors

The etiology of central nervous system tumors (CNSTs) is mainly unknown. Aside from extremely rare genetic conditions, such as neurofibromatosis and tuberous sclerosis, the only unequivocally identified risk factor is exposure to ionizing radiation, and this explains only a very small fraction of cases. Using meta-analysis, gene networking and bioinformatics methods, this dissertation explored the hypothesis that environmental exposures produce genetic and epigenetic alterations that may be involved in the etiology of CNSTs. A meta-analysis of epidemiological studies of pesticides and pediatric brain tumors revealed a significantly increased risk of brain tumors among children whose mothers had farm-related exposures during pregnancy. A dose response was recognized when this risk estimate was compared to those for risk of brain tumors from maternal exposure to non-agricultural pesticides during pregnancy, and risk of brain tumors among children exposed to agricultural activities. Through meta-analysis of several microarray studies which compared normal tissue to astrocytomas, we were able to identify a list of 554 genes which were differentially expressed in the majority of astrocytomas. Many of these genes have in fact been implicated in development of astrocytoma, including EGFR, HIF-1α, c-Myc, WNT5A, and IDH3A. Reverse engineering of these 554 genes using Bayesian network analysis produced a gene network for each grade of astrocytoma (Grade I-IV), and ‘key genes’ within each grade were identified. Genes found to be most influential to development of the highest grade of astrocytoma, Glioblastoma multiforme (GBM) were: COL4A1, EGFR, BTF3, MPP2, RAB31, CDK4, CD99, ANXA2, TOP2A, and SERBP1. Lastly, bioinformatics analysis of environmental databases and curated published results on GBM was able to identify numerous potential pathways and geneenvironment interactions that may play key roles in astrocytoma development. Findings from this research have strong potential to advance our understanding of the etiology and susceptibility to CNSTs. Validation of our ‘key genes’ and pathways could potentially lead to useful tools for early detection and novel therapeutic options for these tumors.

High natural gene expression variation in the reef-building coral Acropora millepora: potential for acclimative and adaptive plasticity

Background: Ecosystems worldwide are suffering the consequences of anthropogenic impact. The diverse ecosystem of coral reefs, for example, are globally threatened by increases in sea surface temperatures due to global warming. Studies to date have focused on determining genetic diversity, the sequence variability of genes in a species, as a proxy to estimate and predict the potential adaptive response of coral populations to environmental changes linked to climate changes. However, the examination of natural gene expression variation has received less attention. This variation has been implicated as an important factor in evolutionary processes, upon which natural selection can act. Results: We acclimatized coral nubbins from six colonies of the reef-building coral Acropora millepora to a common garden in Heron Island (Great Barrier Reef, GBR) for a period of four weeks to remove any site-specific environmental effects on the physiology of the coral nubbins. By using a cDNA microarray platform, we detected a high level of gene expression variation, with 17% (488) of the unigenes differentially expressed across coral nubbins of the six colonies (jsFDR-corrected, p < 0.01). Among the main categories of biological processes found differentially expressed were transport, translation, response to stimulus, oxidation-reduction processes, and apoptosis. We found that the transcriptional profiles did not correspond to the genotype of the colony characterized using either an intron of the carbonic anhydrase gene or microsatellite loci markers. Conclusion: Our results provide evidence of the high inter-colony variation in A. millepora at the transcriptomic level grown under a common garden and without a correspondence with genotypic identity. This finding brings to our attention the importance of taking into account natural variation between reef corals when assessing experimental gene expression differences. The high transcriptional variation detected in this study is interpreted and discussed within the context of adaptive potential and phenotypic plasticity of reef corals. Whether this variation will allow coral reefs to survive to current challenges remains unknown.

Coral Thermal Tolerance: Tuning Gene Expression to Resist Thermal Stress

The acclimatization capacity of corals is a critical consideration in the persistence of coral reefs under stresses imposed by global climate change. The stress history of corals plays a role in subsequent response to heat stress, but the transcriptomic changes associated with these plastic changes have not been previously explored. In order to identify host transcriptomic changes associated with acquired thermal tolerance in the scleractinian coralAcropora millepora, corals preconditioned to a sub-lethal temperature of 3°C below bleaching threshold temperature were compared to both non-preconditioned corals and untreated controls using a cDNA microarray platform. After eight days of hyperthermal challenge, conditions under which non-preconditioned corals bleached and preconditioned corals (thermal-tolerant) maintained Symbiodinium density, a clear differentiation in the transcriptional profiles was revealed among the condition examined. Among these changes, nine differentially expressed genes separated preconditioned corals from non-preconditioned corals, with 42 genes differentially expressed between control and preconditioned treatments, and 70 genes between non-preconditioned corals and controls. Differentially expressed genes included components of an apoptotic signaling cascade, which suggest the inhibition of apoptosis in preconditioned corals. Additionally, lectins and genes involved in response to oxidative stress were also detected. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments; that is, differences in expression magnitude were more apparent than differences in the identity of genes differentially expressed. Our work revealed a transcriptomic signature underlying the tolerance associated with coral thermal history, and suggests that understanding the molecular mechanisms behind physiological acclimatization would be critical for the modeling of reefs in impending climate change scenarios.

Reverse Engineering of Modified Genes by Bayesian Network Analysis Defines Molecular Determinants Critical to the Development of Glioblastoma

In this study we have identified key genes that are critical in development of astrocytic tumors. Meta-analysis of microarray studies which compared normal tissue to astrocytoma revealed a set of 646 differentially expressed genes in the majority of astrocytoma. Reverse engineering of these 646 genes using Bayesian network analysis produced a gene network for each grade of astrocytoma (Grade I–IV), and ‘key genes’ within each grade were identified. Genes found to be most influential to development of the highest grade of astrocytoma, Glioblastoma multiforme were: COL4A1, EGFR, BTF3, MPP2, RAB31, CDK4, CD99, ANXA2, TOP2A, and SERBP1. All of these genes were up-regulated, except MPP2 (down regulated). These 10 genes were able to predict tumor status with 96–100% confidence when using logistic regression, cross validation, and the support vector machine analysis. Markov genes interact with NFkβ, ERK, MAPK, VEGF, growth hormone and collagen to produce a network whose top biological functions are cancer, neurological disease, and cellular movement. Three of the 10 genes - EGFR, COL4A1, and CDK4, in particular, seemed to be potential ‘hubs of activity’. Modified expression of these 10 Markov Blanket genes increases lifetime risk of developing glioblastoma compared to the normal population. The glioblastoma risk estimates were dramatically increased with joint effects of 4 or more than 4 Markov Blanket genes. Joint interaction effects of 4, 5, 6, 7, 8, 9 or 10 Markov Blanket genes produced 9, 13, 20.9, 26.7, 52.8, 53.2, 78.1 or 85.9%, respectively, increase in lifetime risk of developing glioblastoma compared to normal population. In summary, it appears that modified expression of several ‘key genes’ may be required for the development of glioblastoma. Further studies are needed to validate these ‘key genes’ as useful tools for early detection and novel therapeutic options for these tumors.

Identification of genes downstream of endothelin-3 signaling: characterization of WSB1 and SPC12

Signaling of endothelin-3 (Edn3) through its receptor, endothelin receptor B (EdnrB), has been shown to be indispensable for the development of certain neural crest derivatives. Since no research has been directed to investigate what the downstream targets of this signaling pathway are, the purpose of this study was to identify and characterize genes that are transcriptionally regulated by Edn3 signaling. Data from Differential Display RT-PCR of Edn-3 induced cDNA vs. non-induced cDNA obtained from primary neural crest cultures was analyzed. Thirty bands that were differentially expressed were sequenced and submitted for a homology search (BLAST). Among the genes identified were WSB 1 (a member of the SOCS family of negative regulators) and SPC 12 (the smallest subunit, 12kDa, of mammalian signal peptidase). Using whole-mount in-situ hybridization, the expression patterns of EdnrB, WSB 1 and SPC 12 were characterized. WSB 1 and SPC 12 expression patterns were found to overlap with that of EdnrB, suggesting that Edn3 might regulate the transcription of these genes in specific neural crest derived lineages.