William Boswell

Characterization and differential expression of CPD and 6-4 DNA photolyases in Xiphophorus species and interspecies hybrids.

Among the many Xiphophorus interspecies hybrid tumor models are those that exhibit ultraviolet light (UVB) induced melanoma. In previous studies, assessment of UVB induced DNA damage and nucleotide excision DNA repair has been performed in parental lines and interspecies hybrids. Species and hybrid specific differences in the levels of DNA damage induced and the dark repair rates for cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine pyrimidine photoproducts (6-4PPs) have been reported. However, UVB induced DNA lesions in Xiphophorus fishes are thought to primarily be repaired via light dependent CPD and 6-4PP specific photolyases. Photolyases are of evolutionary interest since they are ancient and presumably function solely to ameliorate the deleterious effects of UVB exposure. Herein, we report results from detailed studies of CPD and 6-4PP photolyase gene expression within several Xiphophorus tissues. We determined photolyase gene expression patterns before and after exposure to fluorescent light in X. maculatus, X. couchianus, and for F1 interspecies hybrids produced from crossing these two parental lines (X. maculatus Jp 163 B×X. couchianus). We present novel results showing these two photolyase genes exhibit species, tissue, and hybrid-specific differences in basal and light induced gene expression.

Sex-specific molecular genetic response to UVB exposure in Xiphophorus maculatus skin.

In both Xiphophorus fishes and humans, males are reported to have a higher incidence of melanoma than females. To better understand sex-specific differences in the molecular genetic response to UVB, we performed RNA-Seq experiments in skin of female and male Xiphophorus maculatus Jp 163 B following UVB doses of 8 or 16kJ/m(2) exposure. Male X. maculatus differentially express a significantly larger number of transcripts following exposure to 16kJ/m(2) UVB (1293 genes) compared to 8kJ/m(2) UVB (324 genes). Female skin showed differential gene expression in a larger number of transcripts following 8kJ/m(2) UVB (765) than did males; however, both females and males showed similar numbers of differentially expressed genes at 16kJ/m(2) UVB (1167 and1293, respectively). Although most modulated transcripts after UVB exposure represented the same dominant pathways in both females and males (e.g., DNA repair, circadian rhythm, and fatty acid biosynthesis), we identified genes in several pathways that exhibited opposite modulation in female vs. male skin (e.g., synaptic development, cell differentiation, wound healing, and glucose metabolism). The oppositely modulated genes appear related through uncoupling protein 3 (UCP3) that is involved with the regulation of fatty acid oxidation and serves to balance glucose and lipid metabolism. Overall, these results identify gender-specific differences in UVB-induced genetic profiles in the skin of females and males and show female and male X. maculatus respond to UVB differently through pathways involved in reactive oxygen species, wound healing, and energy homeostasis.

Molecular genetic analysis of the melanoma regulatory locus in Xiphophorus interspecies hybrids

Development of spontaneous melanoma in Xiphophorus interspecies backcross hybrid progeny, (X. hellerii × [X. maculatus Jp 163 A × X. hellerii]) is due to Mendelian segregation of a oncogene (xmrk) and a molecularly uncharacterized locus, called R(Diff), on LG5. R(Diff) is thought to suppresses the activity of xmrk in healthy X. maculatus Jp 163 A parental species that rarely develop melanoma. To better understand the molecular genetics of R(Diff), we utilized RNA‐Seq to study allele‐specific gene expression of spontaneous melanoma tumors and corresponding normal skin samples derived from 15 first generation backcross (BC1) hybrids and 13 fifth generation (BC5) hybrids. Allele‐specific expression was determined for all genes and assigned to parental allele inheritance for each backcross hybrid individual. Results showed that genes residing in a 5.81 Mbp region on LG5 were exclusively expressed from the X. hellerii alleles in tumor‐bearing BC1 hybrids. This observation indicates this region is consistently homozygous for X. hellerii alleles in tumor bearing animals, and therefore defines this region to be the R(Diff) locus. The R(Diff) locus harbors 164 gene models and includes the previously characterized R(Diff) candidate, cdkn2x. Twenty‐one genes in the R(Diff) region show differential expression in the tumor samples compared to normal skin tissue. These results further characterize the R(Diff) locus and suggest tumor suppression may require a multigenic region rather than a single gene variant. Differences in gene expression between tumor and normal skin tissue in this region may indicate interactions among several genes are required for backcross hybrid melanoma development.

Comparison of Xiphophorus and human melanoma transcriptomes reveals conserved pathway interactions

Comparative analysis of human and animal model melanomas can uncover conserved pathways and genetic changes that are relevant for the biology of cancer cells. Spontaneous melanoma in Xiphophorus interspecies backcross hybrid progeny may be informative in identifying genes and functional pathways that are similarly related to melanoma development in all vertebrates, including humans. To assess functional pathways involved in the Xiphophorus melanoma, we performed gene expression profiling of the melanomas produced in interspecies BC1 and successive backcross generations (i.e., BC5) of the cross: X. hellerii × [X. maculatus Jp 163 A × X. hellerii]. Using RNA‐Seq, we identified genes that are transcriptionally co‐expressed with the driver oncogene, xmrk. We determined functional pathways in the fish melanoma that are also present in human melanoma cohorts that may be related to dedifferentiation based on the expression levels of pigmentation genes. Shared pathways between human and Xiphophorus melanomas are related to inflammation, cell migration, cell proliferation, pigmentation, cancer development, and metastasis. Our results suggest xmrk co‐expressed genes are associated with dedifferentiation and highlight these signaling pathways as playing important roles in melanomagenesis.

C3HeB/FeJ MiceMimic many aspects of Gene Expression and Pathobiological Features of Human Hepatocellular Carcinoma: C3HeB/FeJ Mice Mimic Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Transcriptome assembly and candidate genes involved in nutritional programming in the swordtail fish Xiphophorus multilineatus

Background Nutritional programming takes place in early development. Variation in the quality and/or quantity of nutrients in early development can influence long-term health and viability. However, little is known about the mechanisms of nutritional programming. The live-bearing fish Xiphophorus multilineatus has the potential to be a new model for understanding these mechanisms, given prior evidence of nutritional programming influencing behavior and juvenile growth rate. We tested the hypotheses that nutritional programming would influence behaviors involved in energy homeostasis as well gene expression in X. multilineatus. Methods We first examined the influence of both juvenile environment (varied in nutrition and density) and adult environment (varied in nutrition) on behaviors involved in energy acquisition and energy expenditure in adult male X. multilineatus. We also compared the behavioral responses across the genetically influenced size classes of males. Males stop growing at sexual maturity, and the size classes of can be identified based on phenotypes (adult size and pigment patterns). To study the molecular signatures of nutritional programming, we assembled a de novo transcriptome for X. multilineatus using RNA from brain, liver, skin, testis and gonad tissues, and used RNA-Seq to profile gene expression in the brains of males reared in low quality (reduced food, increased density) and high quality (increased food, decreased density) juvenile environments. Results We found that both the juvenile and adult environments influenced the energy intake behavior, while only the adult environment influenced energy expenditure. In addition, there were significant interactions between the genetically influenced size classes and the environments that influenced energy intake and energy expenditure, with males from one of the four size classes (Y-II) responding in the opposite direction as compared to the other males examined. When we compared the brains of males of the Y-II size class reared in a low quality juvenile environment to males from the same size class reared in high quality juvenile environment, 131 genes were differentially expressed, including metabolism and appetite master regulator agrp gene. Discussion Our study provides evidence for nutritional programming in X. multilineatus, with variation across size classes of males in how juvenile environment and adult diet influences behaviors involved in energy homeostasis. In addition, we provide the first transcriptome of X. multilineatus, and identify a group of candidate genes involved in nutritional programming.

Expression signatures of early-stage and advanced medaka melanomas

Melanoma is one of the most aggressive tumors with a very low survival rate once metastasized. The incidence of newly detected cases increases every year suggesting the necessity of development and application of innovative treatment strategies. Human melanoma develops from melanocytes localized in the epidermis of the skin to malignant tumors because of deregulated effectors influencing several molecular pathways. Despite many advances in describing the molecular changes accompanying melanoma formation, many critical and clinically relevant molecular features of the transformed pigment cells and the underlying mechanisms are largely unknown. To contribute to a better understanding of the molecular processes of melanoma formation, we use a transgenic medaka melanoma model that is well suited for the investigation of melanoma tumor development because fish and human melanocytes are both localized in the epidermis. The purpose of our study was to gain insights into melanoma development from the first steps of tumor formation up to melanoma progression and to identify gene expression patterns that will be useful for monitoring treatment effects in drug screening approaches. Comparing transcriptomes from juvenile fish at the tumor initiating stage with nevi and advanced melanoma of adults, we identified stage specific expression signatures and pathways that are characteristic for the development of medaka melanoma, and are also found in human malignancies.

Characterization of basal gene expression trends over a diurnal cycle in Xiphophorus maculatus skin, brain and liver

Evolutionarily conserved diurnal circadian mechanisms maintain oscillating patterns of gene expression based on the day-night cycle. Xiphophorus fish have been used to evaluate transcriptional responses after exposure to various light sources and it was determined that each source incites distinct genetic responses in skin tissue. However, basal expression levels of genes that show oscillating expression patterns in day-night cycle, may affect the outcomes of such experiments, since basal gene expression levels at each point in the circadian path may influence the profile of identified light responsive genes. Lack of knowledge regarding diurnal fluctuations in basal gene expression patterns may confound the understanding of genetic responses to external stimuli (e.g., light) since the dynamic nature of gene expression implies animals subjected to stimuli at different times may be at very different stages within the continuum of genetic homeostasis. We assessed basal gene expression changes over a 24-hour period in 200 select Xiphophorus gene targets known to transcriptionally respond to various types of light exposure. We identified 22 genes in skin, 36 genes in brain and 28 genes in liver that exhibit basal oscillation of expression patterns. These genes, including known circadian regulators, produced the expected expression patterns over a 24-hour cycle when compared to circadian regulatory genes identified in other species, especially human and other vertebrate animal models. Our results suggest the regulatory network governing diurnal oscillating gene expression is similar between Xiphophorus and other vertebrates for the three Xiphophorus organs tested. In addition, we were able to categorize light responsive gene sets in Xiphophorus that do, and do not, exhibit circadian based oscillating expression patterns.