Browsing by Subject "Germ Cells"
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Item BMP Signaling Regulates Germ Cell Pluripotency, Sexual Differentiation, and Cancer Susceptibility(2016-10-14) Sanchez, Angelica; Castrillon, Diego H.; Amatruda, James F.; Cobb, Melanie H.; Cleaver, OndineTesticular germ cell tumors are the most common malignancy found in young men between the ages of 14-40. While these tumors are highly curable with cisplatin based combined chemotherapies, the treatments come with very detrimental side effects and ultimately fail in up to 15% of patients. These patients have no other avenues of treatment and often times succumb to the disease. Very little is currently known about the biology of the tumors but risk factors highlight possible mechanisms of action. For example, patients with Disorders of Sex Development (DSD) have increased risk for developing malignant germ cell tumors. DSDs manifest at birth and present with atypical gonadal or anatomical sex as well as chromosomal aberrations[1, 2]. Some cases are explained by the presence of chromosomal aberrations, but the cause of many others remains unknown. Such syndromes thus highlight the potential links between germ cell pluripotency, sexual differentiation and cancer susceptibility. A recent GWAS study [3] identified association of BMP7 with testicular dysgenesis syndrome; however the molecular mechanisms behind this association remain unknown. Previously, we described the development of testicular germ cell tumors in zebrafish carrying a mutation in bmpr1bb, a BMP family receptor, and demonstrated that human GCTs have defects in BMP signaling. Here I use this model and next-generation sequencing analysis in a cross-species comparative oncology approach to identify genes and pathways with fundamental importance to the development of the human disease. I further defined the role of specific BMP ligands in mediating germ cell differentiation and identified reciprocal somatic- and germ cell BMP signaling events that regulate germ cell differentiation and maturation. Using genetic crosses to further impair BMP signaling, I found that zebrafish doubly heterozygous for mutations in bmpr1bb and bmp2b, bmp7a or smad5 have profoundly impaired gonadogenesis and altered male:female sex ratios. Affected fish also exhibit markedly abnormal gonadal differentiation, including the presence of undifferentiated gonadal tissue and the occurrence of biphenotypic gonads, as well as greatly increased germ cell tumor susceptibility. Our findings implicate defective BMP pathway signaling as a potential factor in DSDs and GCT susceptibility. Our goals are to identify biological mechanisms that govern germ cell differentiation and to understand how defects in this process cause human disease.Item Identification of Developmental Signaling Pathways with a Novel Role in Regulating Zebrafish Primordial Germ Cell Migration(2013-07-01) Boldt, Clayton Ryan; Lum, Lawrence; Amatruda, James F.; Cobb, Melanie H.; Buszczak, MichaelNormal cell migration is critical for embryonic patterning, organ development and immune response. Primordial germ cell (PGC) migration in zebrafish has proven a valuable model for the study of cell motility. In zebrafish, PGC migration is guided principally by the chemokine Sdf-1a and its receptors Cxcr4b in germ cells, and Cxcr7b in somatic cells. While a role has also been suggested for PI3K signaling, the possible contribution of other signaling pathways in PGC migration is not fully understood. In this study, I used inhibitors of early developmental signaling pathways to identify those with a novel role in PGC migration. Among these compounds, the most significant effects were from the Tankyrase inhibitor IWR-1, which blocks β-catenin-dependent Wnt signaling. IWR-1 treatment during periods of active PGC migration results in fewer germ cells reaching the gonad. Treatment with the Porcupine inhibitor IWP-L6, which blocks Wnt production, did not result in substantial effects on PGC migration, suggesting that the effects of IWR-1 may not be dependent on β-catenin activity, but on the direct IWR-1 target, Tankyrase. Treatment with XAV939, an additional Tankyrase inhibitor, phenocopied the effects of IWR-1. Germ cell-targeted inhibition of Wnt signaling did not phenocopy the effects of IWR-1 and XAV939, arguing against a germ cell-autonomous role for Wnt signaling in PGC migration. We observed significant changes in the expression and patterning of sdf-1a, cxcr4b and cxcr7b following IWR-1 treatment, but not Wnt inhibition. Thus, I conclude that Tankyrase has an important role in patterning the PGC migratory environment during early development. I also sought to determine the eventual fate of ectopic PGCs in zebrafish. I created a transgenic line with GFP-labeled PGCs that has enabled us to follow ectopic germ cells during the first two weeks of development. Daily monitoring of ectopic PGCs revealed that they are not cleared, as in mice, but persist in their ectopic locations. In several instances, I observed ectopic germ cells undergoing morphological changes, followed by loss of GFP expression. The results of these observations lend credibility to the hypothesis that extragonadal germ cell tumors could arise from PGCs that do not migrate properly during development.Item A Novel Transgenic Rat Model for the Study of Germ Cell Biology(2005-08-11) Cronkhite, Jennifer T.; Garbers, David L.With over one million publications in scientific journals, the rat is a very important biological model in science. Unfortunately, since the introduction of genetic manipulation technology in the mouse, extension of this technology to the rat has proven to be very difficult. In an attempt to generate a transgenic line of rats expressing GFP in all cells of the body, a serendipitous integration of a ROSA-EGFP transgene resulted in exclusive expression of EGFP in the germ cells of both sexes. EGFP expression was uniform and robust in cleavage stage embryos beginning at the late 2-cell stage and continuing through blastocyst development where expression became restricted to cells of the inner cell mass. Subsequent analysis showed high EGFP expression exclusively in primordial, embryonic, and adult germ cells. This unique expression pattern makes this EGFP marked locus the first molecular marker of the germline lineage in both sexes in mammals. FISH was used to localize the transgene insertion to chromosome 11q11-q12, proximal to Grik1 and in close proximity to Ncam2. Analysis of the region did not identify known germ cell-specific genes but did identify 19 ESTs or transcribed loci present in testes, ovary, or pre-implantation libraries from mice or rats. The unique germ cell specific expression of EGFP in these transgenic rats makes them an excellent novel tool to study germ cell origin, development, and differentiation. To evaluate the utility of the transgenic line for germ cell transplantation studies, non-selected, freshly isolated seminiferous tubule cells were transferred to the testis of recipient males. The donor cell population colonized the testis at a surprisingly high efficiency within 30 days following transfer. Since EGFP is a vital marker, the colonization process can be followed in vivo and the extent of colonization quantified. This assay was then used to define when developing germ cells first acquire apparent stem cell activity, and to assess the plasticity of adult SP bone marrow cells to enter the germ lineage.