Identification of Developmental Signaling Pathways with a Novel Role in Regulating Zebrafish Primordial Germ Cell Migration

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Normal 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.

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The file named "BOLDT-DISSERTATION-2013.pdf" is the primary dissertation file. Four (4) supplemental videos are also provided (in Audio Video Interleaved format).
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