Molecular mechanisms governing primordial germ cell migration in zebrafish

In most sexually reproducing organisms primordial germ cells (pGCs) are specified early in development in places that are distinct from the region where the somatic part of the gonad develops. From their places of specification they have to migrate towards the site where they associate with somatic gonadal cells and differentiate to the gametes. The question conceming the molecular mechanisms that guide PGCs during their migration and allow them to reach their target is the focus of this work. The process was investigated in zebrafish, where the extrauterine development of the embryos their translucency allows monitoring cell migration at high resolution.Previous studies showed that zebrafish PGCs are specified in four different positions. From these positions the cells perform distinct migration steps until they arrive at their target by the end of the flTst day of development. During their migration the cells are guided by cues provided by somatic tissues.To identify the actual molecules that function as the guidance cues, a largescale antisense-oligonucleotide-based screen was carried out. In this screen, a chemokine receptor, CXCR4b and its ligand, the chemokine SDFla, were identified as proteins required for guided PGC migration. This pair of molecules had previously been shown to guide cell migration in other model organisms in a variety of developmental processes and disease. For example, SDF-l/CXCR4 signalling guides leul(Ocytes to the sites of inflarnmation or metastatic tumour ce lis to sites where they form secondary tumors.We found that in zebrafish embryos, the receptor CXCR4b is expressed in the migrating PGCs and its ligand, SDFla, is expressed in the tissues along which the PGCs migrate. Knocking down either CXCR4b or SDFla impairs PGC directed migration, which becomes evident by the inability of the cells to reach their target. Furthermore, when SDFla was expressed in ectopic sites in the embryos, PGCs arrived at these sites thus demonstrating the instructive role of this chemokine in PGC migration. Together, these results strongly suggest that SDFla provides the directional cue for PGC migration in zebrafish. These findings have since been generalized to mouse and chicken, where it was shown that CXCR4 and SDF-I play an essential role in PGC migration as weIl.Interestingly, in Drosophila a different biochemical pathway was shown to be important for providing directional cues for migrating PGCs, namely, the cholesterol/isoprenoid biosynthesis pathway. To determine whether this pathway plays a similar role in zebrafish, a 'block and rescue' pharmacogenetic approach was employed. Small chemical compounds were utilized to inhibit distinct steps in the cholesterol and isoprenoid synthesis pathway and the effect on PGC migration was examined. Using this approach, we showed that blocking HMGCoAR reductase (an enzyme that catalyses the rate limiting step in cholesterol synthesis) results in slower PGC migration. As a consequence, PGCs in treated embryos were frequently found in Abnormal locations. We then determined which components of the Cholesterol/isoprenoid biosynthetic pathway that act downstream of HMGCoAR are involved in this process. We could demonstrate that Geranylgeranyl transferase I (GGTI) activity in the isoprenoid branch of the pathway is important for optimal PGC motility.