PGC migration in Drosophila

As mentioned in the section 1.1.2, Drosophila PGCs first undergo passive involution inside the embryo and after gastrulation can be found in the posterior midgut pocket surrounded by midgut epithelium. During these stages of embryonic development, PGCs are tightly associated with each other and with somatic cells. Several data suggest that PGCs have migratory ability shortly after their formation, but adhesion might prevent active PGC migration (Santos and Lehmann, 2004a; Kunwar et al., 2006). When PGCs arrive in the posterior midgut, they form tight cluster with each other, but form little contact with the surrounding epithelium. In this cluster cells become organized in a radial manner and are polarized with a leading edge facing outwards. One of the key regulators involved in PGC polarization is Trapped in endoderm 1 (TRE1). The corresponding mRNA is maternally supplied and encodes a G-protein coupled receptor which belongs to the rhodopsin family.

Most likely, it acts through the small GTPase RhoI, but the exact molecular mechanism of

TRE1 function in PGCs is not known (Richardson and Lehmann, 2010). Since TRE1 is provided maternally, an unknown ligand coming from the surrounding tissues was suggested to initiate PGC polarization and subsequent migration (Kunwar et al., 2006). Polarization by a TRE1-dependent mechanism could cause redistribution of the Gβ subunit of the heterotrimeric G-protein, Rho1 and adherent junction components (E-cadherin and catenins) from the periphery to the cell rear, facing the inside of the PGC cluster. Polarized PGCs start to extend cellular protrusions at the leading edge towards the surrounding midgut epithelium. Transition to active migration is associated with a loss of contacts between the PGCs. They begin to disperse from the cluster and migrate as individual cells through the posterior midgut (Fig. 2a1) (Richardson and Lehmann, 2010). Epithelium cells of the posterior midgut also undergo reorganization. They lose apical junctions that results in the formation of the gaps between the cells. This process was shown to be critical for the passage of the migrating PGCs (Jaglarz and Howard, 1994).

Once PGCs pass through the midgut, they reorient on its surface and migrate dorsally along the epithelium. Later they leave the midgut and migrate towards the posterior mesoderm. This migration depends on surrounding tissues and is regulated by two related proteins with redundant functions, Wunen (Wun) and Wunen2 (Wun2). Wun and Wun2 are expressed in most ventral regions of posterior midgut and other tissues that PGCs normally avoid during their migration. Knock-down of the Wunens causes loss of directionality during PGC migration, while their overexpression in mesoderm prevents PGC migration towards this region (Kunwar et al., 2006). Both wun and wun2 genes encode homologs of mammalian lipid phosphate phosphatases (LPPs), transmembrane exoenzymes that have catalytic phosphatase domain on the cell surface. Although in vitro experiments helped to identify several phospholipid substrates for LPPs and Wunens, in vivo targets for both classes of proteins remain unknown (Fig. 2a2) (Richardson and Lehmann, 2010). It has been shown that LPPs and Wunens are not only responsible for the hydrolysis of the phospholipids, but also facilitate uptake of dephosphorylated lipids by Wunen- or LPP-expessing cells (Roberts and Morris, 2000). The exact mechanism of Wunens’ function in PGC migration remains unclear, but two possible models were suggested. According to one model, Wun and Wun2 expressing cells produce PGC repellent, that facilitates migration of PGCs away from Wun/Wun2-positive cells. The alternative model suggests that PGC migration and/or survival might depend on certain extracellular factor that can be processed by Wun/Wun2 expressing cells. According to the second model, somatic Wun/Wun2-producing cells locally deplete a putative attraction factors creating an inverse gradient and forcing PGCs to migrate away from this region (Richardson and Lehmann, 2010).

Migration of Drosophila PGCs to the somatic gonad precursors in the mesoderm is also regulated by the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR, also known as Columbus) pathway. Expression of HMGCoAR in the mesoderm is required for directional PGC migration towards this tissue and association of PGCs with somatic gonad precursors. In addition, ectopic expression of HMGCoAR in ectoderm or nervous tissue causes abnormal PGC migration towards these regions (Van Doren et al., 1998). This

suggests that the HMGCoAR pathway is involved in the production of yet unidentified signal necessary and sufficient to attract PGCs. HMGCoAR regulates several pathways within animal cells, mainly cholesterol synthesis and protein isoprenylation. However, genes necessary for cholesterol synthesis are not present in Drosophila genome and this pathway is not active in these species. On the other hand, mutation in other genes involved in the isoprenylation pathway revealed a requirement of this pathway for Drosophila PGC migration (Santos and Lehmann, 2004b). This suggests two possibilities how HMGCoAR could contribute to directional PGC migration. According to one hypothesis, the isoprenylation pathway is directly involved in a modification of the hypothetical attractant guiding PGCs. Alternatively, the isoprenylation pathway could be rate limiting for expression or secretion of the attractant, for example by modifying small GTPases like Ras and Rabs (Fig.

2a3) (Kunwar et al., 2006).

Fig. 2. Molecular regulation of active PGC migration in Drosophila, zebrafish and mice. (a) Initiation and transepithelial migration of Drosophila PGCs through the midgut is controlled by the G-protein coupled receptor (GPCR) TRE-1. Upon polarization of PGCs, there is redistribution of Rho1 to the cell rear, which can act as downstream target of TRE-1. Wun and Wun2 are expressed at sites that PGCs avoid, like ventral midgut. They may participate in PGC repulsion, or hydrolyse putative phospholipid attractant or survival factor to create a gradient for PGC migration. Migration of PGCs to the somatic gonads depends on the isoprenylation branch of HMGCoAR pathway. It may act in the attachment of geranyl-geranyl (GG) group to a putative chemoattractant, or factors necessary for its secretion, such as MDR49. (b) In zebrafish, PGCs are guided by a gradient of the chemoattractant SDF-1α. They express the receptor CXCR4b that also belongs to the GPCR family. Another somatically expressed GPCR, CXCR7B, promotes the internalization and degradation of SDF-1^α that might lead to proper gradient formation and precise targeting of PGCs. (c) In mice, PGC migration to the genital ridges is also controlled by CXCR4 and SDF1. SDF1 is expressed by the somatic cells of the genital ridge and PGCs express CXCR4. Integrin β1 is also required for this step. PGC motility and survival requires the receptor tyrosine kinase c-Kit and its ligand Steel. Steel is expressed by somatic cells surrounding PGCs throughout migration (taken from Richardson and Lehmann, 2010).

In the final step of the active migration, Drosophila PGCs divide into two clusters within the mesoderm and associate with somatic gonadal precursors. PGCs initially associate with posterior gonadal precursor clusters and then move anteriorly during germ band retraction. At this point, PGCs round up, loose their motility and form tight contacts with each other and with somatic cells in a process known as coalescence (Jaglarz and Howard, 1994). This process correlates with the level of HMGCoR expression in somatic gonadal precursors, since ectopic expression of HMGCoAR leads to an arrest of PGC migration in HMGCoAR-expressing tissues (Van Doren et al., 1998). This can be explained by the highest concentration of potential attractant in this region. Interestingly, formation of cell clusters by somatic gonadal precursors could occur even in the absence of PGCs (Brookman et al., 1992). Formation of contacts between the cells requires expression of Fear of intimacy (Foi) and Drosophila E-cadherin (DE-cadherin, also known as Shotgun). Foi encodes a conserved transmembrane protein, which belong to a family of putative zinc transporters. Based on the role of this family of proteins in cell migration, it has been proposed that Foi may be involved in the regulation of adhesion molecule expression, for example in the regulation of E-cadherin levels (Santos and Lehmann, 2004a). However, additional factors might mediate coalescence, since initial interactions between PGCs and somatic cells are not affected in foi or DE-cadherin mutants.