Signaling pathways involved in PGC migration in Xenopus

In order to migrate directionally, blebs should form primarily on the leading edge of the cell. Most of the cells that migrate via bleb-associated mechanisms are guided by chemoattractants. For the amoeba Dictyostelium discoideum such attractants are nutrients and cAMP, for neutrophils – complement factor C5a, platelet-activating factor (PAF) and formylated Met-Leu-Phe (fMLP) (Charras and Paluch, 2008; Fackler and Grosse, 2008). As discussed above, PGCs derived from zebrafish, chicken and mouse are guided by a system consisting of the chemoattractant stem cell derived factor 1 (SDF-1), also known as Chemokine C-X-C motif ligand 12 (CXCL12), and its receptor C-X-C chemokine receptor type 4 (CXCR4) (Doitsidou et al., 2002; Molyneaux et al., 2003; Stebler et al., 2004).

In the case of Xenopus PGCs, cell migration is guided by chemoattractants, originating from the dorsal part of the embryo. Isolated Xenopus laevis PGCs can be polarized and migrate towards extracts prepared from stage 30-31 embryos lacking endoderm (Fig. 5) (Tarbashevich et al., 2011). Interestingly, expression of chemoattractant SDF-1 in X. laevis embryos at the tailbud stage can be observed in dorsal and anterior structures: mid- and hindbrain, otic vesicles and eyes, the dorsal fin, and the posterior heart anlage (Braun et al., 2002). Expression of the receptor CXCR4 can be observed in PGCs at tailbud stages 24-40 (Nishiumi et al., 2005). Overexpression of SDF-1 in the embryos upon knock-down of its repressor IRX5 leads to mislocalization of PGCs due to a loss of directionality in their migration within the endodermal cell mass (Bonnard et al., 2012). In addition, interference with endogenous SDF-1 or CXCR4 expression results in a decreased number of PGCs arriving at the genital ridges (Takeuchi et al., 2010). Thus, even though it seems highly likely that this signaling system has a role in PGC polarization and migration, an exact guidance mechanism for Xenopus PGCs remains to be determined.

CXCR4, as well as other receptors involved in chemotaxis, belongs to G-protein coupled receptor (GPCR) family. In the context of cell migration, activated heterotrimeric G-proteins activate various downstream pathways, including calcium flux (Dutt et al., 1998;

Blaser et al., 2006), Phospholipase C (PLC) and Phosphatidylinositide 3-kinase (PI3-kinase)

Fig. 5. PGC polarization and migration on fibronectin towards the dorsal extract source. (A) PGCs were isolated form tailbud stage embryos injected at the two-cell stage with EGFP_GRPI_PH_DE mRNA. This chimeric RNA encodes PIP3 sensor fused GFP, and also contains Dead end localization element to target expression specifically in PGCs. Isolated cells were transferred on fibronectin-coated Petri dish in DFA buffer. One sector of the dish was covered with agarose gel. Tailbud stage embryos were dissected to obtain homogenized dorsal (DEx) and ventral (VEx) extracts. These extracts were injected into the opposite corners of the agarose sector. (B) Snapshots from the time-lapse movie illustrating polarization and migration of PGCs on the fibronectin towards the dorsal protein extract (neuro-mesodermal extract, NME) source in the DFA-medium. White arrows depict PIP3-enriched bleb-like protrusions formed by the cells. The red arrow indicates the PGC migrating towards the chemoattractant source (according to Tarbashevich et al, 2011).

(Wang et al., 2000). Activation of PI3-kinases was reported to be one of the major events for polarization of many migratory cells (Chung et al., 2001; Iijima et al., 2002). PI3-kinases generate inositol phospholipids that bind to a subset of PH domain-containing molecules thus recruiting them to the membrane. There are three clases of enzymes in PI3-kinase family (Katso et al., 2001). Class I PI3-kinases catalyze the phosphorylation of the 3’ hydroxyl subunit of PIP2 converting it into PIP3. This class of enzymes was shown to be involved in the regulation of cellular polarization by the localization of PIP3 to the leading edge of the cell (Meili et al., 1999; Haugh et al., 2000; Servant et al., 2000; Merlot and Firtel, 2003; Dumsteri et al., 2004). Asymmetrical localization of PIP2 and PIP3 in the cell facilitates the recruitment of PIP3-specific PH domain-containing proteins, primary effectors of PI3-kinase signaling pathway, to the leading edge. The restriction of PIP3 to the leading edge of the cell is also influenced by the function of PI3K antagonist PTEN (phosphatase and tensin homolog), a phosphoinositide 3’-specific phosphatase that dephospharylates PIP3 to PIP2 (Maehama and Dixon, 1998). Studies in Dictyostelium have revealed that, in resting cells, PTEN is localized to the plasma membrane and is uniformly distributed all over the cell (Funamoto et al., 2002;

Iijima and Devreotes, 2002). In chemotaxing cells, PTEN is downregulated at the leading edge, but persists at the sides and the rear of the cell. Thus, PTEN prevents the accumulation of PIP3 exclusively at these places, resulting in cellular polarization. In addition, distribution of PIP2 and PIP3 can influence formation of the bleb-like protrusions, as discussed above (see section 1.4.2).

In vitro migration of murine PGCs was reported to be activated by the Kit ligand (KL) as a guiding cue, and also described to depend on the PI3-kinase pathway (Farini et al., 2007). In the zebrafish model system, the in vivo motility of PGCs depends on appropriate PIP3 levels, but a polarized distribution of PIP3 was not observed. It was proposed that PI3K activity might be linked to substrate adhesion, rather than to polarization (Dumstrei et al., 2004). Polarization of Xenopus PGCs correlates with asymmetries in respect to the intracellular PIP3 distribution (Tarbashevich et al., 2011). PIP3 is found to be enriched in the bleb-like protrusions formed by isolated PGCs at migratory stage. Downregulation of endogenous PIP3 levels leads to a decrease in PGC number and to abnormal PGC migration.

Loss of endogenous PIP3 is also coupled to a loss of plasma membrane blebbing. One of the molecules involved in generating PIP3 asymmetries in Xenopus PGCs is the kinesin KIF13B.

The corresponding maternal mRNA localizes to the germ plasm and can be later detected in PGCs at the tailbud stages of development. Knock-down of KIF13B leads to the loss of PIP3 enrichment and inhibits formation of bleb-like protrusions in PGCs isolated from stage 30-32 embryos. In contrast, KIF13B overexpression leads to increased plasma membrane blebbing and PIP3 enrichment throughout the plasma membrane (Tarbashevich et al., 2011). Thus, similar to what is observed upon reducing the cellular levels of PIP3, knock down of KIF13B results in a decrease of PGC number and abnormal PGC migration. In good correlation, KIF13B was previously shown to be involved in the polarization of hippocampal neurons prior to axonal growth. It was suggested that KIF13B might contribute to the local enrichment of PIP3 at the tip of growing neurites by directional transport of PIP3-containg

vesicles, mediated by the interaction with a PIP3-binding protein (PIP3BP or Centaurin-α1) (Venkateswarlu et al., 2005; Horiguchi et al., 2006). However, the exact molecular mechanism by which KIF13B influences the PIP3 distribution in Xenopus PGCs remains to be determined.

As outlined above, PIP3 polarization was described to be relevant for a number of different cells migrating via a bleb-associated mechanism, including Dictyostelium amoeba and vertebrate neutrophils (Franca-Koh et al., 2006; Franca-Koh et al., 2007; Yoo et al., 2010). In the case of Dictyostelium, however, multiple parallel pathways appear to be involved in chemotaxis, with each individual one, such as the PI3K pathway, being dispensable (Hoeller and Kay, 2007; King and Insall, 2008; Van Haastert and Veltman, 2007).

Several observations suggest that multiple pathways might also be involved in regulating the directional migration of Xenopus PGCs. One of these comes from experiments characterizing the Xenopus glutamate receptor interacting protein 2 (XGRIP2). Similar to KIF13B, XGRIP2 is also encoded by germ plasm specific maternal mRNA. GRIP multi-PDZ domain family proteins usually serve as adaptor proteins and they are involved in various processes, including cell–matrix interactions during embryogenesis in mammals and control of directional migration of embryonic muscle cells in Drosophila (Takamiya et al., 2004; Swan et al., 2004). Knock-down of XGRIP2 resulted in decelerated PGC migration at tailbud stages and a decrease in the average number of PGCs. PGCs in XGRIP2 morphants were mislocalized to more posterior positions at stage 33/34 (Tarbashevich et al., 2007). These observations on PGC migration in XGRIP2 morphant embyos were confirmed in a second, independent study, also revealing that the ability of the PGCs to enter dorsal mesentery was also significantly impaired (Kirilenko et al., 2008).

Another pathway involved in regulation of PGC migration in Xenopus is the one depending on Notch/Suppressor of Hairless [Su(H)]. The Notch receptor and its ligands Delta and Serrate are single-pass transmembrane proteins. When Notch binds to one of these ligands, the Notch intracellular domain (NICD) is released and translocates into the nucleus, where it forms a complex together with Su(H) and regulates gene expression (Lai, 2004).

Suppression or activation of this pathway in the endoderm of Xenopus embryos starting from stage 18, as well as PGC-specific activation, resulted in defects in PGC migration in the endoderm. In this case PGCs failed to reach the dorsal mesentery by stage 41 and were found ectopically in the endoderm. Similar results were observed upon knock-down of X-Delta-2, one of the ligands of Notch (Morichika et al., 2010). Activation of the Notch pathway leads to the induction of the expression of HES and HES-related genes that encode basic helix-loop-helix type transcriptional repressors (Iso et al., 2003). Activation of such genes in the PGCs, as well as in somatic endodermal cells was observed upon activation of Notch signaling in the endoderm of Xenopus embryos. Altogether these data suggest that proper levels of Notch/Su(H) activity in the endoderm are required for normal PGC migration.

Perturbation of this pathway may affect directionality, motility and/or adhesion of PGCs (Morichika et al., 2010).