Role of cell adhesion in Xenopus PGC migration

For the translocation of the cell body, directionally migrating cells need to interact with their environment. Unlike lamellipodia-based migration, where cells rely on adhesion to generate traction force (Giannone et al., 2007; Le Clainche and Carlier, 2008), cells migrating via bleb-associated mechanism are usually characterized by a decrease in adhesive activity (Fackler and Grosse, 2008). Interaction with a substrate can be achieved through weak adhesion to the surrounding cells or to the extra-cellular matrix (Fig. 6A). Conversely, blebbing cells can exert forces on the surrounding environment perpendicular to the direction of movement (Fig. 6B). In this latter case, a cell can squeeze itself forward without or with little adhesion to the substrate (Charras and Paluch, 2008). Interestingly, some cell types, like lymphocytes and cancer cells, can switch between lamellipodia-based migration and bleb-associated motility depending on the extracellular environment. Migration via lamellipodia that relies on the adhesion to the substrate and actin polymerization at the leading edge to generate traction force, is more efficient for the migration along the rigid environment, like basal membrane, or extracellular matrix filaments. In contrast, migration via a blebbing-associated mechanism is beneficial when cell has to pass through a randomly oriented 3D environment and cannot form specific adhesion contacts (Charras and Paluch, 2008; Fackler and Grosse, 2008).

Fig. 6. Models of bleb-associated motility. (A) In two-dimensional (2D) cultures, in order to translate polarized blebbing into movement, the cell must adhere to the substrate. When a new bleb is formed and comes in contact with the substrate, new cell–substrate adhesions are formed and the cell mass can stream forward. The pink dots indicate cell–substrate attachment points. (B) When the cell is in a confined environment (for example, between two glass coverslips or in a thin microfluidic channel), it can move in the absence of cell–substrate adhesions. Instead, the cell exerts forces perpendicularly to the substrate and can squeeze itself forward. (C) When the cell is migrating in an extracellular matrix (ECM) gel (three-dimensional (3D) matrix), it can move by a combination of the mechanisms described. The fluid nature of growing blebs enables the cell to squeeze through the ECM network mesh. The dashed line indicates the position of the leading edge before bleb nucleation, arrows indicate the forces that are exerted by the cells on the extracellular environment and dashed arrows indicate the streaming of cytoplasm (taken from Charras and Paluch, 2008).

As mentioned above, active PGC migration in Xenopus starts after stage 24, when the cluster, formed by these cells at earlier stages, starts to disperse. In addition, PGCs also change their morphology, from a spherical and poorly blebbing to a motile elongated form (Fig. 3) (Nishiumi et al., 2005; Terayama et al., 2012). In normal Xenopus embryos, although PGCs migrate as a cohort, they do not form direct contacts with one another. Analysis of sectioned tailbud (stage 28-36) and early tadpole (stage 42-45) stage embryos showed that the area of contact between PGCs and somatic cells is relatively small, leaving gaps between these cells. In contrast, somatic cells surrounding PGCs, both in vivo and in vitro, exhibit a high degree of cell-cell contact formation (Heasman and Wylie, 1978; Kamimura et al., 1976;

1980).

Independent observations coming from the analysis of different germ plasm associated RNAs have suggested that regulation of cell adhesion is involved in PGC development in Xenopus embryos. The Germes protein, encoded by one of these RNAs, contains two leucine zipper motifs and putative calcium binding EF-hand domain, but doesn’t show substantial homology to other known proteins (Berekelya et al., 2003). It co-localizes with two dynein light chains (dlc8a and dlc8b) and is suggested to regulate germ plasm formation and development. Overexpression of Germes results in a decrease of the average number of PGCs at the tailbud stage (stage 33/34). Furthermore, the remaining PGCs failed to migrate laterally and were found deep in the endoderm. This phenotype might either be a direct result of Germes-mediated effect on cytoskeletal motor complexes in the PGCs, or an indirect one through alterations in germ plasm organization (Berekelya et al., 2007).

Clustering in the endoderm and subsequent loss of the PGCs was also observed upon knock-down of Dead end (Dnd) and Xdazl. These RNA-binding proteins are also encoded by maternal germ plasm specific mRNAs, and their function in PGC development was discussed previously (see section 1.2.5). The role of Dnd in the initiation of PGC migration was already described in zebrafish (see section 1.3.2). However, the molecular mechanisms that might link Xdazl, Germes and Dnd functions in the context of PGC migration in fish and frogs remains to be defined.

After they reach the dorsal body wall, PGCs leave the endoderm and migrate to the gonads via the dorsal mesentery, a thin stripe of connective tissue that links the dorsal body wall and the gut. From the mesentery, PGCs then migrate laterally to the forming genital ridges, enter the gonads, and differentiate into germ line stem cells capable of forming the gametes (Wylie and Heasman, 1976; 1993). Similar to the cells migrating within the endoderm, PGCs isolated from Xenopus laevis embryos at stage 42-45 show plasma membrane blebbing on artificial substrates (Wylie and Roos, 1976; Heasman et al., 1977).

When seeded on a monolayer of amphibian mesentery cells, such spherical PGCs attach to the substrate and form filopodia-like protrusions (Fig. 3). Similar to bleb-associated migration, translocation is mediated by the contraction of the cell body and cytoplasm being pushed forward, resulting in an elongation of the cell. Following detachment of the rear,

cells return to a spherical shape upon arrival in the new position. Similar cell shapes of PGCs were observed in sectioned embryos (Heasman et al., 1977; Heasman and Wylie, 1978).

Adhesion of PGCs to somatic mesentery cells was discovered to be mediated by fibronectin, a large extracellular protein that can bind to extracellular matrix components and membrane-spanning receptor proteins called integrins (Heasman et al., 1981; Pankov and Yamada, 2002). Mesentery cells produce large amounts of fibronectin both in vivo and in vitro. Isolated PGCs, in contrast, do not secrete detectable quantities of fibronectin in vitro, but are able to adhere to fibronectin and also fibronectin-producing cells, regaining migratory and invasive ability (Heasman et al., 1981; Wylie and Heasman, 1982; Brustis et al., 1984). Interestingly, fibrils of fibronectin formed by mesentery cells in vitro were demonstrated to be often co-linear with microfilament bundles within the cells (Heasman et al., 1981; Wylie and Heasman, 1982). As PGCs frequently formed filopodia-like protrusions and were elongated in the same direction as underlying cells, it was suggested that somatic cells might influence directionality of PGC migration through the dorsal mesentery. For the future, it remains a major challenge to define the regulation of these events on a molecular level and to tie them to the function of the regulators of PGC development and migration in Xenopus, in particular the set of RNA binding proteins that are already known to play a major role in this context.