Cultivation of X. laevis PGCs on the artificial substrates

As discussed previously, migration of X. laevis PGCs during tailbud stage of embryonic development occurs via a bleb-associated mechanism. In order to migrate, cells need to interact with their environment to generate the traction force necessary for translocation.

Fig. 7. Labeling and isolation of X. laevis PGCs. X. laevis embryos at 2-cell stage were injected with in vitro transcribed GFP_DELE mRNA, consisting of green fluorescence protein (GFP) open reading frame (ORF) fused to Dead end localization element (DELE). Injections were performed in the vegetal side of both blastomeres. Schematic representation of 2-cell stage embryo corresponds to a lateral view; animal pigmented side is up, vegetal is down. Embryos were cultivated till neurula (St.17-19; ventral (left) and dorsal (right) view, anterior side is to the right) or tailbud (St.28-30; lateral view, anterior side is to the right) stages of embryonic development. Endodermal explants, highlighted by the white dashed line, containing PGCs and somatic endodermal cells were dissected from the embryos and dissociated on the agarose-coated Petri dish by enzymatic treatment with mild protease (accutase). Dissociated cells can be distinguished as GFP-positive PGCs and GFP-negative somatic endodermal cells.

There are two major models describing how plasma membrane bleb-like protrusions can contribute to directional migration. One model is based on the formation of specific contacts between the migrating cell and the substrate. In the alternative model, a traction force is generated by compressive forces applied by the cell on the substrate. In the latter case, adhesion is not required, but the cell should be surrounded by at least two resistant surfaces (Charras and Paluch, 2008; Fig. 6). To establish an in vitro migration assay on artificial substrates, both of these models were taken in consideration. Isolated PGCs, therefore, were either cultivated on top of, or embedded into the substrate.

First, PGCs isolated from tailbud stage embryos were cultivated in the presence of extracellular matrix (ECM) that contained mainly laminin, collagen type IV, heparan sulfate proteoglycan and entactin. Cells were either premixed with ECM, or applied on top of the polymerized substrate. Homogenized dorsal extract was added in the pocket made in the agarose gel on one side of the culture dish (Fig. 10). Cells were cultivated up to 24 hours.

Fig. 8. Cultivation of isolated PGCs within the endodermal cell mass did not facilitate active migration.

(A) Ventral explants were dissected from tailbud stage embryos injected with GFP_DELE mRNA to label PGCs. Explants were dissociated via enzymatic treatment. Isolated PGCs (green) were transferred in between two layers of endodermal cells (grey). These layers were obtained by dissociation of endodermal explant from uninjected embryos; isolated cells were pre-cultivated on top of the fibronectin-coated culture dish, or fibronectin-coated glass cover slip. Fibronectin coating was used for the adhesion and spreading of the endodermal cells. (B) Representative time-lapse images of PGCs (green) recorded 1 hour after cultivation in the endodermal cell mass. The upper row represents images taken under UV light to identify GFP-positive PGCs. The middle row corresponds to the images taken at the same time under brightfield transmitted light. The lower panel represents merged images. Despite active cellular dynamics, migration of PGCs was not observed. Cells were cultivated up to 24 hours, but after several hours of cultivation cellular dynamics was significantly decreased. Relative time after the first recorded image (hours : minutes : seconds) is indicated in the upper left corner of the images, scale bar: 50 µm.

Within first few hours, PGCs formed bleb-like protrusions, and some of the cells started to contract from one side in order to migrate, but could not move from their point of origin (Fig. 10C). Later, formation of protrusions and overall cellular dynamics were decreased.

Cultivation in different buffer conditions, including cultivation in DFA, DMEM and 0.8x MBSH, did not show any difference in PGC behavior. Similar results were obtained upon cultivation of PGCs in the agarose gel. PGCs were either cultivated on top, or covered with the second layer of the gel (Fig. 11). Variation in the concentration of agarose in the range from 0,1 to 0,5% (m/v) did not affect the results. However, if concentration of the covering agarose gel exceeded 0,5%, PGCs gote smashed.

In a similar approach, known as under-agarose migration assay (Tranquillo et al., 1988), isolated PGCs were placed on the bottom of the culture dish underneath a layer of agarose gel. In this approach, few cells could initiate migration via the bleb-associated mechanism within first several hours (Fig. 12). Later, similar to the cultivation in the other tested conditions, PGCs decreased formation of the bleb-like protrusions and were unable to migrate. Application of homogenized dorsal extract did not lead to an increased motility of the cells. Although in this assay many PGCs showed migratory behavior, characterized by elongated shape and polarized waves of the cell body contraction, only few cells could initiate active migration. This can be explained by the ‘stickiness’ of the plastic surface of the culture dish that ‘anchored’ the cells and prevented translocation of the cell body. To improve efficiency of migration, culture dishes were coated with either fibronectin, or bovine serum albumin (BSA). Coating with fibronectin did not alter the efficiency of

Fig. 9. Isolated PGCs cultivated on mammalian cell substrate formed bleb-like protrusions, but did not exhibit migratory behavior. (A) Ventral explants were dissected from tailbud stage embryos injected with GFP_DELE mRNA to label PGCs. Explants were dissociated via enzymatic treatment. Isolated PGCs (green) were seeded on top of a monolayer of a mammalian cell line, HEK 293, and cultivated in DFA buffer up to 24 hours. (B) Representative time-lapse images of a PGC (GFP-positive, green) recorded approximately 1 hour after the cells were seeded on the surface of mammalian cells. PGC formed bleb-like protrusions (white arrows), but did not reveal high cellular dynamics and did not initiate migration. Merged images are generated from UV and normal transmitted light channels. Relative time after the first recorded image (hours : minutes : seconds) is indicated in the upper left corner of the images, scale bar: 50 µm.

migration, while on BSA-coated culture dishes migration was increased from 1-2 cells per experiment to almost 10% of the analyzed PGCs (Fig. 16A).