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Dev Growth Differ
2013 Feb 01;552:217-28. doi: 10.1111/dgd.12018.
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Developmental regulation of locomotive activity in Xenopus primordial germ cells.
Terayama K
,
Kataoka K
,
Morichika K
,
Orii H
,
Watanabe K
,
Mochii M
.
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Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species-specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re-examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz-like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re-aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re-aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F-actin, myosin II activity and RhoA/Rho-associated protein kinase (ROCK) signaling.
Figure 1. Localization of primordial germ cells (PGCs) in tailbud embryos. (a�d) Transverse section of embryos at stage 24 (a), 28 (b), 33/34 (c) and 41 (d). PGCs were labeled with anti-Xenopus Daz-like protein antibody (magenta; a�d). Nuclei were labeled with Hoechst 33342 (green; a′�d′) 3D images of the in vivo localization of PGCs at stage 24 (e), 28 (f), 33/34 (g) and 41 (h) were constructed with Delta Viewer. PGCs, red. Dorsal side up in all photos, with anterior to the left. (e′�h′) Schematic diagrams of localization of PGCs in tailbud embryos. Very small Dazl-positive fragments did not contain nuclei. PGCs, magenta. Endodermal region, yellow. Scale bars: 100 μm.
Figure 2. Morphology of isolated primordial germ cells (PGCs) and somatic endodermal cells. (a�f) PGCs isolated from embryos at stages 24 (a), 28 (b), 33/34 (c) and 41 (d), and endodermal cells isolated from embryos at stages 24 (e) and 28 (f) were cultured in the fibronectin-coated culture chamber. Insets show fluorescence images of the cells. The PGCs were labeled with Venus fluorescence. Arrows indicate the small blebs. Arrowheads indicate the large clear blebs. Images were taken after cells adhered on the substrate. Scale bars: 20 μm.
Figure 3. Time-lapse observation of isolated primordial germ cells (PGCs) and somatic cells. (a�f) A PGC isolated from stage 24 (a), 28 (b), 33/34 (c) or 41 (d), or an endodermal cell from stage 24 (e) or 28 (f) was cultured in the fibronectin-coated culture chamber. Arrows indicate small blebs. Arrowheads indicate the large clear blebs. Photos were taken at 10 s intervals after PGCs adhered to the substrate. Scale bars: 20 μm.
Figure 5. Localization of F-actin and roles of cytoskeletons in migrating primordial germ cells (PGCs). (a�d) Localization of F-actin in isolated PGCs. A representative PGC isolated from an embryo at each stage was stained with rhodamine-phalloidin. Arrows in (a) indicate the small blebs. Arrows in (b) indicate the rear region. Arrowheads in (b) indicate the front of an elongated PGC. Arrows in (c) indicate the large bleb. (e�h) Elongated PGCs at stage 28 were treated with indicated inhibitors. Photos were taken 30 min after adding the chemicals. Scale bars: 20 μm
Figure 6. Effects of inhibiting Rho family GTPase signals on primordial germ cell (PGC) migration. (a�d) Lateral views of stage 41 embryos injected with Venus-DS mRNA (460 pg) (a), or co-injected with Venus-DS mRNA (460 pg) and mRNA for RhoA-DN (460 pg) (b), Rac1-DN (460 pg) (c) or Cdc42-DN (2.3 ng) (d). Dorsal side up and anterior to the left. (e, f) Isolated PGCs from stage 28 embryos injected with RhoA-DN (460 pg) or Rac1-DN (460 pg) were cultured on fibronectin-coated petridishes. (g, h) Elongated PGCs isolated from stage 28 embyos were incubated with Y27632 (ROCK inhibitor) or NSC27632 (Rac1 inhibitor). Photos were taken 30 min after adding the chemicals. Arrowheads indicate the PGCs in the normal region. Arrows indicate the ectopic PGCs. Scale bars: 1 mm in d; 20 μm in (e�h). (i) Effects of DN-small GTPases on PGC localization in vivo. Venus-DS mRNA (460 pg) was injected into all the embryos for PGC labeling, and additionally indicated mRNAs were injected. PGC localization was observed externally from both sides at stage 41. Graph shows the percentages of embryos with indicated PGCs. Values are includes results of three experiments. (j, k) Effects of DN-small GTPases on elongation (j) and locomotion (k) of isolated PGCs. Time-lapse images of the stage 28 PGCs were captured at 1 min intervals for 30 min after 1 h of cultivation. If a PGC showed an elongated shape during the culture period, it was counted as an elongated PGC. If a PGC moved more than 100 μm for 30 min, it was counted as a locomoting PGC. Values are results of three experiments. C; control injected with only Venus-DS mRNA. *P < 0.05, **P < 0.001.
