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First published online 31 August 2004
doi: 10.1242/jcs.01362

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Signaling pathways controlling primordial germ cell migration in zebrafish

Germ Cell Development, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37070 Göttingen, Germany

View larger version (108K): [in a new window]   Fig. 1. Expression of pertussis toxin (PTX) affects directional PGC migration but not cell identity or motility. (A,B) Expression of PTX results in PGCs localized to ectopic positions by 24 hpf (arrowheads in B), as compared with control embryos where PGCs are found in their proper position (arrow in A). Yet, the ectopic PGCs properly stabilize the GFP-nos1-3'UTR RNA and express GFP. (C,D) Ectopic PTX-expressing PGCs show normal expression of nos1 mRNA (arrowheads in D) similar to control PGCs (arrow in C). (E,F) 6-somite-stage embryos that had been injected with PTX-nos1-3'UTR show normal somatic development as determined by the expression of sdf-1a mRNA (red stain). At this stage, the majority of PGCs (marked by nos1 mRNA, blue stain) in PTX-injected embryos are located outside sdf-1a-expressing regions (arrowheads in F) in contrast to control embryos (E), where the majority of PGCs are located within regions of sdf-1a expression. (G,H) PTX-expressing PGCs are motile. Images taken from time-lapse movies of control embryos (G; see also Movie S1 in supplementary material) and PTX-injected embryos (H; see also Movie S2 in supplementary material) at three different time points encompassing 2 hours of development starting at the end of gastrulation. In H, three individual PGCs are marked using red, green and blue arrows to illustrate the motility of these cells with respect to somatic tissue. Embryos are shown in a dorsal view with anterior up.

View larger version (19K): [in a new window]   Fig. 4. The localization of PHCRAC-GFP to the membrane of PGCs is independent of SDF-1a/CXCR4b signaling. Localization of PHCRAC-GFP in (A) control PGCs, (B) cells expressing dnPI3K, (C) cells expressing CXCR4b morpholino (MO) or (E) PTX, as well as in PGCs in (D) an ody/ody mutant embryo. All cells also express the Vasa-GFP fusion protein, which labels the perinuclear granules. Arrows in A-E indicate the perinuclear granules.

View larger version (58K): [in a new window]   Fig. 2. Synergistic interaction between pertussis toxin and CXCR4b. (A) The severity of PGC migration phenotype induced by PTX RNA or CXCR4b morpholino (MO) is concentration dependent. Different amounts either of control RNA and control morpholino (turquoise), CXCR4b morpholino (green) or PTX RNA (blue) were injected. The arrow indicates the amount used for the synergism experiment. (B) Injections of control RNA and control morpholino (turquoise, n=67 embryos), CXCR4b morpholino (green, n=64 embryos) or PTX-nos1-3'UTR RNA (blue, n=84 embryos) result in 0.1, 1.3 and 2.3 average number of ectopic PGCs per embryo at 24 hpf, respectively. Injection of both CXCR4b morpholino and PTX-nos1-3'UTR RNA (red, n=110 embryos) results on average in 7.3 ectopic PGCs per embryo. (C) Representative images of 24 hpf embryos from the experiment presented in (B).

View larger version (69K): [in a new window]   Fig. 3. Localization of the PH-GFP fusion proteins in wild-type PGCs. (A,B,C) Images are taken from 63x time-lapse movie showing PGCs labeled with PHCRAC-GFP at the indicated time points. A PGC in (A) shows stable localization of PHCRAC-GFP to the membrane (Movie S3 in supplementary material). (B) No differential membrane localization can be observed either prior to the formation of a protrusion (indicated by an arrow) or at the site of the protrusion itself (indicated by an arrowhead; Movie S3 in supplementary material). (C) No polarized membrane localization of PHCRAC-GFP can be seen in a PGC undergoing directed migration relative to somatic cells (Movie S4 in supplementary material). The arrow marks the direction of migration of the PGC whereas the white symbol marks a neighboring somatic cell serving as a reference point. (D) A PGC showing a uniform membrane localization of PHAkt-GFP, whereas the PGC in (E) shows weak and spotty membrane expression of PHGRP1-GFP. The PGC in (F) shows uniform distribution of PHTAPP1-GFP. (G) Non-confocal images of PGCs labeled with PHAkt-GFP. The arrow marks a forming protrusion showing no increased fluorescence. Brighter fluorescence is mostly apparent at regions of cell-cell contact (Movie S5 in supplementary material).

View larger version (90K): [in a new window]   Fig. 5. Expression of dnPI3K in PGCs results in their localization to ectopic positions. (A,B) Expression of dnPI3K-nos1-3'UTR results in PGCs in ectopic positions at 24 hpf (arrowheads in B) as compared with control embryos where PGCs are located in their proper positions (arrow in A). Embryos were co-injected with GFP-nos1-3'UTR to label the PGCs. (C,D) dnPI3K-nos1-3'UTR-injected embryos show normal morphology (D) as demonstrated by in situ hybridizations of embryos at the 6-7-somite stage stained with sdf-1a (red) and nos1 (blue) and compared with a control embryo (C). High-magnification inset in C shows PGCs in regions of sdf-1a expression in control embryos whereas inset in D shows a subset of PGCs in a region of non-sdf-1a expression in dnPI3K-injected embryos. Arrows in B,D and high-magnification inset in F indicate the presence of PGCs in their proper position in dnPI3K-injected embryos. (E,F) PGCs located in ectopic positions in dnPI3K-injected embryos show normal expression of nos1 mRNA (arrowheads in F) similar to PGCs in control embryos (arrow in E). (G) Graph illustrating the percentages of embryos with ectopic PGCs. 2% of control embryos (green bar) had 2 PGCs located in ectopic locations at 24 hpf as compared with 63% of dnPI3K-nos1-3'UTR injected embryos (red bar) showing 2 ectopic PGCs by 24 hpf. (H) Graph illustrating the average speed of PGC migration in control embryos (green bar) and dnPI3K-nos1-3'UTR-injected embryos (red bar). Control PGCs migrate at an average speed of 1.7 µm/min (n=23) compared with PGCs that express dnPI3K, which migrate at a rate of 1.3 µm/min (n=21; P<0.05).

View larger version (79K): [in a new window]   Fig. 6. PI3K activity affects PGC morphology and filopodia stability. (A) The outline of 8 control PGCs and (B) 8 PGCs expressing dnPI3K. The outlines of PGCs were generated as described in the Materials and Methods. Expression of dnPI3K in PGCs reduces filopodia stability and length (D; Movie S7 in supplementary material), as compared with control embryos (C; Movie S6 in supplementary material). Arrows in C and D indicate filopodia. The area demarcated in red is enlarged in the right panels.

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