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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02522

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Transition from non-motile behaviour to directed migration during early PGC development in zebrafish

¹ Germ Cell Development, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
² Department for Developmental Biology, Institute for Biology I, Freiburg University, 79104 Freiburg, Germany
³ Current address: Biozentrum, Department of Cell Biology, Klingelbergstrasse 70, 4056 Basel, Switzerland
⁴ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP10142, 67404 Illkirch Cedex, France

View larger version (61K): [in a new window]   Fig. 1. Early labelling of PGCs in live embryos. (A) Schematic representation of the kop promoter region fused to EGFP-F-nos1-3'UTR that was used to generate transgenic fish. (B) Whole mount in situ hybridization of progeny of transgenic females showing the distribution of egfp RNA. The inset included in the image depicting a 22 hpf embryo shows a magnification of the region where the PGCs normally reside. (C) Specific expression of EGFP-F in the zebrafish germline starting at the earliest stages following its formation. No specific EGFP-F expression prior PGC specification (e.g. 2.75 hpf) can be detected.

View larger version (50K): [in a new window]   Fig. 2. Marked alterations in migratory behaviour are detectable during earliest stages of PGC development. (A) Snapshots from a low magnification time-lapse movie recorded over 4 hours of zebrafish development. The area marked in the top left panel is magnified in the rest of the panels. The germ cells remain in their positions for about 1.5 hours following PGC specification and start to migrate actively around dome stage (4.5 hpf, supplementary material Movie 1, n=4 embryos). An asterisk marks a PGC, which leaves the cell cluster. (B-E) High-magnification snapshots from time-lapse movies of wild-type embryos (see supplementary material Movies 2-5). (B) At 3 hpf, PGCs exhibit simple morphology. (C,D) At 3.7 hpf (C) up to 4.3 hpf (D) the cells develop small, as well as long complex protrusions extended in random directions, yet are not polarized and do not migrate. At these stages, the cells show round morphology during cell divisions (D). (E) Soon after 4.3 hpf, PGCs exhibit polarized elongated cell morphology and extend pseudopodia in the direction of migration. (F) Snapshots from a low magnification 1-hour long time-lapse movie of PGCs from a 12 hours donor embryo in a 3 hpf host embryo. The transplanted cells migrate in the early host (supplementary material Movie 6, three cells in two embryos examined). (G) Transplanted cells similar to those in F show polarized elongated cell morphology and migration behaviour similar to PGCs after 4.3 hpf (supplementary material Movie 7, 40x magnification). The white scale bars represent 10 µm and the white arrowheads indicate the direction of migration.

View larger version (89K): [in a new window]   Fig. 3. Zygotically transcribed genes are essential for PGC polarization and motility. (A) Embryos treated with -amanitin (lower panels) exhibit gastrulation defects, most pronounced is the inhibition of epiboly (arrowheads). (B) Transcription of the zygotically expressed genes [represented here by RT-PCR analysis for no tail (ntl)] is inhibited by -amanitin (lower panel) whereas the level of maternally provided transcripts [represented here by RT-PCR analysis for vasa (vas)] is unaffected. (C) Snapshots from low-magnification time-lapse movies recorded over 2 hours of zebrafish development showing a representative PGC cluster. The germ cells in control embryos (upper panels, one cell labelled with an asterisk) migrate actively and leave the cluster following the transition whereas PGCs in -amanitin treated embryos (lower panels) remain clustered and immotile. (D) Snapshots from high-magnification time-lapse movies of control (upper panels) and -amanitin treated PGCs (lower panels). PGCs treated with -amanitin remain arrested in the second phase of their differentiation extending protrusions in all directions and fail to polarize (supplementary material Movies 8 and 9). (E) Snapshots from high-magnification time-lapse movies. 12 hpf old wild-type PGCs transplanted into an -amanitin treated host embryo are not able to migrate relative to host cells, but show cell morphology characteristic of cells of their age. (F) 7 hpf old -amanitin treated PGCs transplanted into a 5.3 hpf wild-type host remain arrested in the second phase of their maturation displaying extensions in all directions and fail to polarize and migrate. The apparent movement of the cluster in the lower panels is a result of passive movement together with somatic cells. (G) At 4.3 hpf, Dead end knockdown (dnd-MO) PGCs behave like wild-type cells of a similar age. At 4.7 hpf, dnd-MO treated germ cells do not polarize and migrate but are capable of extending small protrusions and divide (supplementary material Movies 10 and 11). Bars, 10 µm.

View larger version (65K): [in a new window]   Fig. 4. E-cadherin is dynamically regulated during early stages of PGC development. (A) The expression level of E-cadherin protein is altered during early development in wild-type PGCs (top row, blue, the contours of the relevant cells are delineated with white dots), but remains constant in PGCs knocked down for Dead end (bottom row). (B) A graphic representation of the quantitative analysis examining the relative E-cadherin level on PGCs. Regions in PGC and somatic cell membranes were selected for analysis and the average pixel intensity obtained from the PGC membrane (the inner half of the membrane, red) was divided by the average pixel intensity of the somatic cell membrane (the inner half of the membrane, yellow). (C) Wild-type PGCs at 5.3 hpf show significantly reduced E-cadherin levels relative to earlier stages (comparing 5.3 hpf with 3 hpf). dnd-MO treated PGCs showed no such change in E-cadherin levels on the membrane. (D) PGCs in which full-length E-cadherin is forced expressed show a strong reduction in cell motility (e.g. the cell marked with the green arrow). The PGCs exhibit extensive non-polarized protrusive activity (high magnification snapshots). (E) PGC migration speed is severely reduced compared with wild-type PGCs. (F) Embryos forced expressing E-cadherin (122 embryos examined) in PGCs show 30% ectopic germ cells in comparison to 5% of control embryos (120 embryos examined). (G) In 22 hpf embryos in which E-cadherin is forced expressed in the PGCs, ectopic PGCs can be observed (black arrowheads, cells labelled using a GFP probe). In (C,E) n is the number of cells analysed, the error bars represent the standard error of the mean (s.e.m.), asterisk signifies P<0.001.

View larger version (61K): [in a new window]   Fig. 5. The progression of PGC responsiveness to SDF-1a. (A) PGCs (green) do not respond to SDF-1a expressed by transplanted cells (red), from the time of their specification until 4.5 hpf. (B) At 5.5 hpf, the PGCs (green) migrate actively towards the SDF-1a expressing transplant (red) and remain in close proximity to it. Two PGCs migrating toward the SDF-1a source are marked with an asterisk and an arrowhead. (C) A control experiment shows that migrating PGCs (green) remain indifferent to transplanted cells (red) that do not express SDF-1a. The images were obtained from time-lapse movies generated for each experiment (see supplementary material Movies 12-14). Bars, 100 µm.

View larger version (17K): [in a new window]   Fig. 6. Differentiation stages during early PGC development leading to motility, polarization and guided migration. Following their specification, a process which is controlled by maternally provided germ plasm (Hashimoto et al., 2004; Knaut et al., 2000; Yoon et al., 1997), the PGCs appear morphologically similar to somatic cells, yet they show expression of characteristic markers and can be specifically labelled with GFP. During the next stage, the PGCs assume a complex non-polarized morphology, express high levels of E-cadherin on their membrane and do not respond to guidance cues provided by the chemokine SDF-1a. Further differentiation depends on de novo transcription in the zygote since inhibition of transcription by -amanitin leads to a complete developmental arrest of PGCs at the second phase. The transition to the third stage relies on the function of the Dead end protein. This stage is characterized by moderate down regulation of E-cadherin and the competence of PGCs to polarize and migrate in response to directional cues.

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