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Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target

¹ Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
² Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch cedex, CU de Strasbourg, France

View larger version (59K): [in a new window]   Fig. 1. Zebrafish PGCs form bilateral clusters in the anterior trunk during early somitogenesis. (A,B) Dorsal views of zebrafish embryos depicting the movements (arrows) of PGCs that result in bilateral PGC cluster formation. The PGCs (red) are drawn relative to the adaxial cells, the somites and the lateral edges of the trunk mesoderm. (A) At the end of gastrulation, most PGCs have accumulated in two medial-to-lateral lines at the head-trunk border, while the rest aligns along the lateral trunk mesoderm borders in more posterior regions. During early somitogenesis, both groups of cells migrate towards the lateral mesoderm of the anterior trunk (steps IV and V). (B) At the six-somite stage, bilateral clusters of PGCs have formed in the anterior trunk, while the posterior trailing PGCs continue to migrate towards the anterior. (C-L) Fluorescent pictures taken at 16 minute intervals from a time-lapse movie showing migrating PGCs between the bud- and seven-somite stage on the right side of a wild-type embryo injected with GFP-nos1-3'UTR. Dorsal views, anterior is upwards. Medially located PGCs migrate laterally (one cell is marked by a green arrow) to join those PGCs that are already located in lateral positions. The forming cluster follows the general convergence movements medially towards the midline. A single ectopic anterior cell (red arrow) migrates posteriorly and laterally into the cluster. Note that on this side of the embryo no posterior trailing PGCs can be seen.

View larger version (66K): [in a new window]   Fig. 2. Ectopic anterior PGCs migrate posteriorly towards the main clustering position in spt mutant embryos. (A-J) Fluorescent pictures of embryos injected with GFP-nos1-3'UTR. Dorsal views, anterior is upwards. (A) In wild-type embryos, most PGCs are located in two medial-to-lateral lines at the head-trunk border at the one-somite stage. (B-J) Time-lapse cinematography of a spt mutant embryo starting at early somitogenesis. During the 3.5 hours shown, the embryo was kept at 25°C, thus it developed to approximately the six-somite stage. On each side of the embryo, a pair of PGCs that were initially located close to each other in ectopic anterior regions is marked by arrowheads and arrows. Note that two of these cells end up in the ectopic anterior cluster (arrowheads), while the others migrate over a considerable distance posteriorly towards the main clustering position (arrows).

View larger version (152K): [in a new window]   Fig. 3. Zebrafish PGCs migrate actively. (A-F) Fluorescent pictures taken at the indicated intervals from a time-lapse movie showing migrating PGCs during early somitogenesis in a wild-type embryo injected with GFP-nos1-3'UTR and EGFP-F-globin marking the outlines of somatic cells. The full movie can be found at http://dev.biologists.org/supplemental/ (Movie 1). Individual PGCs are marked by colored asterisks and a single somatic cell by a green arrow. Note that the PGCs move extensively relative to the somatic mesodermal cells. The apparent cell shape changes of somatic cells seen in A-F are largely due to different focal planes at which the pictures were taken to keep the PGCs in focus. (G-I) Fluorescent pictures taken at the indicated intervals from a time-lapse movie showing migrating PGCs during mid-somitogenesis in a wild-type embryo injected with EGFP-F-nos1-3'UTR marking the outlines of PGCs. The full movie can be found at http://dev.biologists.org/supplemental/ (Movie 2). Note the highly dynamic processes extended by the PGC on the left side.

View larger version (56K): [in a new window]   Fig. 4. wt1 is expressed in the putative PGC attraction center of the anterior trunk at early somitogenesis. All pictures shown are dorsal views of flatmounts of whole-mount in situ stained embryos with wt1 in blue in A-C, nos1 in blue in D and other markers in red (see Materials and Methods). (A) At the two-somite stage, wt1 is exclusively expressed in the lateral mesoderm of the anterior trunk with a slight extension into the head, as seen in comparison with myoD in red, which stains the adaxial cells and the forming somites. Expression is confined to the mesoderm, as can be seen in side view (data not shown). Prior to the one-somite stage, no expression can be detected in whole-mount in situ staining. (B) By the six-somite stage, wt1 expression has extended posteriorly to the fifth somite. (C) Beginning at around the 10-somite-stage, wt1 expression starts to extend into the lateral mesoderm of the head and into the anterior halves of the first four somites, which do not express myoD. At about this stage, the PGCs start to migrate posteriorly. (D) The PGC clusters, which form between the one- and six-somite stages, are located within the wt1-expressing region, as seen by comparison with B and shown here for the eight-somite-stage.

View larger version (100K): [in a new window]   Fig. 5. PGC cluster formation correlates with proper differentiation of the target tissue. (A,C,E,G,I,K) Flat-mounts of embryos at the six- to eight-somite stage stained for wt1 in blue. (B,D,F,H,J,L) Flat-mounts of embryos at the same stage stained with nos1 in blue to visualize the PGCs. The position of the main clusters of PGCs in the anterior trunk is marked by an arrow and that of the ectopic anterior clusters by an arrowhead. Embryos have been stained with other markers in red or blue for identification of mutants and for providing positional landmarks (see Materials and Methods). Note that wt1 is expressed at normal or only slightly reduced levels in all embryos that show clustering of PGCs in the anterior trunk (spt, oep and oep;ntl). (M-O) Lateral views of embryos at 24 hpf stained for nos1 in blue. The position of the main PGC clusters is marked by an arrow and that of the ectopic anterior cluster by an arrowhead. PGC main clusters have formed in the correct region at the anterior end of the yolk extension in wild-type (M) and spt (N), but not in spt;ntl (O) double mutants. Note the presence of PGCs in between the ectopic anterior and the main clustering positions in spt;ntl mutants (O).

View larger version (121K): [in a new window]   Fig. 6. The lateral mesoderm of the anterior trunk comprises an intermediate target of PGCs. (A,B) Fate-mapping of the somatic cells that surround the main PGC clusters at early somitogenesis. Cells that contain the uncaged fluorescein lineage tracer are labeled in red and PGCs in blue using nos1 as probe. (A) Flat-mount of an embryo at the six-somite stage, fixed immediately after the uncaging procedure. Note that the uncaged region includes the PGC cluster. (B) Lateral view of a deyolked embryo at 24 hpf. Note that the cells containing the lineage tracer (bracket) and the PGCs (arrow) have separated and that no labeled somatic cells are detectable in the region of the PGCs.

View larger version (41K): [in a new window]   Fig. 7. PGCs actively migrate towards their final target. (A-D) Fluorescent pictures taken at the indicated intervals from a time-lapse movie (the full movie can be found at http://dev.biologists.org/supplemental/ (Movie 3) showing the main cluster of PGCs during late somitogenesis on the right side of an embryo injected with full-vasa-GFP. Dorsal views, anterior is upwards. The Vasa-GFP fusion protein is localized into perinuclear granules in the PGCs, which migrate posteriorly relative to the somites; one somite boundary is marked with a black line.

View larger version (77K): [in a new window]   Fig. 8. Migration of PGCs towards their final target is defective in han mutants. (A-D) Dorsal views of the trunk region of embryos at day 1 of development with the PGCs stained with nos1 in blue. The PGC clusters are dispersed and located more anterior in han mutants (B,D) than in wild-type (A,C) relative to the wt1-expressing glomerulus (arrows in A,B) and relative to the endocrine pancreas stained in red with preproinsulin (C,D). (E,F) Lateral views of wild-type (E) and han mutant (F) embryos at the 25-somite stage stained with pax2.1 in blue. Note that a gap of pax2.1 expression in the pronephric mesoderm is present in wild-type embryos at the anterior end of the yolk extension (bracket in E), while expression in this region is stronger in han mutants (bracket in F).

View larger version (22K): [in a new window]   Fig. 9. A model for the regulation of the final steps of zebrafish PGC migration. Arrows indicate the direction of cell movement. (A) During early somitogenesis the lateral mesoderm of the anterior trunk (blue), which is marked by expression of the wt1 gene, produces signals that attract PGCs. (B) At the six-somite stage PGCs have formed clusters in the attracting region, while in some embryos posterior trailing cells are still migrating anteriorly (step V). (C) At about the 10-somite stage, the attraction center stops to function as such (light blue) or the PGCs no longer respond. The clusters of PGCs and remaining trailing PGCs, which have not yet reached the clusters, start to migrate (downward arrows) towards their final target, located in the lateral mesoderm around somite levels 8 to 10 (green). It is possible that the final target, which presumably gives rise to the somatic tissues of the gonad, also attracts PGCs. (D) At 24 hpf, all PGCs have reached the final target (green), while the cells of the intermediate attraction center (light blue) contribute to formation of the pronephros.