Migration of sea urchin primordial germ cells

Abstract

Background: Small micromeres are produced at the fifth cleavage of sea urchin development. They express markers of primordial germ cells (PGCs), and are required for the production of gametes. In most animals, PGCs migrate from sites of formation to the somatic gonad. Here, we investigated whether they also exhibit similar migratory behaviors using live-cell imaging of small micromere plasma membranes.

Results: Early in gastrulation, small micromeres transition from non-motile epithelial cells, to motile quasi-mesenchymal cells. Late in gastrulation, at 43 hr post fertilization (HPF), they are embedded in the tip of the archenteron, but remain motile. From 43-49 HPF, they project numerous cortical blebs into the blastocoel, and filopodia that contact ectoderm. By 54 HPF, they begin moving in the plane of the blastoderm, often in a directed fashion, towards the coelomic pouches. Isolated small micromeres also produced blebs and filopodia.

Conclusions: Previous work suggested that passive translocation governs some of the movement of small micromeres during gastrulation. Here we show that small micromeres are motile cells that can traverse the archenteron, change position along the left-right axis, and migrate to coelomic pouches. These motility mechanisms are likely to play an important role in their left-right segregation.

Keywords: cortical bleb; filopodia; live cell imaging; migration; motility; phosphoinositides; primordial germ cell; small micromere.

Fig. 1

Small micromeres expressing NTM-mCit move from the tip of the archenteron to the two coelomic pouches between 43 and 54 HPF. Maximum intensity projections of embryos injected with mRNA encoding NTM-mCit (green) shows small micromeres transition from (A, A′) tightly packed cells at 43 HPF to (B, B′) a line of cells on the tip of the archenteron at 49 HPF that (C, C′) begin segregating among the coelomic pouches at 54 HPF. Top panel, confocal snapshots of fluorescent channel (A–C) and DIC overlay with contrast enhanced fluorescent channel (A′–C′) in bottom panel. Scale bars = 20 µm. D: Number of small micromeres on the left coelomic pouch after overexpression of NTM-mCit, Vasa-mChr or after detection by immunolocalization. N>20 embryos from three females (P≥0.05, One-way ANOVA).

Fig. 2

Small micromeres develop cortical blebs and thin filopodia before segregating to the coelomic pouches. Representative confocal sections of embryos with small micromeres expressing NTM-mCit (green) with cortical blebs (white arrowheads) at (A,B) 43 HPF microvilli or thin filopodia from small micromeres (orange arrowheads) and SMCs (blue arrowhead) contacting ectoderm at (C,D) 49 HPF and (E,F) 54 HPF. Scale bars = 10 µm.

Fig. 3

Localization of the apical marker G2a-mCit. Representative confocal images of embryos expressing Vasa-mChr (red) and G2a-mCit (green or gray). Symbols indicate small micromeres (blue asterisks), microvilli (orange arrowheads), lateral membrane (blue arrowheads). A: Whole embryo fluorescence of Vasa-mChr and G2a-mCit accompanied by an overlay at regions with small micromeres for mesenchyme blastula at 22 HPF, mid-gastrula at 36 HPF, and late gastrula at 49 HPF. B: G2a-mCit in small micromeres of the mesenchyme blastula at 22 HPF, early gastrula at 29 HPF, mid-gastrula at 29 HPF, full gastrula at 43–54 HPF. All scale bars = 10 µm.

Fig. 4

Small micromeres move through the tip of the archenteron between 43 and 54 HPF. Embryos expressing NTM-mCit (green) and Vasa-mChr (red) were time lapsed for 120 min by confocal microscopy and tracked (white lines) using mTrackJ. Representative tracks show (A) small micromeres moved around the tip of the archenteron before (B) moving to form a line at the tip of the archenteron and (C) segregating among the left and right coelomic pouches. Scale bar = 10 µm.

Fig. 5

Small micromeres move farther and faster than endoderm or SMCs that produce the coelomic pouch. Line plots display representative tracks of the micrometers traveled over 1 hr in the (A) x–y direction and (B) x–z direction of four small micromeres, SMCs, and endoderm cells between 43 and 54 HPF. Each track was normalized and plotted from a common origin (0,0). Dot plots of (C) total displacement from origin and (D) average cell velocity for all small micromeres, SMCs, and endoderm cells measured after 1 hr. Each dot represents the micrometers traveled from the origin or the average velocity of one cell and the bar represents the mean of at least four cells of each type measured from three different embryos (n≥12 cells). Letters above mean lines denote statistical significance (P≤0.05, two-way ANOVA).

Fig. 6

Small micromeres expressing PH-mCit develop cortical blebs (white arrowheads) and thin filopodia (orange arrowheads). Representative confocal snapshots of embryos containing small micromeres (A, C, E) expressing Vasa-mChr (red) and the PIP₂ marker, PH-mCit (green, B, D, F) or only PH-mCit, show small micromeres produce cortical blebs (A,B) at 43 HPF, and thin filopodia (C,F) between 49–54 HPF. Representative oral view (G) of coelomic pouches at 60 HPF indicates small micromeres produce filopodia after left/right segregation. H: Film strip of confocal time-lapse recording of small micromere expressing PH-mCit overlaid with results of cell tracking in white. Filmstrip shows a small micromere using filopodia to contact ectoderm before migrating to a coelomic pouch. Scale bars = 10 µm.

Fig. 7

Small micromeres produce transient and stable filopodia. Confocal micrographs of representative small micromeres expressing Vasa-mChr (red) and PH-mCit (green) in coelomic pouches at 60 HPF that produce (A) stable filopodia that contact ectoderm for over 45 min and (B) short-lived filopodia that project into the blastocoel and last <10 min. Scale bars = 10 µm. White arrowheads indicate positions of filopodia.

Fig. 8

Small micromeres bleb rapidly in vivo and in vitro. A: Film strips of representative confocal time lapse recordings of small micromeres in vivo at 41 and 48 HPF expressing NTM-mCit (green) produce blebs (white arrowheads) that expand and contract within 1 min. B: Film strips of representative confocal time lapse recordings of small micromeres in vitro expressing NTM-mCit (yellow) and Vasa-mChr (red). White arrowheads at 40 HPF show the expansion and contraction of a cortical bleb within 1 min and at 48 HPF the extension of a filopodium in 140 sec. All scale bars = 10 µm.

Fig. 9

Schematic illustration depicting the phases of small micromere migration shown with arrows indicating the major direction of movement during each phase. During phase 1, small micromeres bleb basolaterally and produce filopodia to release from epithelium and move to the animal pole. In phase 2, small micromeres have taken a mesenchymal morphology, produce numerous filopodia and re-position along the left/right axis as they move to the roof of the archenteron. Phase 3 is characterized by the production of dynamic filopodia during the left/right segregation of small micromeres to the coelomic pouches.