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Fig. 1. Spatiotemporal pattern of accumulation of Hp-gad transcript, determined using whole-mount in situ hybridization.(A,a) Unfertilized eggs. (B,b) Fertilized eggs. (C,c) Morulae. (D,d) Swimming blastulae. (E–G,I) Double-stained whole-mount in situ hybridization. Hp-gad transcripts, red. Primary mesenchyme cells, green. (E) Mesenchyme blastula. (F,F′,e) Early gastrulae. (G,H,f) Late gastrulae. (I,I′,g) Prism larvae. Inset of (I′), higher magnification of the region indicated by a box in (I′). (E′,F′,I′) Hp-gad-transcripts, green. Nuclei, red. Arrows, Hp-gad-transcribing epithelial cells (white) and blastocoelar cells (yellow). Upper-case letters, anti-sense probe. Lower-case letters, sense probe. Scale bars: 50 µm (A–I′,a–g) and 20 µm (I′ inset).
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Fig. 2. Temporal changes in the expression of Hp-Gad during development, as determined by immunoblotting.(A) Immunoblotting pattern of Hp-Gad (GAD). Uf, unfertilized eggs. f, fertilized eggs. sBl, swimming blastulae. mBl, mesenchyme blastulae. eG, early gastrulae. lG, late gastrulae. Prs, prism larvae. 2 dp, two-day post-fertilization (-dpf) two-arm plutei. 10 dp, 10-dpf four-arm plutei. Tm, sea urchin tropomyosin. (B) Relative intensity of immunoreaction of Hp-Gad during development. Abscissa, developmental stage with the same abbreviations as in (A). Vertical bars, s.d. Asterisks indicate significant differences determined using unpaired t-test (P<0.005).
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Fig. 3. Spatiotemporal expression pattern of Hp-Gad (green) during development, determined using double-stained whole-mount immunohistochemistry with nuclei (red; A–C) or Epith-2 (red; D–K′).(A) Unfertilized eggs. (B) Fertilized eggs. (C) Morula. (D) Swimming blastula. Inset, higher magnification of boxed area. (E) Mesenchyme blastula. (F) Early gastrula. (G) Surface of early gastrula. (H) Optical section of early gastrula. (H′) Higher magnification of an optical section at the blastopore region indicated by a box (h′) in (H). (I) Optical section of late gastrula. (J) Optical section of late gastrula. (K) Optical section of prism larva (Prsm). (K′) Higher magnification of a box (k′) in (K). Scale bars: 10 µm (G), 20 µm (H′,K′) and 50 µm (A–F,H–K).
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Fig. 4. Ingression of GAD-expressing cells (GADCs; green) and an increasing number of blastocoelar GADCs during early development, as indicated by double-stained whole-mount immunohistochemistry with Hp-Gad (green) and Epith-2 (red).(A) Optical section of mesenchyme blastula. Box (b–d) is shown with higher magnification from (B) to (D). (B) Enlarged ectodermal GADCs indicated by box (b) in (A). Arrow, GADC. (C) Epith-2 on the surface of GADCs (arrow) of (B). (D) Merged image between (B) and (C). Inset, Optical sagittal section of the ectoderm shows Hp-Gad-positive cytoplasmic dot and the plasma membrane. (E) Animal ectoderm of early gastrula. Pear-shaped GADCs (arrow 1) in animal ectoderm and round GADC in the blastocoel (arrow 2). (F) Higher magnification of vegetal ectoderm of early gastrula of Fig. 3H. GADCs in the ectoderm and basipetal extension of fine filopodia (arrow). Inset, high-contrast image of filopodia (arrow) indicated by an arrow in the mainframe. (G) Prism larva. GADCs on the basal surface of ectoderm (arrow 1) and that showing transient morphology of ingression (arrow 2). (H) Late gastrula. GADCs in the wall of archenteron near the tip region. (I) Prism larva. Spindle-shaped GADCs in the blastocoel. (J) The proportions of GADCs in the ectoderm (blue columns) and in the blastocoel (red columns). Scale bars: 30 µm (A,I), 10 µm (B,D inset,H), 15 µm (E–G). Error bars, s.d. *, statistically significant difference from the one to the immediate left by unpaired t-test (P<0.0001).
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Fig. 5. Proportion of bromodeoxyuridine (BrdU; green)-incorporated Hp-Gad-expressing cells (GADCs; red) in two-dpf two-arm (2aPlut) plutei analyzed by double-stained whole-mount immunohistochemistry.(A) Stacked image of 2aPlut. Arrows, BrdU-positive blastocoelar GADCs. (B) Optical section of 2aPlut. Yellow arrow, BrdU-positive GADCs. White arrows, BrdU-negative blastocoelar GADCs. Scale bars: 20 µm. (C) The proportion of BrdU-incorporated GADCs in 2aPlut is significantly smaller than that of total GADCs (P<0.0005), as determined by an unpaired t-test. Error bars, s.d.
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Fig. 6. Schematic representation of the chimeric 16-cell stage embryo (A) and double-stained whole-mount immunohistochemistry with Hp-Gad (green) and fluorescein (red) of chimeric prism larva (B–E) and two-dpf two-arm plutei (F–H).(A) Schematic of chimera construction between fluorescein-stained mesomeres (red) and unlabelled macro- and micromeres. (B) Fluorescence micrograph of triple-stained prism larva. Red, animal ectoderm. Blue, 4′,6-diamidino-2-phenylindole. Green, Hp-Gad. Green arrow, GADCs. Red arrow, dextran-rhodamine-B-stained cells. Yellow arrow, cells that were stained with Hp-Gad and dextran-rhodamine-B. (C) Four-micrometer-thick optical section of dextran-rhodamine-B-labelled animal ectoderm in prism larva. (D) GADCs in the same larva as in (C). (E) Merged image of (C) and (D). Yellow arrow, mesomere-derived GADC. Green arrow, vegetal ectoderm-derived GADC. Red arrow, animal ectoderm-derived non-GADCs. Vegetal ectoderm is not stained with dextran-rhodamine-B. (F) Ninety-five-micrometer-thick stacked image of two-day post-fertilization two-arm pluteus larva. Boxes (g) and (h) are highly magnified in (G) and (H). (G) Four-micrometer-thick optical section of oral lobe are indicated by the box (g) in (F). Yellow arrows, TRITC-positive GADCs. Orange arrow, TRITC-positive non-GADCs. (H) Forty-micrometer-thick optical section of posterior trunk region indicated by the box (h) in (F). Yellow arrows, TRITC-positive GADCs. Green arrow, TRITC-negative GADC. Orange arrows, GAD-negative TRITC-positive cells. Scale bars: 30 µm.
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Fig. 7. Heterogeneous blastocoelar cells in early gastrulae and expression of multiple proteins in a blastocoelar cell in plutei analysed by double-stained whole-mount immunohistochemistry.(A) Early gastrula. Green, glutamate decarboxylase (GAD)-expressing cell (GADC), Red, primary mesenchyme cell. (B) Late gastrula. Green, GADCs. Red, SM50-expressing cell. (C) Prism larva. Green, GAD. Red, serotonin receptor (5HThpr). (D) High magnification of cells indicated by the box (d) in (C). Scale bars: 20 µm (A), 40 µm (B), 25 µm (C), 10 µm (D).
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