|
Fig. 1. Embryonic development of T. toreumaticus. Embryos observed by light (C,D,F,K,L,N–P) or SEM (B,E,E′,G–J,M). (A) Schematic diagrams of a morula (upper) and blastula (bottom), lateral view. The area inside the black boxes was focused on using SEM. (B) The internal surface at the vegetal pole of an embryo 3.5 h after fertilization. (C) A morula 4 h after fertilization. (D,E) Blastulae 4.5 h after fertilization. The embryos had developed wrinkles (D, arrowheads). Blastomeres were still orbicular in shape (E,E′). E′ shows a higher magnification of area inside white box in E. (F,G) Blastulae 6 h after fertilization. The wrinkles had disappeared (F). At the vegetal pole (G), micromeres-descendants had kept their orbicular shape, but the cells around them had started to produce pseudopod-like structures. Insert, higher magnification of the area inside the white box in G. (H) An embryo 6.5 h after fertilization. As shown in the insert, which show a higher magnification of the area inside the white box, orbicular cells were surrounded by cells that extended pseudopod-like structures toward these cells and there was a ring of ECM at the vegetal pole that seemed to form a hole-like structure. (H′) A schematic diagram of the inside of the vegetal plate. Center, presumptive PMCs; outer, cells with pseudopod-like structures; blue lines, ECM. (I) A hatching blastula 7 h after fertilization. (J) An embryo 8.5 h after fertilization. PMCs migrated into the blastocoel as a mass (white dashed circle). (K–M) Embryos at 10 h (K), 11 h (L) or 11.5 h (M) after fertilization; dorso-ventral (K,L,N–P) or animal views (M). After migration, PMCs moved into a narrow blastocoel at the vegetal side. (N–P) Gastrulae 12.5 h (N), 13 h (O) or 15 h (P) after fertilization. The archenteron with the flat tip elongated toward the apical plate and then formed SMCs (N). The middle part of archenteron was narrower (O) and finally the tip attached to the apical plate (P). At this stage, there were some SMCs with filopodia (arrow). Scale bars: 20 μm in B,E,G–J,M; 50 μm in C,D,F,K,L,N–P.
|
|
Fig. 2. Embryonic development of T. reevesii. Embryos observed by light (B,D,F,H,J–M) or SEM (C,E,G,I). (A) Schematic diagrams of a morula (upper) and blastula (bottom), lateral view. The area inside the black boxes was studied by SEM. (B,C) Morulae 3.5 h after fertilization. On the internal surface at the vegetal pole, blastomeres were adjoined closely to each other by pseudopod-like structures (white arrows). (D) An early blastula 4.5 h after fertilization. (E) The internal surface of the lateral region of an embryo 5.5 h after fertilization. Some cells extended pseudopod-like structures to the neighboring cells. (F,G) Blastula 6 h after fertilization. By observation of the internal surface of the vegetal plate (G), some presumptive PMCs were globular (area encircled by the dashed white line). (H,I) Hatching blastulae 7 h after fertilization. By observation of the internal surface of the vegetal plate (I), cells around the globular cells (area encircled by the dashed white line) extended pseudopod-like structures toward this area. (J) A mesenchyme blastula 11 h after fertilization (lateral view). PMCs were identified at the vegetal area. (K–N) Gastrulae at 12.5 h (K), 14 h (L), 15.5 h (M) or 23 h (N) after fertilization viewed from the dorso-ventral side. The vegetal plate became thick and then invaginated into the blastocoel (K). SMCs were identified near the tip of the archenteron and some SMCs had filopodia (black arrows) (L,M). The tip of archenteron has a diameter larger than that of the blastopore (N). Scale bars: 50 μm in B,D,F,H,J–N; 20 μm in C,E,G,I.
|
|
Fig. 3. Embryonic development of T. hardwickii. Embryos observed by light (D–I) or SEM (B,C). (A) Schematic diagrams of a morula (upper) and blastula (bottom), lateral view. The area inside the black boxes was examined by SEM. (B) The internal surface at the vegetal pole of an embryo 4.5 h after fertilization. Each cell extended pseudopod-like structures (white arrows) toward the neighboring cells. (C) The internal surface at the vegetal pole of an embryo 7 h after fertilization. Some cells had pseudopod-like structures extended toward the adjacent cells (white arrows). Cells enclosed by the dashed white line were identified as the globular cells. (D) A mesenchyme blastula 11 h after fertilization. (E–I) Gastrulae at 13 h (E), 14 h (F), 16 h (G), 17 h (H) or 21 h (I) after fertilization (dorso-ventral views). The invagination is slightly curved (E,F) and then the archenteron became thin (G). The length and shape of the archenteron did not change for a while. SMCs occurred and they moved into the blastocoel and formed filopodia (black arrows) (G,H). Finally, invagination was completed (I). Scale bars: 20 μm in B,C; 50 μm in D–G.
|
|
Fig. 4. Embryonic development of M. globulus. Embryos observed by light (C,I–N) or SEM (B,D–H). (A) Schematic diagrams of a morula (upper) and blastula (bottom), lateral view. The area inside the black boxes was examined by SEM. (B) The internal surface structure at the vegetal pole at 3.5 h after fertilization. Each blastomere adjoined closely. (C–E) Early blastulae 4.5 h after fertilization. Cells at the lateral region with intricate elongated pseudopod-like structures (D). Some specimens had granular structures on the surface of the blastular wall (E, higher magnification of area inside white box in the insert). (F) The internal surface at the lateral region of an embryo 6 h after fertilization. (G,H) The internal surface at the vegetal plate of early mesenchyme blastulae 10 h (G) or 11 h (H) after fertilization. In the area enclosed by the dashed white circle are orbicular cells that have started to ingress into the blastocoel as PMCs (H). (I) A mesenchyme blastula 12 h after fertilization (lateral view). (J–N) Gastrulae 14 h (J), 15 h (K), 16 h (L), 17 h (M) or 21 h (N) after fertilization (dorso-ventral views). The invaginating vegetal plate is shaped like a hemisphere (J). When the archenteron had invaginated by approximately one fifth of the total length of the embryo (L), the tip of archenteron was flat and released SMCs. Some SMCs formed filopodia (black arrows) and moved in the blastocoel (K–M). Without attachment of the archenteron to the apical plate, the invagination finished (N). Scale bars: 20 μm in B,D–G; 50 μm in C,H–M.
|
|
Fig. 5. Pattern of invagination of the archenteron in four temnopleurids. (A) Schematic diagram of a gastrula derived from Fig. 6 for measurements to find the invagination ratio. (B–E) Calculated invagination ratios of T. toreumaticus (B), T. reevesii (C), T. hardwickii (D) and M. globulus (E). Y and X axes show the invagination ratio (%) or the time after initiation of invagination. Gray areas show the secondary invagination. The timing of SMC appearance or SMCs with filopodia show the stage when over 60% of specimens have these features. Error bars represent s.d.
|
|
Fig. 6. Measurement of gastrulae. Each part of the gastrula was measured; the total length of the embryo (A) or the archenteron (B), the total width of the embryo (C), the diameter of the blastopore (D), the outer or inner diameter of the archenteron at the middle part of the total length of the archenteron (E or E′) or at the tip (F or F′).
|
|
Fig. 7. Ratio of the diameter of blastopore to the total width of embryo in four temnopleurids. (A) Schematic diagram of a gastrula for measurement to find the ratio of the blastopore. (B–E) Calculated blastopore-to-width ratios of T. toreumaticus (B), T. reevesii (C), T. hardwickii (D) and M. globulus (E). Y and X axes show the ratio of the blastopore (%) or the time after initiation of invagination. Error bars represent s.d.
|
|
Fig. 8. Diameter of the archenteron and thickness of the archenteron wall in four temnopleurids. (A) Schematic diagram of a gastrula for measurement of the diameter of the archenteron and measurement to find the thickness of the archenteron wall. (B–I) Archentreon diameter (B,D,F,H) and wall thickness (C,E,G,I) of T. toreumaticus (B,C), T. reevesii (D,E), T. hardwickii (F,G) and M. globulus (H,I). X-axis shows the time after initiation of invagination. Error bars represent s.d.
|
|
Fig. 9. The diameter of the tip of the archenteron in four temnopleurids. (A) Schematic diagram of a gastrula for measurement of the outer and inner diameter of the tip of the archenteron. (B–I) Outer (B,D,F,H) and inner (C,E,G,I) diameter tip of T. toreumaticus (B,C), T. reevesii (D,E), T. hardwickii (F,G) and M. globulus (H,I). X-axis shows the time after initiation of invagination. Error bars represent s.d.
|
|
Fig. 10. Summary of blastula and gastrula formation in four temnopleurids. Schematic diagrams of the vegetal part of embryos, lateral view. Blue lines, ECM. Blastulae in T. toreumaticus well-develop ECM at the blastocoelic surface and then ingress orbicular primary mesenchyme cells en masse from a hole-like structure of ECM and finally invaginate continuously. On the other hand, blastulae in T. reevesii, T. hardwickii and M. globulus form blastomeres with pseudopods at the blastular wall and then ingress primary mesenchyme cells separately and finally invaginate stepwise by all or some of factors: change of cell shape, rearrangement, pushing up and towing of cells.
|
