Figure 1.
Life cycle of Tripedalia cystophora.
Schematic diagram illustrating the life cycle of T. cystophora. The planula larva settles on the bottom and undergoes the first metamorphosis into a sessile primary polyp with two tentacles. The primary polyp grows into a fully grown polyp that usually possesses from 7 to 9 tentacles. At this stage much asexual reproduction takes place (not shown). Under optimal conditions the grown polyp undergoes the second metamorphosis and forms juvenile medusa with four primary tentacles. Sexual maturity of the medusae is reached in approximately three months. Fertilization is internal and the diagram shows a pregnant female with bell completely filled by larvae and an adult male with ripe spermatophores (orange spheres).
Figure 2.
Proliferation zones during the metamorphosis of Tripedalia cystophora polyps.
T. cystophora polyps during different stages of metamorphosis stained with DAPI (A′,B′, C′, D′, E′) and S phase cells visualized with EdU (A′′, B′′, C′′, D′′, E′′). (A-A′′′) Non-metamorphosing polyp. The S phase cells in non-metamorphosing polyps are dispersed throughout the entire body including the tentacles (A′′′). (B-B′′′) Polyp in the early stage of metamorphosis (stage of congregating tentacles). High density of S phase cells defining a proliferation zone is seen around the mouth and at the tentacle bases (B′′′). (C-C′′′) Metamorphosing polyp in the early stage of rhopalia formation. A proliferation zone is found in the oral end of the polyp and especially in the forming rhopalia (C′′′). (D-D′′′) Metamorphosing polyp in the late stage of rhopalia formation (forming eyes visible on the rhopalia). The highest number of S phase cells is again in the forming rhopalia but also in the growing medusa tentacles (D′′′). (E-E′′′) Polyp in the late stage of metamorphosis. An additional proliferation zone is observed in the forming manubrium and gastric filaments (E′′′). Scale bar, 300 µm (A) applies to all the pictures.
Figure 3.
Comparison of proliferation zones during metamorphosis in Tripedalia cystophora and Alatina moseri.
(A, B) Dispersed proliferation in the non-metamorphosing polyps. The overall pattern of proliferating cells is the same in two species with labeled cells seen throughout the body. (C, D) Both species show high S phase cells density in the early metamorphosis at the distal end of the body especially where the rhopalia are forming. Still, T. cystophora shows higher concentration of labeled cells (C). (E, F) The most apparent proliferation zone is found in the forming rhopalia and in the area surrounding the mouth where the ring nerve is reorganizing. At the end of the metamorphosis there are some differences between two species. Again A. moseri has fewer labeled S phase cells (F) than T. cystophora. It is also evident that the cells (at least the nuclei) are smaller in T. cystophora than in A. moseri (note the difference in scale bars). (G, H) Negative controls. Scale bars, 300 µm (A) applies to C, E, G; 600 µm (B) applies to D, F, H.
Figure 4.
Proliferation zones in juvenile medusae of Tripedalia cystophora and Alatina moseri.
(A-A′′) The marked area (white dashed line) indicates where DAPI-stained (A) and EdU-stained (A′) nuclei were counted in a pedalium of a juvenile medusa. (B–E) Proliferation zones in juvenile medusae of T. cystophora. The highest rate of proliferation is found in the rhopalia (B) and in the proximal part of the pedalia (before the first ring of nematocytes) (C). The rate of cell proliferation in the manubrium and gastric filaments (D) seems to be as high as in the rhopalia (B) and pedalia (C), but this is due to very high cell density in the manubrium. The proliferation rate is as low as in the bell (see also Table 1 and results section for details on statistics). (F–I) Proliferation zones in juvenile medusae of A. moseri. As seen for the polyps, the overall proliferation pattern is the same for two species, and proliferation zones are again found in the rhopalia (G) and pedalia (H). The manubrium (white dashed line) and gastric filaments (black dashed line) also display many S phase cells (I). PE represents pit eye; SE, slit eye; ULE, upper lens eye; LLE, lower lens eye; St, stalk; Cr, crystal. Scale bars, 50 µm (A, A′, A′′, B); 100 µm (C, D, G, H, I); 300 µm (E, F).
Table 1.
Percentage of S phase cells in predefined areas of juvenile Tripedalia cystophora (see also Figure 4, Figure S2 and Table S1).
Figure 5.
Cell turnover in adult Tripedalia cystophora rhopalia.
(A, B) TEM pictures of the cells in posterior cell sheet. These cells have typical morphology of undifferentiated cells (small nuclei, little cytoplasm) (A). Dividing cells in the posterior cell sheet (B). (C) Vertical peripheral section of adult rhopalium. Labeled cells are after 5 h localized in the posterior cell sheet (white dashed area), gastrodermis (black dashed area) and epidermis. (D) Horizontal central section through LLE of rhopalium. Labeled cells, 24 h (day 1) after treatment, do not change localization and are still observed in the posterior cell sheet (white dashed line), gastrodermis (black dashed line) and epidermis. (E, F) Within one week labeled S phase cells migrated into the retinas of ULE (E, red arrowheads) and LLE (F, red arrowheads). (E) Vertical central section of an adult rhopalium. (F) Horizontal peripheral section through ULE of an adult rhopalium. PE represents pit eye; SE, slit eye; ULE, upper lens eye; LLE, lower lens eye; St, stalk; Cr, crystal. Scale bars, 5 µm (A); 1 µm (B); 200 µm (C–F).
Table 2.
Counts of S phase cells in fully grown rhopalia of Tripedalia cystophora after 5, 24, 72 and 168 h (see also Figure 5).
Figure 6.
Diurnal change in the rate of proliferation in Tripedalia cystophora.
(A, C, E, G) Proliferation during daytime in the bell area midways over the radial channel (A), in the pedalium (C), at the stalk base (E) and in the rhopalium (G). The same areas were likewise labeled during nighttime (B, D, F, H). White dashed squares indicate areas of cell counting (200×200 µm). Black dashed area indicates where the ring nerve enters rhopalial stalk (stalk base). The proliferation in the pedalium (D), stalk base (F) and rhopalium (H) is significantly higher at night than during the day (see result section for details on statistics). PE represents pit eye; SE, slit eye; ULE, upper lens eye; LLE, lower lens eye; Cr, crystal. Scale bars, 300 µm (A–F); 200 µm (G, H).
Table 3.
Counts of S phase cells at daytime and nighttime in predefined area of different body parts of Tripedalia cystophora (see also Figure 6 and Table S2).