In this study, we investigate the role of cell proliferation in medusa growth and morphogenesis, using Cladonema pacificum as a model of hydrozoan jellyfish. We show that cell proliferation occurs evenly across the medusa body, including the umbrella and manubrium, with the exception of the tentacles, where cell proliferation is spatially clustered. Blocking cell-cycle progression with a pharmacological assay inhibits the increase of body size, tentacle branching, and nematocyte differentiation, which suggests that cell proliferation is necessary for growth and tentacle morphogenesis. We further show that cell proliferation is required for tentacle regeneration in Cladonema medusae. Our findings reveal cell proliferation’s critical roles in the development and maintenance of the Cladonema body and appendages and provide a basis for understanding growth-control mechanisms in hydrozoan jellyfish.

(A) Young medusa of Cladonema pacificum . (B) Sexually-matured medusa of Cladonema pacificum . (C) The scheme of Cladonema medusa development. (D–K, N, O) Distribution of S-phase cells in the Cladonema pacificum medusa (1 day old) revealed by EdU staining (20 μM, 24 h incubation). (D, E) Distribution of S-phase cells (EdU+) in a medusa whole body. (F, G) Distribution of S-phase cells (EdU+) in a medusa manubrium. (H, I) Distribution of S-phase cells (EdU+) in a medusa exumbrella. (J, K) Distribution of S-phase cells (EdU+) in a medusa subumbrella. (L, M) Mitotic cells detected by anti-PH3 in a medusa umbrella (8 day old). (N, O) Distribution of S-phase cells (EdU+) in medusa tentacles. (P) Mitotic cells (PH3+) in medusa tentacle bulbs (1 day old). Arrows indicate EdU-positive (H–K) and PH3-positive (L, P) cells, respectively. Scale bars: (A, B) one mm, (D, E, H–K, N, O) 200 μm, (F, G) 100 μm, (L, M, P) 50 μm.

We performed statistical analysis for the proliferating cells’ distribution in umbrellas and tentacles by applying the nearest neighbor distance (NND) test to EdU positive cells ( Table S1 ). Here, we used the images of 1-day-old medusae that had been incubated with EdU 150 μM for 1 h. This analysis was applied to the umbrella area, except for tentacle bulb and manubrium, while the same analysis was applied to the entire main tentacle. The area (S), the signal number (N), and NND in analyzed areas were obtained using ImageJ/Fiji. The average of NND (W), the expectation value of W (E[W]), and the normalized average of NND ( w = W/E[W]) were calculated. In this analysis, w > 1 means that EdU signals are distributed uniformly or randomly. In contrast, w < 1 means that EdU signals are distributed clustered or randomly. The spatial distribution of EdU signals were determined by Z score.

This protocol was adapted from Szczepanek, Cikala & David (2002) : The medusae were anesthetized with 7% MgCl 2 in ASW for 10 min and fixed with 4% PFA in ASW for 1 h. After fixation, the samples were rinsed in 1× PBS and washed three times (10 min each) in 0.1% PBT. The samples were incubated in DAPI (1:500; Polysciences, Inc., Warrington, PA, USA) in PBT for 60 min. After the DAPI incubation, samples were washed four times (10 min each) in PBT and mounted on slides with 70% glycerol in DW. Samples were scanned with a combination of 488 nm excitation and 555 nm emission filter using either Leica SP8 or SP5 confocal microscopes. Using ImageJ, we performed Z-stacks and counted nematocysts. Empty nematocysts were counted manually.

Pictures of medusae were taken with a Nikon D5600, and umbrella size was measured using polygon selections with ImageJ software ( Fig. 3C ). We measured the length and width of medusae under the microscope using an ocular micrometer and multiplied the length and width to generate a value for umbrella size ( Fig. 3M ). Tentacle length was measured daily under the microscope with an ocular micrometer ( Fig. 5H ).

The live medusae were incubated with 10 mM hydroxyurea (HU) (085-06653; Wako, Osaka, Japan) in ASW (ASW only for control) ( Figs. 3 – 5 ; Fig. S3 ). HU incubation was continued for a maximum of 9 days. Medusae were fed every other day, and HU solution or ASW was renewed after feeding. The medusae treated with HU were able to ingest prey like controls, demonstrating that HU treatment had no effect on feeding behavior ( Figs. S3A and S3B ).

The medusae were incubated with 20 μM 5-ethynyl-2′-deoxyuridine (EdU; EdU kit; 1836341; Invitrogen, Carlsbad, CA, USA) in ASW for 24 h ( Figs. 1 – 3 and 6 ) or 150 μM for 1 h ( Fig. S1 ). After EdU treatment, the medusae were anesthetized with 7% MgCl 2 in ASW for 10 min and fixed 4% PFA in ASW for 1 h. After fixation, the samples were rinsed in 1× PBS and washed three times (10 min each) in 0.1% PBT. The samples were incubated with a EdU reaction cocktail (1× reaction buffer, CuSO 4 , Alexa Fluor azide, and 1× reaction buffer additive; all included in EdU kit; 1836341; Invitrogen, Carlsbad, CA, USA) for 30 min in the dark. After the EdU reaction, the samples were washed three times (10 min each) in 0.1% PBT and Hoechst 33342 (1:250; Thermo Fisher Scientific, Waltham, MA, USA) in 0.1% PBT for 1 h in dark. The samples were washed four times (10 min each) in 0.1% PBT and were mounted on slides with 70% glycerol.

The medusae were anesthetized with 7% MgCl 2 in ASW for 10 min and fixed 4% paraformaldehyde (PFA) in ASW for 1 h. After fixation, the samples were rinsed in 1× PBS and washed three times (10 min each) in PBS containing 0.1% Triton X-100 (0.1% PBT). The samples were incubated in primary antibodies in 0.1% PBT overnight at 4 °C. The antibodies used were rabbit anti-Phospho-Histone H3 (Ser10) (1:500; 06–570, Upstate, Lake Placid, NY, USA) and mouse anti-α-Tubulin (1:500; T6199, Sigma-Aldrich, St Louis, MO, USA). After the primary antibody incubation, the samples were washed three times (10 min each) in 0.1% PBT and incubated in secondary antibodies (1:500; ALEXA FLUOR Goat anti-mouse IgG, ALEXA FLUOR Goat anti-rabbit IgG; Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst 33342 (1:250; Thermo Fisher Scientific, Waltham, MA, USA) in 0.1% PBT for 1 h in dark. After four washes (10 min each) in 0.1% PBT, the samples were mounted on slides with 70% glycerol. Confocal images were collected through Leica SP8 or SP5 confocal microscopes. Z-stack images were performed using ImageJ/Fiji software.

(A) Distribution of S-phase cells in the Cytaeis uchidae medusa (30 day old) shown with EdU staining (EdU: 20 μM, 24 h). (B) Distribution of S-phase cells (EdU+) in Cytaeis medusa (11 day old). (C) Mitotic cells (PH3+) in the umbrella of Cytaeis medusa (30 day old). (D) Mitotic cells in Cytaeis medusa tentacle bulbs (30 day old). (E, F) Distribution of S-phase cells (EdU+) in the Rathkea octpunctata juvenile medusa (EdU: 20 μM, 24 h). (G) Mitotic cells (PH3+) in a manubrium of Rathkea juvenile medusa. (H) Mitotic cells (PH3+) in Rathkea juvenile medusa tentacles. Arrows indicate PH3-positive mitotic cells. Scale bars: 100 μm.

(A–D) Tentacle regenerative processes after amputation in an adult medusa. Series of pictures show the growing tentacle over 4 days. (E, F) Mitotic cells (PH3+) in tentacle bulbs of the unremoved control and the dissected medusa. Arrowheads indicate PH3-positive cells. (G) Quantification of proliferative cells in tentacle bulbs for control and after amputation. Control: n = 26, Amputation: n = 11. Error bar: SD. Unpaired two-tailed t -test. t (35) = 6.246, **** p < 0.0001. (H) Quantification of tentacle length after amputation in control (HU−) and 10 mM HU treatment (HU+). Unpaired two-tailed t -test. Day 1 t (46) = 9.227, day 2 t (46) = 10.29, day 3 t (46) = 14.1, day 4 t (46) = 20.5, day 5 t (46) = 22.49, day 6 t (45) = 17.11, day 7 t (45) = 15.36, **** p < 0.0001. Scale bars: (A–D) one mm, (E, F) 100 μm.

(A) Control (HU−) medusa incubated in ASW for 9 days. The picture shows the representative image of medusae with three branched tentacles. (B) The medusa incubated in 10 mM HU (HU+) ASW for 9 days. The picture shows the representative image of medusae with one branched tentacle. (C) Quantification of branching numbers per tentacle at day 0 and day 9. HU+: n = 313, HU− condition: n = 199. Error bars: SD. Unpaired two-tailed t -test. t (510) = 54.49, **** p < 0.0001. (D–G) Nematocytes in tentacles labeled by DAPI (poly-γ-glutamate) in the 8 day old medusa incubated in ASW (HU−) or 10 mM HU ASW (HU+). Arrowheads indicate empty nematocysts. (H) The proportion of empty nematocysts in HU− and HU+ medusa. HU+: n = 19, HU−: n = 18. Unpaired two-tailed t -test. t (31) = 2.869, ** p < 0.01 ( p = 0.0074). Scale bar: (D–G) 50 μm.

(A) Cladonema pacificum newborn medusa (0 day old). (B) Cladonema pacificum juvenile medusa (8 day old). (C) Quantification of umbrella size in control and starved medusae. Control medusae were fed every other day. Error bar: SD. Unpaired two-tailed t -test. Day 1 t (36) = 4.545, day 3 t (36) = 9.888, day 6 t (36) = 12.56, **** p < 0.0001. (D–G) Distribution of S-phase cells in control medusa and starved medusa with EdU staining (20 μM, 24 h incubation). (H) Quantification of the number of S-phase cells (EdU+) in control and starved medusae. Unpaired two-tailed t -test. * p < 0.05 ( p = 0.0127), t = 3.194 d f = 8. (I–L) Distribution of S-phase cells in medusa of control (HU−) and hydroxyurea (HU+) treatment detected by EdU staining (20 μM, 24 h). No S-phase cells were detected in HU+ medusae. (M) Quantification of body size in control and in HU conditions. HU suppresses body-size growth. HU−: control medusae incubated in ASW, HU+: medusae incubated in HU 10 mM ASW. Both HU+ and HU− were fed every other day. Error bar: SD. Unpaired two-tailed t -test. Day 1 t (93) = 3.561, day 2 t (90) = 4.079, day 3 t (81) = 3.657, day 5 t (85) = 6.329, day 6 t (52) = 4.105, day 7 t (79) = 7.319, day 8 t (71) = 9.201, day 9 t (59) = 8.826, *** p < 0.0005, **** p < 0.0001. Scale bars: (A, B) one mm, (D–G and I–L) 100 μm.

(A–H) Distribution of S-phase cells in the Cladonema pacificum medusa (45 day old) shown with EdU staining (20 μM, 24 h incubation). (A, B) Distribution of S-phase cells (EdU+) in a medusa exumbrella. (C, D) Distribution of S-phase cells (EdU+) in a medusa subumbrella. (E, F) Distribution of S-phase cells (EdU+) in medusa tentacles. (G, H) Distribution of S-phase cells (EdU+) in a medusa manubrium. Arrows indicate EdU-positive (A–D) cells. Scale bars: (A–D, G, H) 200 μm, (E, F) 100 μm.

We used Cladonema pacificum (strains 6W and UN2) ( Figs. 1 – 5 ; Figs. S1 – S3 ), Cytaeis uchidae (strain ♀17) ( Fig. 6 ) and Rathkea octopunctata (strain MF-1) ( Fig. 6 ) medusae for this research. The medusae were cultured in plastic cups (V-type container, V-7 and V-8, AS ONE, Osaka, Japan) at 20 °C ( Cladonema and Cytaeis ) or 4 °C ( Rathkea ), and their polyps were maintained in the cups (V-7) at 20 or 4 °C in darkness. Vietnamese brine shrimp (A&A Marine LLC, Elk Rapids, MI, USA) were fed to medusae and polyps every other day ad libitum, with water renewed immediately after feeding. Artificial sea water (ASW) was prepared by SEA LIFE (Marin Tech, Tokyo, Japan). Pictures of medusae were taken through a LEICA S8APO microscope with a Nikon digital camera (D5600).

Results

Cell proliferation is necessary for the control of body size Animal body size increases upon intake of nutrition because nutrition influences cell proliferation and cell growth (Bohnsack & Hirschi, 2004). We first monitored the body size of juvenile medusae by focusing on the size of their umbrella because the umbrella grows in direct proportion with whole body size. Under normal feeding conditions, the medusa umbrella size increased dramatically by 54.8%, from 0.62 ± 0.02 to 0.96 ± 0.02 mm2 during the first 24 h, with a subsequent minor increase observed over the following 5 days (0.98 ± 0.03 mm2) (Figs. 3A–3C). By contrast, under starved conditions, the size of medusa umbrella did not increase, compared to controls, and rather gradually decreased over the following 5 days (Fig. 3C). Moreover, fewer EdU positive cells were detected in the starved medusae than in fed controls (Figs. 3D–3H; Control: 1,240.6 ± 214.3, Starved: 433.6 ± 133, t(8) = 3.194, *p < 0.05 (p = 0.0127)), suggesting that, at the cellular level, nutrition affects cell proliferation in medusae. These results indicate that body-size growth in juvenile medusae depends on available nutrition. To test the hypothesis that uniform cell proliferation in medusae contributes to body-size increase, we performed a pharmacological assay to block cell-cycle progression using HU, a cell-cycle inhibitor that causes G1 arrest (Koç et al., 2004). Under HU treatment, S phase cells detected by EdU staining disappeared from the medusa body (Figs. 3I–3L). By tracking the size of umbrella, we found that HU-treated medusae did not exhibit the size increase that was observed in controls (Fig. 3M). Together, these results suggest that cell-cycle progression affects body size in Cladonema medusae.