GoKawiilAnimal culture
A closed life cycle of M. leidyi based on cydippid reproduction has been established in the laboratory. The animals used to establish the culture were originally collected at the Kristineberg Marine Research Station.
Twenty to 40 individuals were maintained in 3-l Kreisel tanks at 17–19 °C in 27.5 ppt artificial seawater (ASW) (Red Sea Salt, Red Sea Fish Pharm), under a 19–5 light–dark cycle and fed once daily with Brachionus plicatilis (Rotifera). Feeding was performed manually using a Pasteur pipette by adding 3 drops of a concentrated rotifer suspension (100,000–150,000 rotifers per ml) per 3-l tank. Water in the Kreisel tanks was completely changed manually once every 2 weeks. Rotifers were fed with a commercial concentrated microalgae solution (RGcomplete, Reef Nutrition) and cultured according to a previously described protocol15.
For embryo collection, 10–20 cydippids (0.5–0.8 cm in size) were transferred into 200–250 ml beakers before the dark period of the light–dark cycle. Spawning occurred synchronously about 2–2.25 h after the start of the next light cycle. More than 90% of embryos (up to about 99%) developed synchronously. A small fraction of embryos lagged behind by one cleavage division at early stages; however, this difference was no longer detectable by the gastrula stage. After spawning, animals were returned to 3-l Kreisel tanks. Under these conditions, animals spawned daily.
Nematostella vectensis polyps were maintained in 16% ASW at 18 °C in the dark and fed daily Artemia salina nauplii. To induce spawning, the polyps were transferred to a 25 °C illuminated incubator for 10 h. Eggs were fertilized for 30 min, dejellied in 3% l-cysteine–ASW solution and then washed six times in ASW53.
No ethical approval was required for work with invertebrate species (M. leidyi and N. vectensis).
Transplantation experiments
Specific parts of the donor gastrula (around 4 h after fertilization, at 17–19 °C, mid-gastrulation) were excised with a fine scalpel (Feather Sterile MicroScalpel 15 Deg, 72045-15, Feather Safety Razor) and transplanted into a host embryo at the same stage of development. The tissues were transplanted to the lateral side of the host embryo at the tentacular axis. A single incision (around 30 μm deep and 40 μm long) was made in the host embryo, and a donor explant was gently inserted into this slit so that its internal surface lay against the wound of the host embryo. The graft adhered immediately and became firmly attached to the host tissues within the next 5–7 min (Extended Data Fig. 1). Following grafting, embryos were incubated in plastic Petri dishes coated with 2% agarose. The agarose-coated bottom prevented embryo adhesion to the plastic surface and further mechanical damage of the embryos. The results of transplantation were analysed in cydippids at day 2 after fertilization.
To follow the distribution of the grafted tissues in the host animals, we labelled the embryos with the vital membrane dye FM4-64FX (F34653, Invitrogen) or FM1-43FX (F35355, Invitrogen). Before transplantation, the vitelline membrane was manually removed from embryos at the 2–2.5 h after fertilization stage. Embryos were then transferred to a staining solution for 1 h (ASW containing FM4-64FX (or FM1-43FX) at 10 μg ml–1) and incubated in the dark. After staining, embryos were rinsed three times in seawater to remove excess dye and maintained in seawater until the desired developmental stage. Tissue from stained with FM4-64FX embryos was then transplanted into unstained (or stained with FM1-43FX) embryos.
The same grafting methodology was used for xenotransplantation experiments. Specific parts of the donor M. leidyi gastrula were transplanted into the blastocoel of gastrulating N. vectensis embryos. These manipulations were performed in ASW with a salinity of 22–23%. For N. vectensis transplantations, all the manipulations were the same except that they were performed in 16% ASW. After grafting, N. vectensis embryos were incubated in plastic Petri dishes. The embryos were fixed at 58 h after fertilization.
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