Staging of hydrocoel lobe formation
Firstly, the overall process of hydrocoel-lobe formation in auricularia larvae was observed, focusing on morphological characteristics. Fixed specimens were observed under an optical microscope and a confocal laser scanning microscope after staining with 4',6-Diamidino-2-phenylindole (DAPI) (Fig. 2). During the formation process, 4 developmental phases were defined based on the hydrocoel morphology (Table 1).
Phase 1: The hydrocoel primarily emerged at the left side of the digestive tract, showing a spindle-like shape (Fig. 2a-a’’’).
Phase 2: The entire hydrocoel extended up to 100 µm along its longitudinal axis of the spindle shape and became a slightly curved columnar shape (Fig. 2b-b’’’).
Phase 3: The hydrocoel lobes started to form (Fig. 2c-c’’’). The lobes were formed in a line along the AP axis of a larva. Each of 5 minor hydrocoel lobes which will become radial water vascular canals were respectively formed between the 5 major hydrocoel lobes which became water vascular canals of tentacles.
Phase 4: The lobes continuously extended to form tubular structures (Fig. 2d-d’’’). Each hydrocoel formed a tubular structure surrounded by epithelial tissue, and these tubular structures later formed the tubular network of the water vascular system.
There were no other obvious morphological changes in other parts of an auricularia larva, such as the digestive tract and ciliate bands, throughout these phases.
Observations of cell proliferation
To examine whether cell proliferation contributes to the hydrocoel morphogenesis, we observed the distribution patterns of cell-proliferation signals by labelling with 5-Ethynyl-2'-deoxyuridine (EdU) during the process. Larvae were incubated in 10 μM EdU sea water for 3 hours. In all of the observed phases, cell proliferation was detected (Fig. 3).
In phases 1 and 2, the EdU signal was distributed throughout the whole hydrocoel, and observations of both z-stack images and optical sections indicated that the signal was equally localized throughout the hydrocoel (Fig. 3a-b, a’-b’’). The cell proliferation was also observed in phases 3 and 4 (Fig. 3 c-d, c’-d’). Both z-stack images and optical sections showed that the EdU signals were not specifically localized at certain regions including lobe tips (Fig. 3c-d, c’-d’, arrowheads).
To examine whether cell proliferation is required for the hydrocoel-lobe formation, larvae at phase 2 was treated with the cell proliferation inhibitor aphidicolin. Administration of 0.5 µg/mL aphidicolin was shown to completely inhibit the EdU incorporation into the hydrocoel tissues, confirming the effect of aphidicolin (Fig. S1). Then auricularia larvae at phase 2, just prior to the hydrocoel-lobe formation, were treated with 0.5 µg/ml aphidicolin (Fig. 4a). At 20 hours after the onset of treatment, the hydrocoel lobes were successfully formed, as in untreated animals (Fig. 4b). There was no significant difference in the ratio of larvae in which hydrocoel lobes were formed (phase 3 or 4) to larvae in which hydrocoel lobes were not formed yet (phase 2) between the treated and control groups (Two-sided Fisher’s exact test, p = 0.144, Fig. 4c).
Observations on cell arrangement
To observe the changes of cell shape and cell arrangement in the hydrocoel epithelium, the cell membrane of live larvae was stained with bodipy FL C5-ceramide. At phase 1, the hydrocoel was surrounded by a single layer of columnar epithelial cells (Fig. 5a). At phase 2, the hydrocoel epithelium was stratified, which was observed throughout the whole hydrocoel (Fig. 5b). At phase 3, when the lobe formation started, stratified epithelial tissue became single-layered epithelium again at the tip of the hydrocoel lobes (Fig. 5c-d). The tissue between the lobes was still multi-layered and started to be bent. At phase 4, when the hydrocoel lobes extended, the epithelium between the lobes also became single-layered, being sharply bent, and consequently the lobe shape became more constricted (Fig. 5e-f).
The positions of hydrocoel lobes were the same in all of the observed individuals, and all of the hydrocoel lobes formed synchronously (Fig. 5). Although the lobes for water vascular canals of tentacles (i.e., lobe I, II, III, IV and V) were relatively larger than those for radial canals (i.e., lobe ii, iii, and iv) except for lobe i, the transitions in cell arrangement from multi-layered to monolayered epithelium were observed in all of the lobes regardless of lobe size (Fig. 5, 6).