Thyroid hormones reversibly inhibit metamorphic development in ophiuroid larvae

Abstract

The timing of metamorphosis and settlement is critical for the survival and reproductive success of marine animals with biphasic life cycles. Thyroid hormones (THs) regulate developmental timing in diverse groups of chordates, including the regulation of metamorphosis in amphibians, teleosts, lancelets, tunicates and lampreys. Recent evidence suggests a role for TH regulation of metamorphosis outside of the chordates, including echinoderms, annelids and molluscs. Among echinoderms, TH effects on development as well as underlying signaling mechanisms in early embryogenesis have been documented for echinoid (sea urchin) larvae, but we lack information on TH effects on metamorphic development in most other echinoderm groups, including the ophiuroids (brittle stars). Unexpectedly, we found that THs, principally 3,5,3',5'-tetraiodo-l-thyronine (T4), reversibly inhibit metamorphic development and settlement in the daisy brittle star (Ophiopholis aculeata). Exposure to thiourea, an inhibitor of TH synthesis, accelerated metamorphic development. We showed that these effects were highly stage specific, providing evidence for a developmental point-of-no-return in ophiuroid metamorphic development. Furthermore, starvation of O. aculeata accelerated juvenile morphogenesis and settlement. Starvation also prevented the inhibitory effect of thiourea on TH function, suggesting that TH synthesis may play a role in delaying metamorphosis under conditions of high food availability. These findings provide evidence for a function of TH signaling in ophiuroid metamorphic development and suggest that exogenous TH sources may be involved in the regulation of metamorphic timing in O. aculeata. Together with new evidence of TH involvement in metamorphic development in a range of invertebrates, these findings further emphasize the versatile and central role of endocrine signaling in metamorphosis.

Keywords: Ophiopholis aculeata; Echinoderm; Endocrine system; Larva; Metamorphosis; Thyroxine.

Fig. 1.

Metamorphic development in Ophiopholis aculeata staged by hydrocoel morphology. (0) Hydrocoel (green) has two distinct parallel layers. (1) Hydrocoel has begun forming five distinct lobes. Tissue thickness is roughly equal in the five lobes and base of the hydrocoel. (2) Hydrocoel lobes are extended. Base of the hydrocoel dramatically reduces in thickness. The oral lobe may be beginning to bend around the esophagus. (3) Hydrocoel is wrapping around the esophagus. Arm retraction has typically begun. (4) Hydrocoel has finished circularization, both ends of the hydrocoel have joined. The hydrocoel and rudiment are displaying clear pentaradial symmetry. (5) Rudiment has developed distinct and highly skeletonized arms. This is soon followed by separation of the juvenile from the posterolateral arms. (6) Larval arms are fully retracted. Juvenile is separated or separating from the posterolateral arms. h, hydrocoel; e, esophagus; ar, arm retraction; pla, posterolateral arms.

Fig. 2.

Representative images of thyroid hormone-exposed O. aculeata pluteus larvae (fed ad libitum). Polarized light microscopy is used on days 3 and 4 to reveal skeletal structures in the rudiment, and adjacent to the gut. Hydrocoel lobes are marked with arrowheads. (A) Development of control larvae was normal, with metamorphosis and settlement occurring over a 4–5 day period. In Aiii, the hydrocoel can be seen wrapping around the esophagus and developing tube foot buds (indicated with arrows). By Aiv, the ophiuroid has nearly completed metamorphosis and will shortly settle and discard the anterolateral arms (stage 5). (B) Thyroxine-exposed larvae (10^–7 mol l⁻¹ T4) displayed a reduced rate of metamorphic development, with hydrocoel development stalling at stage 1/2. Despite the reduced hydrocoel development, thyroxine-exposed larvae developed skeleton in the somatocoel adjacent to the gut (clearly visible in Biv). (C) Thiourea-exposed larvae (10^–5 mol l⁻¹ TU) metamorphosed and settled more rapidly than the control group. By day 3, the representative larva had a fused hydrocoel, and had nearly complete arm retraction (stage 4). No image is available for day 4, as the larva had settled and attached firmly to the plastic dish (stage 6). No abnormalities in the larvae or metamorphosed juveniles were observed.

Fig. 3.

Thyroid hormones inhibit metamorphosis in O. aculeata larvae under feeding and starvation conditions. (A–C) In the first experiment, five groups of larvae (N=60, n=12) were fed ad libitum and exposed for 4 days to a vehicle control (C), thyroid hormone (T4), an inhibitor of thyroid hormone synthesis (TU) or an inhibitor of MAPK activity (PD98059), or to a combination of T4+TU or PD+T4. (D,E) In the second experiment, larvae were placed under starvation conditions to eliminate exogenous hormones as a factor. Larvae (N=60, n=12) were exposed for 6 days to a vehicle control, thyroid hormones (T4 or T3), an inhibitor of thyroid hormone synthesis (TU) or both TU and T4. (A) T4 inhibited metamorphic development in O. aculeata larvae. (B) TU slightly accelerated metamorphic development, whereas TU+T4 inhibits metamorphic development. (C) PD did not significantly inhibit metamorphic development, whereas PD+T4 inhibited metamorphic development. (D) Unlike fed larvae, starved TU-exposed larvae were not significantly different from the control larvae. T4 inhibited metamorphic development in O. aculeata larvae in both the presence and absence of TU. Starved larvae exposed to both TU and T4 were not significantly different from T4 alone. (E) In starved larvae, T4 and TU+T4 inhibited metamorphic development. In contrast, T3 inhibited metamorphic development. (F) During early observation (<3 days), starved larvae developed more rapidly to metamorphosis. This effect was reduced when comparing starved larvae with TU-exposed fed larvae. Error bars are ±s.e.m.

Fig. 4.

Effects of T4 were reversible when larvae were placed in hormone-free water. After 5 days of exposure to T4, larvae staged to stage 1 of metamorphic development were washed and moved to two separate conditions: with and without T4 (N=24, n=12). Larvae that were removed from the T4 underwent normal metamorphic development faster than the T4-exposed group. Error bars are ±s.e.m.

Fig. 5.

Effect of T4 was not detected after metamorphic stage 2 (coelom wraparound). Larvae were placed in either a vehicle control or T4 (10⁻⁷ mol l⁻¹) at stage 2 and observed for 6 days (N=24, n=12). We did not detect a significant difference in development to metamorphosis in T4-exposed larvae that were exposed after stage 2 (coelom wraparound). Error bars are ±s.e.m.