Wynen H et al. (2022), Thyroid hormone-induced cell death in sea urchi...

ECB-ART-51155

J Exp Biol 2022 Dec 01;22523:. doi: 10.1242/jeb.244560.

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Thyroid hormone-induced cell death in sea urchin metamorphic development.

Wynen H , Taylor E , Heyland A .

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Thyroid hormones (THs) are important regulators of development, metabolism and homeostasis in metazoans. Specifically, they have been shown to regulate the metamorphic transitions of vertebrates and invertebrates alike. Indirectly developing sea urchin larvae accelerate the formation of juvenile structures in response to thyroxine (T4) treatment, while reducing their larval arm length. The mechanisms underlying larval arm reduction are unknown and we hypothesized that programmed cell death (PCD) is linked to this process. To test this hypothesis, we measured larval arm retraction in response to different THs (T4, T3, rT3, Tetrac) and assessed cell death in larvae using three different methods (TUNEL, YO-PRO-1 and caspase-3 activity) in the sea urchin Strongylocentrotus purpuratus. We also compared the extent of PCD in response to TH treatment before and after the invagination of the larval ectoderm, which marks the initiation of juvenile development in larval sea urchin species. We found that T4 treatment results in the strongest reduction of larval arms but detected a significant increase of PCD in response to T4, T3 and Tetrac in post-ingression but not pre-ingression larvae. As post-ingression larvae have initiated metamorphic development and therefore allocate resources to both larval and the juvenile structures, these results provide evidence that THs regulate larval development differentially via PCD. PCD in combination with cell proliferation likely has a key function in sea urchin development.

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	Fig. 1. Effect of thyroid hormone (TH) treatment on arm length in pre- and post-ingression sea urchin larvae. Larvae were treated with seawater alone (control), reverse-triiodothyronine (rT3; TH control), triiodothyronine (T3), thyroxine (T4) or tetraiodothyroacetic acid (Tetrac). (A) T4 (thyroxine) and Tetrac reduce post-oral (PO) arm length in post-ingression larvae but not pre-ingression larvae. (B–D) In post-ingression larvae, post-oral (PO; B), anterolateral (AL; C) and post-dorsal (PD; D) arms are reduced in response to T4 and Tetrac treatment; however, Tetrac consistently shows an inverse dose–response relationship with respect to arm length. Data are means±1 s.e.m. *Significance of P<0.05.
	Fig. 2. T4 treatment results in increased levels of early apoptosis measured by YO-PRO-1 and propidium iodide in post-ingression sea urchin larval arms. (A–C and A′–C′) Representative images from post-ingression larvae (20–22 days post-fertilization, dpf), exposed to one of five hormone treatments (control, rT3, T3, T4 or Tetrac; see Materials and Methods for details), featuring the cell categories (ciliated, mesenchymal, epithelial) quantified in this study using YO-PRO and propidium iodide (PI) staining, imaged under ×300 magnification (scale bars: 5 µm). (A) Apoptotic ciliated cells: arrows point to two small green cells with no overlapping red signal (Tetrac treatment). (A′) Apoptotic ciliated cells with necrosis: arrows indicate a cluster of four apoptotic bodies. It is difficult to distinguish between early apoptotic ciliated cells, necrotic ciliated cells and apoptotic bodies; however, apoptotic bodies and necrotic ciliated cells fluoresce when stained with PI. Additionally, apoptotic bodies are often found in tightly packed clusters (Tetrac treatment). (B) An apoptotic mesenchymal cell associated with skeletal rods (arrow) with no overlapping PI signal (control treatment). (B′) An apoptotic mesenchymal cell associated with a skeletal cell (arrow) that is not in the early stages of apoptosis as the cell shows a positive PI signal, indicating that it is either necrotic or past the early stages of apoptosis. Several necrotic cells can also be seen in the background. (C) Apoptotic epithelial cells (arrows): larger tightly packed cells with no overlapping red signal, with some necrotic cells in background (T3 treatment). (C′) Five epithelial cells with PI staining. These cells are either dead or in the very last stages of apoptosis, because they feature a clear PI signal and apoptotic bodies (arrows). (D–M) Representative images of early apoptosis in response to different TH treatments (D,I: control; E,J: rT3; F,K: T3; G,L: T4; H,M: Tetrac) for 24 h before staining with YO-PRO-1, PI and Hoechst pre-ingression (D–H; 10–11 dpf) and post-ingression (I–M; 20–22 dpf) and imaged at ×100 magnification (scale bars: 10 µm). Arms are outlined with white lines. The strongest YO-PRO-1 signal was detected in the T4 treatment post-ingression, and the strongest PI signal was detected in Tetrac treatment post-ingression. Cells stained with YO-PRO-1 are green, cells stained with PI are red, cells stained with Hoechst are blue.
	Fig. 3. T3, T4 and Tetrac result in increased levels of early apoptosis measured by YO-PRO-1 and PI in ciliated and epithelial cells in larval arms of post-ingression but not pre-ingression larvae. Cells stained with YO-PRO-1 and PI were acquired and processed for intensity quantification and cell count. Cell density for apoptotic ciliated (A), skeletal (B) and epithelial (C) cells was calculated by subtracting the necrotic signal (PI) from the apoptotic signal (YO-PRO-1) in these cell types and dividing by the surface area. In order to validate this approach, apoptotic cells were also counted manually and divided by the manual count of all nuclei within the area (D). Larvae were treated for 24 h in all experiments: control, rT3, T3, T4 and Tetrac. Data are means±1 s.e.m. *Significance of P<0.05.
	Fig. 4. T4 and Tetrac treatment result in the largest number of apoptotic cells (late-stage cell death) in the PO arms of sea urchin larvae, as detected by TUNEL staining. Representative images of TUNEL (A–L) and TUNEL plus Hoechst (A′–L′) staining in response to TH treatment [A,A′,G,G′: control; B,B′,H,H′: positive control (heat-shock treatment); C,C′,I,I′: rT3; D,D′,J,J′: T3; E,E′,K,K′: T4; F,F′,L,L′: Tetrac] in the larval arms of pre-ingression (A,A′–F,F′; 10–11 dpf) and post-ingression (G,G′–L,L′; 20–22 dpf) larvae. Cells stained with TUNEL are green and cells stained with Hoechst are blue. Scale bars in all images: 10 μm. Larval arms are outlined with white lines. For quantitative results, see Fig. 5.
	Fig. 5. T4 and Tetrac treatment result in increased levels of apoptosis (late-stage cell death) in post-ingression compared with pre-ingression larvae, and in PO arms compared with PD arms, post-ingression, as detected by TUNEL staining. (A) Apoptotic cell density increased in larvae treated with Tetrac in pre-ingression larvae, and T4 treatment resulted in increased apoptosis levels post-ingression. (B) Post-ingression, we found higher levels of apoptosis measured by TUNEL in PO arms compared with PD arms in response to T4 treatment. We also found that Tetrac treatment resulted in increased apoptosis levels post-ingression. Data are means±1 s.e.m. *Significance of P<0.05.
	Fig. 6. T4 and Tetrac result in the highest number of caspase-3-positive cells compared with controls and T3 treatment. (A,B) Representative images of caspase-3 immunohistochemistry, indicating cell-associated staining (A; arrows indicate positive staining) and staining that is not associated with cells (B; arrows indicate non-specific staining). (C–N) Representative images of caspase-3 immunohistochemistry in arms of pre-ingression (C–H) and post-ingression (I–N) larvae, exposed to control (C,I: unexposed control; D,J: positive heat shock control; E,K: rT3 control) or TH (F,L: T3; G,M: T4; H,N: Tetrac) treatment for 24 h before staining using caspase-3 antibody (green) and Hoechst (blue) and imaged at ×900 magnification. Scale bar in all images: 10 μm. Larval arms are outlined with white lines.
	Fig. 7. Caspase-3 protein expression increases in response to T4 and Tetrac treatment in PO arms post-ingression but not pre-ingression. (A) Caspase-3 immunohistochemistry measured at the per-cell level in PO arms (number of signals per caspase-positive cell) increases in T4 and Tetrac treatments compared with control and rT3 treatment. (B) Measurement of average caspase-3 signal density results a in similar pattern to that in A. Data are means±1 s.e.m. *Significance of P<0.05.

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