Abstract
Due to similarities in iodine concentrations and peroxidase activities, the thyroid in vertebrates is considered to originate from the endostyle of invertebrate chordates even though it is a glandular (mucus-producing) organ for aquatic suspension feeding. Among chordates with an endostyle, urochordates are useful evolutionary research models for the study of vertebrate traits. The ascidian Ciona intestinalis forms an endostyle with specific components of glandular- and thyroid-related elements, and molecular markers have been identified for these components. Since we previously examined a simple endostyle in the larvacean Oikopleura dioica, the expression of the thyroid-related transcription factor genes, Ciona Nkx2-1 and FoxE, was perturbed by TALEN-mediated gene knockout in the present study to elucidate the shared and/or divergent features of a complex ascidian endostyle. The knockout of Ciona Nkx2-1 and FoxE exerted different effects on the morphology of the developing endostyle. The knockout of Nkx2-1 eliminated the expression of both glandular and thyroidal differentiation marker genes, e.g., vWFL1, vWFL2, CiEnds1, TPO, and Duox, while that of FoxE eliminated the expression of the differentiation marker genes, TPO and CiEnds1. The supporting element-related expression of Pax2/5/8a, Pax2/5/8b, FoxQ1, and β-tubulin persisted in the hypoplastic endostyles of Nkx2-1- and FoxE-knockout juveniles. Although the gene regulation of ascidian-specific CiEnds1 remains unclear, these results provide insights into the evolution of the vertebrate thyroid as well as the urochordate endostyle.
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Acknowledgements
We thank Dr. Nori Satoh and Dr. Yutaka Satou for providing the Ciona cDNA clones. We thank the laboratory members at the Shimoda Marine Research Center at the University of Tsukuba for their technical support with TALEN-mediated knockout experiments. We also thank Drs. Yutaka Satou, Manabu Yoshida, Reiko Yoshida, and Satoe Aratake and other members working under the National Bio-Resource project (AMED) for supplying Ciona adults and the constructs of plasmid vectors for TALEN-mediated knockout experiments. We thank Ms. Mizuki Ito, Mr. Ippei Yamamura, Ms. Haruka Yamaguchi, Ms. Honoka Nakadai, and the laboratory members of our group for their assistance with the laboratory culture of Ciona juveniles.
Funding
This work was supported by JSPS KAKENHI grant number 20K07221 to M. O., and also by Grants-in-Aid for Scientific Research (KAKENHI) from the JSPS and MEXT Japan to Y. S. (Grant Number 19H03262).
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Y.S., T.O., and M.O. designed the experiments. M.Y. performed ISH and histochemical analyses. T.H. and A.H. performed the knockout and knockdown of genes with the aid of Y.S. M.O. and all coauthors wrote the manuscript.
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Yamagishi, M., Huang, T., Hozumi, A. et al. Differentiation of endostyle cells by Nkx2-1 and FoxE in the ascidian Ciona intestinalis type A: insights into shared gene regulation in glandular- and thyroid-equivalent elements of the chordate endostyle. Cell Tissue Res 390, 189–205 (2022). https://doi.org/10.1007/s00441-022-03679-w
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DOI: https://doi.org/10.1007/s00441-022-03679-w