Fu Liezhen, Wen Luan, Shi Yun-Bo
Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA.
Methods Mol Biol. 2018;1801:247-263. doi: 10.1007/978-1-4939-7902-8_20.
The amphibian Xenopus laevis has long been used as a model for studying vertebrate cell and developmental biology largely due to the easiness to manipulate this system in vivo and in vitro. While most of the developmental studies have been on Xenopus embryogenesis, considerable efforts have been made to understand its metamorphosis, a process mimicking postembryonic development in mammals when many organs mature into their adult forms in the presence of high levels of thyroid hormone (T3). Amphibian metamorphosis is totally dependent on T3 and offers a number of advantages for experimental analyses compared to the late stage, uterus-enclosed mammalian embryos. Earlier studies on metamorphosis in Xenopus laevis have revealed dual functions of T3 receptors (TR) during premetamorphic development and metamorphosis as well as important roles of TR-interacting corepressors and coactivators during these two periods, respectively. The development of gene-editing technologies that functions in amphibians in recent years has made possible for the first time to study function of endogenous TRs, especially in the highly related diploid anuran species Xenopus tropicalis. Here, we first review the current mechanistic understanding of the regulation of metamorphosis by T3 and TR, and then describe a detailed method to use TALEN to knock out TRα for studying its role in gene regulation by T3 in vivo and Xenopus development.
两栖动物非洲爪蟾长期以来一直被用作研究脊椎动物细胞和发育生物学的模型，这主要是因为在体内和体外操纵该系统都很容易。虽然大多数发育研究都集中在非洲爪蟾的胚胎发生上，但人们也付出了相当大的努力来了解其变态过程，这一过程类似于哺乳动物的胚后发育，即在高水平甲状腺激素（T3）存在的情况下，许多器官成熟为成体形式。两栖动物的变态完全依赖于T3，与晚期子宫内的哺乳动物胚胎相比，它为实验分析提供了许多优势。早期对非洲爪蟾变态的研究揭示了T3受体（TR）在变态前发育和变态过程中的双重功能，以及TR相互作用的共抑制因子和共激活因子在这两个时期的重要作用。近年来在两栖动物中起作用的基因编辑技术的发展，首次使得研究内源性TR的功能成为可能，特别是在高度相关的二倍体无尾目物种热带爪蟾中。在这里，我们首先综述目前对T3和TR调节变态的机制的理解，然后描述一种使用TALEN敲除TRα以研究其在体内T3基因调控和爪蟾发育中作用的详细方法。
