Local Thyroid Hormone Activation or Inactivation by Deiodinases Regulates Intestinal Remodeling and Tail Resorption during Xenopus Metamorphosis

Abstract

Background:

Amphibian metamorphosis involves dramatic tissue remodeling, such as intestinal remodeling and tail resorption, driven by thyroid hormones (THs). Among these hormones, T3, the major active form of TH, is locally generated from T4 by type I and type II deiodinases (Dio1 and Dio2), while T3 is inactivated to T2 primarily by type III deiodinase (Dio3). Tissue-specific regulation of T3 availability is thus crucial for orchestrating TH-dependent developmental events. However, little is known about how spatiotemporal regulation of TH activation and inactivation by deiodinases influences tissue remodeling and degradation during frog metamorphosis.

Methods:

We analyzed the spatiotemporal distribution of deiodinase mRNAs at single-cell resolution using hybridization chain reaction RNA-fluorescent in situ hybridization (HCR RNA-FISH) during metamorphosis. To investigate the role of local TH activation or inactivation in vertebrate development, we generated mosaic Dio2- and Dio3-knockdown (KD) Xenopus laevis using CRISPR-Cas9 genome editing. Molecular and morphological analyses were then carried out to determine whether local TH conversion is required for intestinal remodeling and tail resorption.

Results:

dio2 mRNA expression peaked at stage 61, whereas dio3 mRNA expression showed delayed upregulation, peaking at stage 64 during intestinal remodeling in wild-type animals. dio2 mRNA appeared in fibroblasts located just beneath the epithelial layer at stage 60 and increased in proliferating fibroblasts at stage 62. Interestingly, dio3 expression was colocalized with lgr5 (an adult intestinal stem cell marker)-positive epithelial cells at stage 61. During tail resorption, dio2 and dio3 expressions peaked at stages 62 and 60, respectively. Following gene KD, Dio2KD animals exhibited delayed tail resorption and intestinal remodeling with reduced expression of multiple TH target genes, whereas Dio3KD animals completed metamorphosis more rapidly.

Conclusions:

Spatiotemporal regulation of TH activation and inactivation by deiodinases is essential for the timing and progression of organ-specific metamorphic events in Xenopus laevis.