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Thyroid Hormone Regulation of Vertebrate Postembryonic Development

• Yun-Bo Shi, PhD, Head, Section on Molecular Morphogenesis
• Liezhen Fu, PhD, Staff Scientist
• Nga Luu, MS, Biologist
• Lingyu Bao, MS, Visiting Fellow
• Suresh Babu Munipalli, PhD, Visiting Fellow
• Yuta Tanizaki, PhD, Visiting Fellow
• Shouhong Wang, PhD, Visiting Fellow
• Emeric Louis, MS, Graduate Student
• Zhaoyi Peng, BS, Graduate Student

The laboratory investigates the molecular mechanisms of thyroid hormone (TH) function during postembryonic development, a period around birth in mammals when plasma TH levels peak. The main model is the metamorphosis of pseudo-tetraploid Xenopus laevis and diploid Xenopus tropicalis, two highly related species that offer unique but complementary advantages. The control of this developmental process by TH offers a paradigm to study gene function in postembryonic organ development. During metamorphosis, different organs undergo vastly different changes. Some, like the tail, undergo complete resorption, while others, such as the limb, are developed de novo. The majority of the larval organs persist through metamorphosis but are dramatically remodeled to function in a frog. For example, tadpole intestine is a simple tubular structure consisting primarily of a single layer of larval epithelial cells. During metamorphosis, through specific larval epithelial cell death and de novo development of the adult epithelial stem cells followed by their proliferation and differentiation, it is transformed into an organ with a multiply folded adult epithelium surrounded by elaborate connective tissue and muscles. The wealth of knowledge from past research and the ability to manipulate amphibian metamorphosis both in vivo, by using genetic approaches or hormone treatment of whole animals, and in vitro in organ cultures offer an excellent opportunity to (1) study the developmental function of TH receptors (TRs) and the underlying mechanisms in vivo and (2) identify and functionally characterize genes that are critical for organogenesis, particularly, the formation of the adult intestinal epithelial stem cells, during postembryonic development in vertebrates [Reference 1]. A major recent focus has been to make use of the TALEN and CRISPR/Cas9 technologies to knock out the endogenous genes for functional analyses. In addition, the recent improvements in Xenopus tropicalis genome annotation allow us to carry out RNA-seq and chromatin-immunoprecipitation (ChIP)-seq analyses at the genome-wide level. They also allow us to adapt single-cell sequencing technology to study how TH induces cell transformations during vertebrate development. Thus, in recent years, we have focused our research on the diploid Xenopus tropicalis. We also complement our frog studies by investigating the genes found to be important for frog intestinal stem-cell development in the developing mouse intestine by making use of the ability to carry out conditional knockout.

Organ-specific effects on target binding due to knocking out thyroid hormone receptor α Xenopus metamorphosis

There are two TR genes, TRα and TRb, in all vertebrates. We previously generated TRα knockout (Xtr.thra^tmshi) tadpoles and showed that TRα is important for TH–dependent intestinal remodeling and hindlimb development, but not tail resorption, during metamorphosis. To investigate the underlying molecular basis, we earlier used chromatin immunoprecipitation-sequencing (ChIP-seq) to identify genes bound by TR in the intestine and hindlimbs of pre-metamorphic wild-type and Xtr.thra^tmshi tadpoles with or without TH treatment. We have now carried out similar analyses on the tail and compared the findings with those in the intestine and hindlimb [Reference 1]. We found that the tail had far fewer genes bound by TR or affected by TRα knockout. Bioinformatics analyses revealed that, among the genes bound by TR in wild-type but not Xtr.thra^tmshi organs, fewer gene-ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail than in the intestine and hindlimb; the difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue-specific roles in regulating TH-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis.

Comparative analysis of transcriptome profiles reveals distinct and organ-dependent genomic and nongenomic actions of thyroid hormone in Xenopus tropicalis tadpoles.

TH is essential for development and organ metabolism in all vertebrates. TH has both genomic and nongenomic effects on target cells. While much has been learnt on its genomic effects via TRs during vertebrate development, mostly through TR–knockout and knockin studies, little is known about the effects of TH on gene expression in animals in the absence of TR. By using the recently generated TR double-knockout (TRDKO) Xenopus tropicalis animals, we compared the effects of TH on global gene expression in tadpole tissues in the presence or absence of TR [Reference 2]. We carried out RNA-seq analyses on gene expression in tadpole tail and intestine of wild-type and TRDKO tadpoles with or without TH treatment. We observed that removing TRs reduced the number of genes regulated by TH in both organs. Gene Ontology (GO) analysis revealed that TH affected distinct biological processes and pathways in wild-type and TRDKO tadpoles. Many GO terms were enriched among genes regulated in wild-type tissues and are likely involved in mediating the effects of TH on metamorphosis, e.g., those related to development, stem cells, apoptosis, and cell cycle/cell proliferation. However, such GO terms and pathways were not enriched among TH–regulated genes in TRDKO tadpoles. Instead, in TRDKO tadpoles, GO terms and pathways related to “metabolism” and “immune response” were highly enriched among TH–regulated genes. We further observed strong divergence in the TR–independent, nongenomic effects of TH in the intestine and tail. Our data suggest that TH has distinct and organ-dependent effects on gene expression in developing tadpoles. The TR–mediated effects are consistent with the metamorphic changes, in agreement with the fact that TR is necessary and sufficient to mediate the effects of TH on metamorphosis. TH appears to have a major effect on metabolism and immune response via TR–independent nongenomic processes [Reference 2].

Thyroid hormone receptor knockout prevents the loss of Xenopus tail regeneration capacity at metamorphic climax.

Animal regeneration is the natural process of replacing or restoring damaged or missing cells, tissues, organs, and even the entire body to full function. Studies in mammals have revealed that many organs lose regenerative capacity soon after birth when TH level is high. This suggests that TH plays an important role in organ regeneration. Intriguingly, plasma TH level peaks during amphibian metamorphosis, which is very similar to postembryonic development in humans. In addition, many organs, such as heart and tail, also lose their regenerative ability during metamorphosis, making frogs a good model in which to address how the organs gradually lose their regenerative ability during development and what roles TH may play in this process. Early tail-regeneration studies have been done mainly in the tetraploid Xenopus laevis (X. laevis), which does not lend itself easily to gene knockout studies. We used the highly related but diploid anuran X. tropicalis to investigate the role of TH signaling in tail regeneration with gene knockout approaches. We discovered that X. tropicalis tadpoles could regenerate their tail from pre-metamorphic stages up to the climax stage 59, whereupon they lose regenerative capacity as tail resorption begins, just as is observed for X. laevis. To test the hypothesis that the TH–induced metamorphic program inhibits tail regeneration, we used TR double-knockout (TRDKO) tadpoles lacking both TRa and TRb, the only two receptor genes in vertebrates, for tail regeneration studies [Reference 3]. Our results showed that TRs were not necessary for tail regeneration at any stage. However, unlike wild-type tadpoles, TRDKO tadpoles retained regenerative capacity at the climax stages 60/61, likely in part by increasing apoptosis during the early regenerative period and enhancing subsequent cell proliferation. In addition, TRDKO animals had higher levels of amputation-induced expression of many genes important for tail regeneration, compared with the non-regenerative wild-type tadpoles at stage 61. The high level of apoptosis in the remaining uncut portion of the tail, as wild-type tadpoles undergo tail resorption after stage 61, appeared to also contribute to the loss of regenerative ability. For the first time, our findings revealed evolutionary conservation in the loss of tail regeneration capacity at metamorphic climax between X. laevis and X. tropicalis. Our studies with molecular and genetic approaches demonstrated that the TR–mediated, TH–induced gene regulation program is responsible not only for tail resorption but also for the loss of tail regeneration capacity [Reference 3]. Further studies using the model should uncover how TH modulates the regenerative outcome, and should offer potential new avenues for regenerative medicines for human patients.

Competitive PCR with dual fluorescent primers enhances the specificity and reproducibility of genotyping animals generated from genome editing.

Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, leading to DNA repair through homologous recombination or non-homologous end joining (NHEJ). DNA repair through the NHEJ mechanism is an error-prone process, which often results in point mutations or stretches of indels (insertions and deletions) within the targeted region. Such mutations in embryos are germline-transmissible, thus providing an easy means to generate organisms with gene mutations. However, point mutations and short indels are difficult to genotype, often requiring labor-intensive sequencing to obtain reliable results. We developed a single-tube competitive PCR assay with dual fluorescent primers that allows simple and reliable genotyping. While we used Xenopus tropicalis as a model organism, the approach should be applicable to genotyping of any organism.

Upregulation of the protooncogene Ski by thyroid hormone in the intestine and tail during Xenopus metamorphosis

TH affects frog metamorphosis through TH receptor (TR)–mediated regulation of TH response genes, where TR forms a heterodimer with RXR (9-cis retinoic acid receptor) and binds to TH response elements (TREs) in TH response genes to regulate their transcription. To study how TH regulates intestinal stem-cell development and/or proliferation, we previously identified many putative direct TH response genes in Xenopus tropicalis tadpole intestine by using ChIP (chromatin immunoprecipitation)-on-chip assays. Among them is the proto-oncogene Ski, which encodes a nuclear protein with complex functions in regulating cell fate. We showed that Ski is upregulated in the intestine and tail of pre-metamorphic tadpoles upon TH treatment, and its expression peaks at stage 62, the climax of metamorphosis [Reference 4]. We further discovered a TRE in the first exon that can bind to TR/RXR in vitro and mediate TH regulation of the promoter in vivo. These data demonstrate that Ski is activated by TH through TR binding to a TRE in the first exon during Xenopus tropicalis metamorphosis, implicating a role of Ski in regulating cell fate in this process.

Liver development during Xenopus tropicalis metamorphosis is controlled by TH activation of WNT signaling.

Many mammalian organs and tissues, including erythrocytes, mature into their adult forms during postembryonic development when plasma TH level peaks, resembling amphibian metamorphosis. TR mutations/deletions can cause hematopoietic dysfunction, suggesting that TH plays a role in erythropoiesis during development. We recently generated TR double knockout (TRDKO) Xenopus tropicalis as a model in which to study TH function during postembryonic development. Our analyses of TRDKO tadpoles during metamorphosis revealed that they exhibited characteristics similar to human iron-deficiency anemia. Given that the liver is the hematopoietic organ, our finding suggests a defect in liver development in TRDKO tadpoles. We analyzed liver metamorphosis in wild-type and TRDKO tadpoles and found that wild-type liver metamorphosis involved increased cell proliferation, hepatocyte hypertrophy, and activation of urea-cycle gene expression, a key feature of adult/mature liver in vertebrates [Reference 5]. Interestingly, the TRDKO liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate the expression of urea-cycle genes. To reveal the molecular pathways regulated by TH during liver remodeling, we performed RNA-seq analysis and found that TH activated the canonical Wnt pathway in the liver. Wnt11 was particularly activated in both fibroblasts and hepatic cells and, in turn, likely acted to promote stem-cell development and/or proliferation and maturation of hepatocytes [Reference 5]. Our findings also resemble those from studies on liver regeneration in mammals. Thus, analyses of liver metamorphosis have the potential to bring new insights not only into how TH regulates liver development but are also a potential means to improve liver regeneration.

The l-type amino acid transporter 1 (LAT1) in hypothalamic neurons in mice maintains energy and bone homeostasis.

To regulate cellular processes, TH has to be actively transported into cells, a process that is mediated by several different types of transporters. One of our previously identified TH–response genes in Xenopus intestine, LAT1, encodes the light chain of a heterodimeric system L type of TH transporter, which also transports several amino acids. Interestingly, LAT1 is highly upregulated at the climax of metamorphosis in the tadpole intestine, coinciding with the formation and rapid proliferation of adult intestinal stem cells. Further, we found that LAT1 was also highly expressed in the mouse intestine during the neonatal period when the mouse intestine matured into the adult form, a process that appears also to involve TH–dependent formation and/proliferation of adult intestinal stem cells. In a collaborative study, we generated a mouse line with the LAT1 gene floxed, which allows conditional knockout of LAT1 upon expression of the Cre recombinase. We are currently analyzing the effect of LAT1 knockout specifically in the mouse intestine by expressing Cre under the control of the intestinal epithelial-specific villin promoter. In another collaborative study, we discovered LAT1 (also known as Slc7a5) expression in hypothalamic neurons, which regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). Importantly, we found that LAT1 in hypothalamic leptin receptor (LepR)–expressing neurons was important for systemic energy and bone homeostasis. We observed LAT1–dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by Slc7a5) in LepR–expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR–expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR–expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient in Slc7a5 in LepR–expressing cells. We found that the mechanistic target of rapamycin complex-1 (mTORC1) is a crucial mediator of the LAT1–dependent regulation of energy and bone homeostasis. These results suggest that the LAT1–mTORC1 axis in LepR–expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of amino-acid sensing by hypothalamic neurons in body homeostasis.

Additional Funding

• FY23 NICHD Early Career Award for Lingyu Bao

Publications

1. Tanizaki Y, Zhang H, Shibata Y, Shi Y-B. Organ-specific effects on target binding due to knocking out thyroid hormone receptor α during Xenopus metamorphosis. Dev Growth Differ 2023 65:23–28.
2. Wang S, Shibata Y, Tanizaki Y, Zhang H, Yan W, Fu L, Shi Y-B. Comparative analysis of transcriptome profiles reveals distinct and organ-dependent genomic and nongenomic actions of thyroid hormone in Xenopus tropicalis tadpoles. Thyroid 2023 33:511–522.
3. Wang S, Shibata Y, Fu L, Tanizaki Y, Luu N, Bao L, Peng Z, Shi Y-B. Thyroid hormone receptor knockout prevents the loss of Xenopus tail regeneration capacity at metamorphic climax. Cell Biosci 2023 13(1):40.
4. Fu L, Liu R, Ma V, Shi Y-B. Upregulation of protooncogene Ski by thyroid hormone in the intestine and tail during Xenopus metamorphosis. Gen Comp Endocrinol 2022 328:114102.
5. Tanizaki Y, Wang S, Zhang H, Shibata Y, Shi Y-B. Liver development during Xenopus tropicalis metamorphosis is controlled by T3-activation of WNT signaling. iScience 2023 26:106301.

Collaborators

• Caroline Esnault, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD
• Eiichi Hinoi, PhD, Kanazawa University Graduate School, Kanazawa, Japan
• James Iben, PhD, Molecular Genomics Core, NICHD, Bethesda, MD
• Tianwei Li, PhD, Molecular Genomics Core, NICHD, Bethesda, MD
• Fabio Rueda Faucz, PhD, Molecular Genomics Core, NICHD, Bethesda, MD
• Bingyin Shi, MD, Xi’an Jiaotong University School of Medicine, Xi'an, China
• Guihong Sun, PhD, Wuhan University School of Medicine, Wuhan, China
• Peter Taylor, PhD, University of Dundee, Dundee, United Kingdom
• Chuan Wu, MD, PhD, Experimental Immunology Branch, NCI, Bethesda, MD
• Henry Zhang, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD

Contact

For more information, email shi@helix.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/shi.

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