We investigate the molecular mechanisms of thyroid hormone function during postembryonic development. The main model is the metamorphosis of the amphibians Xenopus laevis, a species well studied as a developmental model, and Xenopus troplicalis, a species more recently developed as a model system and whose shorter life cycle and sequenced genome offers some unique advantages. The control of postembryonic development by thyroid hormone (TH) offers a unique paradigm to study gene function in postembryonic organ development. During metamorphosis, various organs undergo vastly different changes. Some, such as 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 in Xenopus is a simple tubular structure consisting primarily of a single layer of larval epithelial cells. During metamorphosis, tadpole intestine is transformed into an organ with a multiply folded adult epithelium surrounded by elaborate connective tissue and muscles; the process involves specific larval epithelial cell death and de novo development of adult epithelial stem cells, which then proliferate and differentiate. The wealth of knowledge from earlier research and the ability to manipulate amphibian metamorphosis both in vivo,by using transgenesis or hormone treatment of whole animals, and in vitro in organ cultures offer an excellent opportunity to study the developmental function of TH receptors (TRs) and underlying mechanisms in vivo and to identify and functionally characterize genes that are critical for postembryonic organ development in vertebrates. Our studies have revealed likely conserved mechanisms of adult intestinal stem cell development in vertebrates, prompting us to adapt the mouse model, which complements the amphibian system, in which gene knockout is not yet available.
Based on TR expression profiles and the receptor's molecular properties, we previously proposed a dual-function model for TR during frog development. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes in vivo. In premetamorphic tadpoles, the heterodimers repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. When TH is present, either from endogenous synthesis during development or exogenous addition to the rearing water of premetamorphic tadpoles, TR/RXR heterodimers activate TH-inducible genes to initiate metamorphosis. Our studies over the last several years have provided molecular and genetic support for this model. Furthermore, we revealed important roles of co-repressor and co-activator complexes in TR action during metamorphosis. Given that these cofactor complexes can modify histone acetylation and methylation levels, we were interested in determining whether gene regulation by TR involves alterations in histone modifications, especially considering that little is known about whether and how histone modifications change upon gene regulation by TR during development of any vertebrate. Using chromatin immunoprecipitation (ChIP) assay and the intestinal metamorphosis in Xenopus tropicalis as a model, we demonstrated for the first time in vivo during vertebrate development that liganded TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, changes in histone acetylation and methylation induced by liganded TR indicate that some histone activation and repression marks for gene regulation during vertebrate development differ from those assigned based on correlations with mRNA levels in cell cultures, suggesting the importance of tissue- and developmental context in the roles of histone modifications in gene regulation. Our findings further provide important mechanistic insights into how chromatin remodeling affects developmental gene regulation in vivo.
The complexity of metamorphic changes in different organs argues for the presence of distinct gene regulation programs regulated by TR. Knowledge of this systematic gene regulation will help identify not only molecular markers but also important cellular pathways or critical genes for future mechanistic studies. Thus, we have begun to use the recently developed Xenopus laevis cDNA array to analyze genome-wide gene expression changes associated with TH-induced intestinal remodeling, a process that involves selective degeneration of the larval epithelium through apoptosis and de novo development of the adult epithelium. Our earlier analysis using total intestinal RNA revealed co-expressed genes involved in essential cell processes such as apoptosis and proliferation. Furthermore, we showed that most of the genes highly induced at metamorphic climax are also upregulated in the mouse intestine around birth, the postembryonic period resembling metamorphosis, an observation that supports conservation of the underlying molecular pathways. More recently, we carried out cDNA arrays by using isolated epithelium and non-epithelium tissues of the intestine at various stages during metamorphosis. Preliminary analysis suggested the existence of distinct epithelial and non-epithelial genes that are likely involved in the development of adult intestinal stem cells.
We showed earlier that adult intestinal stem cells originate from differentiated larval epithelial cells in the Xenopus laevis intestine. To determine whether TH signaling in the epithelium alone is sufficient for inducing adult stem cells, we performed tissue-recombinant culture experiments by using transgenic Xenopus laevis tadpoles that express a dominant positive TH receptor (dpTR) under a control of heat shock promoter. We isolated wild-type (WT) or dpTR transgenic (Tg) larval epithelium (Ep) from the tadpole intestine, recombined with homologous or heterologous non-epithelial tissues (non–Ep), and then cultivated them in the absence of TH but with daily heat shocks to induce transgenic dpTR expression. Adult epithelial progenitor cells expressing sonic hedgehog became detectable on day 5 in both the recombinant intestine of Tg Ep and Tg non–Ep (Tg/Tg) and that of Tg Ep and Wt non–Ep (Tg/WT). However, in Tg/WT intestine, the cells did not express other stem cell markers such as Musashi-1 and never generated adult epithelium expressing a marker for absorptive epithelial cells. Our results indicate that, while the adult progenitor cells may be predetermined in the larval epithelium, TR–mediated gene expression in the surrounding tissues other than the epithelium is required for the cells to develop into adult stem cells, suggesting the importance of TH–inducible epithelial-connective tissue interactions in establishing the stem cell niche in the amphibian intestine.
As indicated above, TH action in the larval epithelium can induce some larval epithelial cells to de-differentiate into adult progenitor cells via a cell-autonomous function. Interestingly, the TR–co-activator PRMT1 is strongly induced in these progenitor cells. More important, our in vivo studies showed that transgenic overexpression of PRMT1 leads to increased population of adult stem cells while knockdown of endogenous PRMT1 reduces the number of stem cells. In addition, PRMT1 is upregulated during zebrafish and mouse development in the equivalent postembryonic period when thyroid hormone levels are high and adult intestinal stem cells develop, suggesting that PRMT1 plays an evolutionarily conserved role in the development of adult intestinal stem cells.
To regulate gene transcription in the nucleus, TH is taken up from the circulating plasma by cells by active transport. Several transporters for TH have been identified over the years. We previously identified one of TH's direct target genes in the metamorphosing intestine as the light chain of a heterodimeric transporter, the L-type amino acid transporter-1 (LAT1), and showed that it is capable of transporting TH into cells. LAT1 complexes with the heavy chain CD98, which is a multifunctional protein CD98 (CD98hc, Slc3a2) that can also associate with integrin b1 through its cytoplasmic and transmembrane domains. It has been shown that CD98hc–mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc–null mice exhibit early post-implantation lethality similar to integrin b1–null mice, supporting the importance of its interaction with integrin b1. On the other hand, the extracellular domain of CD98hc interacts with LATs and is essential for appropriate cell-surface distribution of LATs. LATs mediate the transport of both TH and branched chain amino acids. In this respect, CD98hc may also affect development via these transporters.As indicated above, TH is important for the maturation of the adult intestine in mouse, and many TH–target genes identified during Xenopus metamorphosis are similarly regulated during mouse intestinal development. To investigate the role of TH in adult intestinal stem cell development, we are interested in carrying out gene knockout experiments in mouse, which is not yet possible in frogs. Thus, in collaboration with Chuxia Deng's, we generated a mutant mouse line from an embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hcD/+). Expression of the CD98hc mutant allele results in DCD98hc-b geo fusion protein, in which the extracellular C-terminal 102 amino acids of CD98hc are replaced by b geo. Analyses of PST080 ES cells and of reconstituted frog oocytes demonstrated that the DCD98hc-b geo fusion protein preserved its ability to interact with integrin b1 even though the mutant protein was hardly localized on the cell surface. These findings suggest that DCD98hc-b geo protein can mediate integrin signaling but cannot support TH or amino acid transport through LATs. CD98hcD/+ mice were normal. Although some implantation sites lacked embryonic component at E9.5, all implantation sites contained embryonic component at E7.5. Thus, CD98hcD/D embryos are likely to die between E7.5 and E9.5. Considering that CD98hc-complete-knockout (CD98hc−/−) embryos reportedly die shortly after implantation, our findings suggest potential stage-specific roles of CD98hc in murine embryonic development. CD98hc may be essential for early post-implantation development by regulating integrin-dependent signaling while the other function of CD98hc as a component of TH and amino acid transporters may be required for embryonic development at later stages. The availability of this mouse line will allow us to investigate, in the near future, whether TH transport plays a role in intestinal stem cell development.
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