EPITHELIAL-CONNECTIVE TISSUE INTERACTIONS INDUCED BY THYROID HORMONE RECEPTOR ARE ESSENTIAL FOR ADULT STEM CELL DEVELOPMENT IN THE XENOPUS LAEVIS INTESTINE. We have previously shown that the 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 the adult stem cells, we have 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. Wild-type (Wt) or dpTR transgenic (Tg) larval epithelium (Ep) was isolated from the tadpole intestine, recombined with homologous or heterologous non-epithelial tissues (non-Ep), and then cultivated 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, they did not express other stem cell markers such as Musashi-1 and never generated the 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 them to develop into adult stem cells, suggesting the importance of TH-inducible epithelial-connective tissue interactions in establishment of the stem cell niche in the amphibian intestine. PROTEIN ARGININE METHYLTRANSFERASE 1 (PRMT1) PLAYS AN EVOLUTIONARILY CONSERVED ROLE IN ADULT INTESTINAL STEM CELL DEVELOPMENT DURING POSTEMBRYONIC DEVELOPMENT. As indicated above, TH action in the larval epithelium can induce some larval epithelial cells to dedifferentiate into adult progenitor cells via a cell autonomous function. Interestingly, the TR-coactivator, PRMT1, is strongly induced in these progenitor cells. More importantly, 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. Additionally, PRMT1 is also 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 evolutionally conserved role in the development of adult intestinal stem cells. EXTRACELLULAR DOMAIN OF A TH TRANSPORTER SUBUNIT CD98HC IS REQUIRED FOR EARLY MURINE DEVELOPMENT. To regulate gene transcription in the nucleus, TH is taken up from the circulating plasma by cells via active transport. A number of transporters for TH have been identified over the years. We have previously identified one of the TH direct target genes in the metamorphosing intestine as the light chain of a heterodimeric transporter, the L-type amino acid transporter-1 (LAT1) and shown 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 associates with integrin β1 through its cytoplasmic and transmembrane domains. It has been shown that the CD98hc-mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc-null mice exhibit early post-implantation lethality similar to integrin β1-null mice, supporting the importance of its interaction with integrin β1. 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 thyroid hormone 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 to make sure of the ability to carry out gene knockout in mouse, which is not possible at the moment in frogs. Thus, in collaboration with Dr. C Dengs laboratory at NIDDK, NIH, we have generated a mutant mouse line from an embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hcΔ/+). Expression of the CD98hc mutant allele results in ΔCD98hc-βgeo fusion protein where extracellular C-terminal 102 amino acids of CD98hc are replaced with βgeo. Analyses of PST080 ES cells as well as reconstituted frog oocytes demonstrated that ΔCD98hc-βgeo fusion protein preserved its ability to interact with integrin β1 although this mutant protein was hardly localized on the cell surface. These findings suggest that ΔCD98hc-βgeo protein can mediate integrin signaling but cannot support TH or amino acid transport through LATs. CD98hcΔ/+ mice were normal. Although some of the implantation sites lacked embryonic component at E9.5, all the implantation sites contained embryonic component at E7.5. Thus, CD98hcΔ/Δembryos are likely to die between E7.5 and E9.5. Considering that CD98hc complete knockout (CD98hc-/-) embryos are reported to 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 whether TH transport plays a role in intestinal stem cell development in the near future.
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