Atsuko Ishizuya-Oka

In the Xenopus laevis intestine during metamorphosis, stem cells appear and generate the adult epithelium analogous to the mammalian one. We have previously shown that connective tissue cells surrounding the epithelium are essential for the stem cell development. To clarify whether such cells correspond to mammalian Foxl1-expressing mesenchymal cells, which have recently been shown to be a critical component of intestinal stem cell niche, we here examined the expression profile of Foxl1 in the X. laevis intestine by using RT-PCR and immunohistochemistry. Foxl1 expression was transiently upregulated only in connective tissue cells during the early period of metamorphic climax and was the highest just beneath the proliferating stem/progenitor cells. In addition, electron microscopic analysis showed that these subepithelial cells are ultrastructurally identified as telocytes like the mammalian Foxl1-expressing cells. Furthermore, we experimentally showed that Foxl1 expression is indirectly upregulated by thyroid hormone (TH) through Shh signaling and that TH organ-autonomously induces the Foxl1-expressing cells concomitantly with appearance of the stem cells in the tadpole intestine in vitro. The present results suggest that intestinal niche cells expressing Foxl1 are evolutionally conserved among terrestrial vertebrates and can be induced by TH/Shh signaling during amphibian metamorphosis for stem cell development.

During amphibian intestinal remodeling, thyroid hormone (TH) induces adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian one. We have previously shown that hyaluronan (HA) is newly synthesized and plays an essential role in the development of the stem cells via its major receptor CD44 in the Xenopus laevis intestine. We here focused on HA synthase (HAS) and examined how the expression of HAS family genes is regulated during natural and TH-induced metamorphosis. Our quantitative RT-PCR analysis indicated that the mRNA expression of HAS2 and HAS3, but not that of HAS1 and HAS-rs, a unique Xenopus HAS-related sequence, is upregulated concomitantly with the development of adult epithelial primordia consisting of the stem/progenitor cells during the metamorphic climax. In addition, our in situ hybridization analysis indicated that the HAS3 mRNA is specifically expressed in the adult epithelial primordia, whereas HAS2 mRNA is expressed in both the adult epithelial primordia and nearby connective tissue cells during this period. Furthermore, by treating X. laevis tadpoles with 4-methylumbelliferone, a HA synthesis inhibitor, we have experimentally shown that inhibition of HA synthesis leads to suppression of TH-upregulated expression of leucine-rich repeat-containing G protein-coupled 5 (LGR5), an intestinal stem cell marker, CD44, HAS2, HAS3, and gelatinase A in vivo. These findings suggest that HA newly synthesized by HAS2 and/or HAS3 is required for intestinal stem cell development through a positive feedback loop and is involved in the formation of the stem cell niche during metamorphosis.

During Xenopus metamorphosis, most tadpole organs remodel from the larval to adult form to prepare for adaptation to terrestrial life. Organ culture serves as an important tool for studying larval-to-adult organ remodeling independent of the effects of other parts of the body. Here, I introduce a protocol for organ culture in vitro using the Xenopus laevis tadpole intestine before metamorphic climax. During culture in the absence of exogenous 3,3′,5-triiodo-l-thyronine (T3), the most potent natural thyroid hormone, the intestine remains in its larval state without any metamorphic changes. In contrast, when T3 is added to the culture medium, the larval epithelium undergoes apoptosis, whereas adult stem cells appear, actively proliferate, and finally generate the differentiated adult epithelium within a week. At the same time, the surrounding nonepithelial tissues also develop. Thus, this culture model is useful for clarifying the control mechanisms of apoptosis in larval tissues, formation of adult stem cells, and cell proliferation and differentiation of adult tissues, all of which occur in harmony during natural metamorphosis. Moreover, a procedure for tissue recombination combined with organ culture provides a platform for investigating complex tissue interactions during organ remodeling. Such tissue recombination experiments will help to reveal the important role of nonepithelial tissues in larval epithelial apoptosis and/or adult stem cell development in the X. laevis intestine.

In the amphibian intestine during metamorphosis, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into stem cells, which generate the adult epithelium analogous to the mammalian intestinal epithelium. We have previously shown that the canonical Wnt signaling pathway is involved in adult epithelial development in the Xenopus laevis intestine. To understand the function of this pathway more precisely, we here focused on CD44, a major Wnt target, which has been identified as a TH response gene in the X. laevis intestine. Our in situ hybridization analysis indicated that CD44 mRNA is detectable in adult epithelial primordia consisting of the adult stem/progenitor cells and is strongly expressed in the connective tissue (CT) cells surrounding them. Interestingly, when the expression of CD44 mRNA is the highest, hyaluronan (HA), a principle ligand of CD44, is newly synthesized and becomes most abundantly distributed in the CT just beneath the adult epithelial primordia that are actively proliferating. Thereafter, as the adult primordia differentiate into the simple columnar epithelium, the expression of CD44 mRNA is gradually downregulated. More importantly, using organ cultures of the X. laevis tadpole intestine in the presence of TH, we have experimentally shown that inhibition of HA synthesis by 4-methylumbelliferone suppresses development of not only the CT but also the epithelial stem cells, resulting in failure to generate the AE. Our findings strongly suggest that TH-upregulated HA/CD44 signaling plays an essential role in formation of the intestinal stem cell niche during vertebrate postembryonic development.

During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including β-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear β-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling.

Intestinal remodeling during amphibian metamorphosis has long been studied as a model for the formation of the adult organs in vertebrates, especially the formation of adult organ-specific stem cells. Like all other processes during metamorphosis, this process is controlled by thyroid hormone (T3), which affects cell fate and behavior through transcriptional regulation of target genes by binding to T3 receptors (TRs). Earlier studies have shown that Sonic hedgehog (Shh) is induced by T3 in the developing adult stem cells and that the Shh receptor and other downstream components are present in the connective tissue and at lower levels in the muscles at the climax of intestinal remodeling. However, no in vivo studies have carried out to investigate whether Shh produced in the adult cells can regulate the connective tissue to promote intestinal maturation. We have addressed this issue by treating tadpoles with Shh inhibitor cyclopamine. We showed that cyclopamine but not the structurally related chemical tomatidine inhibited the expression of Shh response genes BMP4, Snai2, and Twist1. More importantly, we showed that cyclopamine reduced the cell proliferation of both the developing adult stem cells as well as cells in the other intestinal tissues at the climax of metamorphosis, leading to delayed/incomplete remodeling of the intestine at the end of metamorphosis. We further revealed that both Snai2 and Twist1 were strongly upregulated during metamorphosis in the intestine and their expression was restricted to the connective tissue. Our results suggest that Shh indeed signals the connective tissue whereby it can increase adult stem cell proliferation and promote formation of the adult intestine.

Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis. Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation. Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.