Amphibian organ remodeling during metamorphosis: Insight into thyroid hormone-induced apoptosis

Authors


Author to whom all correspondence should be addressed.
Email: a-oka@nms.ac.jp

Abstract

During amphibian metamorphosis, the animal body dramatically remodels to adapt from the aquatic to the terrestrial life. Cell death of larval organs/tissues occurs massively in balance with proliferation of adult organs/tissues, to ensure survival of the individuals. Thus, amphibian metamorphosis provides a unique and valuable opportunity to study regulatory mechanisms of cell death. The advantage of this animal model is the absolute dependence of amphibian metamorphosis on thyroid hormone (TH). Since the 1990s, a number of TH response genes have been identified in several organs of Xenopus laevis tadpoles such as the tail and the intestine by subtractive hybridization and more recently by cDNA microarrays. Their expression and functional analyses, which are still ongoing, have shed light on molecular mechanisms of TH-induced cell death during amphibian metamorphosis. In this review, I survey the recent progress of research in this field, focusing on the X. laevis intestine where apoptotic process is well characterized at the cellular level and can be easily manipulated in vitro. A growing body of evidence indicates that apoptosis during the intestinal remodeling occurs not only via a cell-autonomous pathway but also via cell–cell and/or cell–extracellular matrix (ECM) interactions. Especially, stromelysin-3, a matrix metalloproteinase, has been shown to alter cell–ECM interactions by cleaving a laminin receptor and induce apoptosis in the larval intestinal epithelium. Here, I emphasize the importance of TH-induced multiple apoptotic pathways for massive and well-organized apoptosis in the amphibian organs and discuss their conservation in the mammalian organs.

Introduction

The amphibian organs extensively change from larval to adult form during a short period of metamorphosis. Rapid degeneration of larva-specific tissues/organs occupying a large portion of tadpoles has attracted biological researchers as the subject of cell death for a long time. At the cellular level, Kerr et al. (1972, 1974) first identified cell death in the tail epidermis and muscles during amphibian metamorphosis as “apoptosis” by electron microscopy (EM). Thereafter, by using EM and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods for the detection of DNA fragmentation (Gavrieli et al. 1992), unequivocal apoptosis has also been observed not only in larva-specific organs but also in larval-to-adult remodeling organs including the brain (Decker 1976; Wahnschaffe et al. 1987) and the intestine (Ishizuya-Oka & Shimozawa 1992; Ishizuya-Oka et al. 2010). Apoptosis is now widely accepted as the major cell death during amphibian metamorphosis, although it still remains possible that lysosome-mediated autophagy may be partly involved in the degeneration (Levine & Klionsky 2004; Lockshin & Zakeri 2004). Characteristically, the amphibian apoptosis occurs massively and in a spatiotemporally well-regulated manner to survive after metamorphosis. Especially, in the remodeling organ, apoptosis of larval tissues/cells proceeds in balance with development of adult ones. Thus, it serves as a unique model to study ingenious mechanisms by which apoptosis progresses adequately in the whole organ.

Amphibian metamorphosis is generally known to be trigged by a single hormone, thyroid hormone (TH) (Dodd & Dodd 1976; Kikuyama et al. 1993; Shi 1999). This implies that, in this animal, clarification of the expression cascade of TH response genes is an effective approach to investigate regulatory mechanisms of apoptosis at the molecular level. So far, a number of TH response genes have been identified in several organs such as the tail, the limb, and the intestine of the African clawed frog, Xenopus laevis (Buckbinger & Brown 1992; Shi & Brown 1993; Wang & Brown 1993; Denver et al. 1997; Das et al. 2006; Buchholz et al. 2007; Suzuki et al. 2009). Among them, the intestine is a typical remodeling organ, where apoptosis has been well characterized at the cellular level and has been shown to be experimentally induced by TH in vitro (Ishizuya-Oka & Shimozawa 1992). In this organ, functional analyses of TH response genes involved in apoptosis are currently underway both in vivo and in vitro and increasingly accumulate evidence that TH induces apoptosis via multiple pathways including cell–cell or cell–extracellular matrix (ECM) interactions. Here, I will give an overview of the study of apoptosis in X. laevis intestine and then discuss how far the apoptotic pathways in this model are conserved among other amphibian and mammalian organs.

Morphological aspects of apoptosis during intestinal remodeling

The herbivorous tadpole intestine during prometamorphic larval period is long and consists of a single layer of primary (larval) epithelium, the immature connective tissue, and thin layers of muscles (Figs 1, 2A). During metamorphosis, in the adaptation to the carnivorous life, the larval epithelium is replaced by the secondary (adult) one with the rapid shortening of the tadpole intestine (Hourdry & Dauca 1977; McAvoy & Dixon 1977). The adult epithelium after metamorphosis acquires a cell-renewal system along the trough-crest axis of intestinal folds (McAvoy & Dixon 1977; Shi & Ishizuya-Oka 1996), analogous to the mammalian crypt-villus axis (Cheng & Bjerknes 1985; Madara & Trier 1994).

Figure 1.

 Larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis. The long tadpole intestine has a single fold and consists of the larval epithelium (LE), the immature connective tissue (CT), and muscles. During metamorphic climax, when plasma thyroid hormone (TH) levels are high, the tadpole intestine shortens and is remodeled into a more complex frog one. Larval epithelial cells rapidly undergo apoptosis labeled by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (arrows; lower panel), whereas progenitor cells of the adult epithelium (AE) appear as islets stained strongly red with methyl green-pyronin Y (MG-PY) (arrowheads; upper panel). They proliferate and completely replace larval ones by the end of metamorphosis (stage 66). The adult epithelium acquires a cell-renewal system along the axis of intestinal folds (IF), where apoptosis is localized in their tips. Bars, 20 μm.

Figure 2.

 Apoptosis of the larval epithelium (LE) and its correlation with the basal lamina modification during natural metamorphosis (A–G) and in experimental conditions (H–L). (A, B) Throughout pre- and prometamorphosis, the larval epithelium remains simple columnar, and the basal lamina (bl) is thin and continuous. (C–E) At the onset of metamorphic climax (stages 60/61), apoptotic bodies (ab), which contain nuclear fragments characterized by condensed chromatin close to the nuclear membrane, suddenly increase in number in the larval epithelium but not in the adult one (AE). The basal lamina entirely becomes thickened and then amorphous. Through the modified basal lamina, macrophages (Mφ) migrate from the connective tissue (CT) into the larval epithelium. (F, G) Thereafter, the macrophages become larger by engulfing the apoptotic bodies and are finally extruded into the lumen (lu). (H, I) In the intact intestine cultured in vitro, TH induces apoptosis of the larval epithelium and the basal lamina modification. (J) TH also induces apoptosis in the larval epithelium cultured alone in vitro. (K, L) In the transgenic (Tg) intestine overexpressing ST3 in the absence of TH, some epithelial cells undergo apoptosis, and the basal lamina becomes amorphous or absent. Bars, 1 μm.

We previously identified the cell death of the larval epithelium as apoptosis (Ishizuya-Oka & Ueda 1996), which are ultrastructurally characterized by the condensation of chromatin close to the nuclear membrane and subsequent fragmentation of nuclei and cytoplasm into apoptotic bodies (Kerr et al. 1974; Wyllie et al. 1980). Apoptotic cells suddenly increase in number around stage 60 (the onset of metamorphic climax; Nieuwkoop & Faber 1967) (Figs 1, 2C), concomitantly with the appearance of a small number of adult progenitor cells, which are conventionally stained strongly red with pyronin-Y and express markers for mammalian intestinal stem cells such as Musashi-1 (Ishizuya-Oka et al. 2003; Ishizuya-Oka & Shi 2007). Interestingly, when the apoptotic larval cells coexist with the adult cells, macrophages rapidly increase in number only in the larval epithelium undergoing apoptosis. These intraepithelial macrophages become enlarged by engulfing the apoptotic bodies derived from larval epithelial cells and are finally extruded into the intestinal lumen (Fig. 2F,G; Ishizuya-Oka & Shimozawa 1992). In this way, the larval epithelial cells are rapidly removed from the intestine during stages 60–62, whereas the adult progenitor cells actively proliferate during this period. Then, the adult cells completely replace the larval ones at stage 63 and differentiate into the adult absorptive epithelium as the progress of formation of intestinal folds.

Although it remains unknown how apoptosis of the larval epithelium proceeds concomitantly with adult epithelial development, findings of previous EM studies provide an important clue to its mechanisms. The amphibian intestinal epithelium is separated from the connective tissue by a basal lamina, which comprises ECM components such as laminin, type IV collagens, and various kinds of proteoglycans (Hay 1991), and is usually thin and continuous before and after metamorphosis (Fig. 2B; Ishizuya-Oka & Shimozawa 1987). When the larval epithelium begins to undergo apoptosis around stage 60, the basal lamina beneath the epithelium becomes suddenly thickened by vigorous folding and then amorphous (Fig. 2D). Through such modified basal lamina, the macrophages often migrate from the connective tissue into the degenerating larval epithelium (Fig. 2E) but not into the adult one, which often makes contacts with sub-epithelial fibroblasts through the modified basal lamina (Ishizuya-Oka & Shimozawa 1987). Thereafter, the basal lamina beneath the larval epithelial cells remains amorphous until they finally disappear through apoptosis, whereas the basal lamina beneath the adult epithelial cells becomes thin again as they differentiate into the absorptive epithelium. This close correlation between the larval epithelial apoptosis and the basal lamina modification strongly suggests the involvement of the basal lamina ECM in the apoptosis.

TH-induced apoptosis in the intestinal epithelium in vitro

To investigate experimentally the regulatory mechanisms of apoptosis, we first established an organ culture system, where the apoptotic processes mentioned above can be reproduced by the inductive action of TH (Ishizuya-Oka & Shimozawa 1992). In the intestine isolated from the X. laevis tadpole at stage 57 (prometamorphosis 4–5 days before metamorphic climax) and cultured in vitro, apoptotic cells of the larval epithelium become detectable after 2 days of TH-treatment and are most numerous after 3–5 days (Fig. 2H), concomitantly with active proliferation of adult progenitor cells. Then, after 7 days, the adult epithelial cells completely replace the larval ones and differentiate into the absorptive epithelium. Again, as during natural metamorphosis, apoptosis of the larval epithelium is followed by the basal lamina modification (Fig. 2I). In contrast, in the absence of TH, the epithelium of the tadpole intestine is well maintained as larval-type throughout 7 days of cultivation. These results support the idea that molecular mechanisms of the larval apoptosis can be clarified by analyzing TH response genes endogenously expressed in the X. laevis intestine.

Using this culture system, the direct action of TH on the epithelium was also examined. When the epithelium is isolated from the stage 57-tadpole intestine and cultured alone on a matrigel, a genuine reconstituted basal lamina, the epithelium is well maintained as larval-type in the absence of TH over 10 days. In contrast, in the presence of TH, apoptotic cells become detectable after 3 days (Fig. 2J), although their number is smaller than that in organ cultures of the intact intestine (Ishizuya-Oka, unpubl. data, 1992). In addition, by using primary cell cultures, it has been shown that TH induces apoptosis of some epithelial cells isolated from the tadpole intestine and cultured separately on plastic dishes. However, various components of ECM coated on the dishes make an inhibitory effect on the epithelial apoptosis (Su et al. 1997b). Notably, although all of the epithelial cells finally degenerate after 4 days on the plastic dishes, TH causes proliferation of other cells during the early period of cultivation (Su et al. 1997b). The conclusions drawn from these culture studies are that (i) TH induces apoptosis of the epithelium cell-autonomously in a cell type-dependent manner, and that (ii) TH-induced apoptosis of epithelial cells can be inhibited by components of the basal lamina ECM, which may function as a spatiotemporal regulator of the epithelial apoptosis. To understand their molecular bases, I will next survey TH response genes related to apoptosis in the X. laevis intestine.

Molecular aspects of apoptosis during intestinal remodeling

Thyroid hormone receptors and a cell-autonomous pathway

In the first step of TH action, TH binds to the TH receptor (TR), which is one of the nuclear hormone receptors. By using transgenic (Tg) X. laevis tadpoles expressing dominant-positive (Buchholz et al. 2004) and dominant-negative mutants of TR (Schreiber et al. 2001; Buchholz et al. 2003; Schreiber & Brown 2003), previous studies have shown that TR is necessary and sufficient to mediate TH-inducible metamorphic events, including apoptosis of the larval intestinal epithelium.

There are two subtypes of TR; TRα and TRβ, and only TRβ has been identified as the TH response gene in amphibian organs. The expression of TRβ mRNA is upregulated during X. laevis metamorphosis, simultaneously with the increase of the plasma TH level, while the expression of TRα mRNA is upregulated during premetamorphosis when the TH level is still low (Yaoita & Brown 1990; Yaoita et al. 1990). Thus, apoptosis during amphibian metamorphosis is considered to be mediated predominantly by TRβ. In the X. laevis intestine, previous in situ hybridization (ISH) analysis showed that the expression of TRβ mRNA is highly upregulated in the larval epithelium itself just before its apoptosis (Shi & Ishizuya-Oka 1997), providing circumstantial evidence for the existence of a cell-autonomous apoptotic pathway. Since TRβ is a transcription factor, TRβ binds to TH response elements (TRE) located in the direct TH response genes and regulates their expression by interacting with coactivators or corepressors in a TH-dependent manner. Some of their encoded proteins further regulate the expression of other genes, leading to the cell-autonomous progress of apoptosis. Although only fragmentary data are available about apoptosis-related genes in this pathway, the expression of caspases including caspase-3 and -9 is known to be upregulated in the X. laevis intestine during metamorphic climax (Nakajima et al. 2000). In addition, a previous culture study showed that TH-induced apoptosis of intestinal epithelial cells is inhibited by Cyclosporine A, which blocks the mitochondrial permeability transition pore (MPTP) opening (Su et al. 1997a), suggesting the involvement of mitochondrial apoptotic machinery as known in the mammalian apoptosis.

Cell-ECM interactions mediated by MMPs

Among TH response genes isolated from the X. laevis intestine (Shi & Brown 1993; Amano & Yoshizato 1998; Shimizu et al. 2002; Buchholz et al. 2007; Heimeier et al. 2010), there are a number of genes encoding matrix metalloproteinases (MMPs) including stromelysins, collagenases, and gelatinases, which belong to a superfamily of Zn-dependent proteases capable of degrading various components of ECM (Shi & Ishizuya-Oka 2001). They are noteworthy in correlation with modification of the basal lamina described above. Previous ISH analyses showed different expression profiles of different MMPs during the X. laevis intestinal remodeling, suggesting their distinct roles (Shi et al. 2007). Of particular interest is stromelysin-3 (ST3; MMP-11), since the expression profile of ST3 mRNA is most closely related to the epithelial apoptosis and the basal lamina modification (Patterton et al. 1995). The expression of ST3 is directly upregulated by TH in fibroblasts through the TR-TREs binding (Fu et al. 2006), and ST3 protein encoded is secreted into the ECM as an active protease (Pei & Weiss 1995). To investigate roles of ST3 in apoptosis, an antibody against the catalytic domain of ST3 was used to block ST3 function in vitro. In organ cultures of the X. laevis intestine, we have shown that the addition of the anti-ST3 antibody to the culture medium inhibits both TH-induced apoptosis of the larval epithelium and the basal lamina modification in a dose-dependent manner (Ishizuya-Oka et al. 2000). In addition, the function of ST3 was investigated in vivo by using Tg tadpoles expressing ST3 under the control of a heat shock-inducible promoter. Overexpression of ST3 in the premetamorphic tadpole has been shown to cause precocious apoptosis of the intestinal epithelium followed by modification of the basal lamina in the absence of exogenous TH (Fu et al. 2005) (Fig. 2K,L). This result indicates that ST3 is sufficient to induce some cells to undergo apoptosis, possibly through altering interactions between the epithelial cells and the basal lamina ECM. However, the number of apoptotic cells in the Tg intestine is smaller than that in the wild-type intestine during natural and TH-induced metamorphosis, possibly because of the lack of cell-autonomous apoptotic pathway. On the other hand, in TH-treated Tg tadpoles expressing ST3-driven dominant negative thyroid hormone receptor, the intestinal epithelium appeared fragmented and pycnotic in histological sections (Schreiber et al. 2009). This suggests that TH causes cell death irrespective of ST3 expression. Taken together, it appears likely that either one of the two pathways, the cell-autonomous or the ST3-mediated ones, is sufficient to induce apoptosis in at least some of the epithelial cells.

As one of the physiological substrates of ST3, a laminin (LR) receptor has been identified in the X. laevis intestine. ST3 cleaves LR, which is localized to the basal surface of epithelial cells, at two distinct sites between the transmembrane domain and the laminin binding sequence (Amano et al. 2005; Fiorentino et al. 2009). Thus, one possible pathway could be that TH upregulates the fibroblast-specific expression of ST3, which is secreted into the basal lamina and alters interactions between the epithelial cells and laminin, a major component of the basal lamina ECM, resulting in signaling to cause apoptosis (Shi et al. 2007) (Fig. 3).

Figure 3.

 Model of epithelial apoptotic pathways during Xenopus laevis intestinal remodeling. T3, whose activity is regulated by deiodinases (D1–D3), initiates multiple apoptotic pathways. In a cell-autonomous pathway (1; red), T3 directly affects larval epithelial cells via thyroid hormone receptor (TR) and upregulate the expression of genes involved in apoptosis. In an extracellular pathway (2; blue), T3 first induces fibroblasts to express matrix metalloproteinases (MMPs) such as ST3, which is secreted into the basal lamina (bl) and alters cell-extracellular matrix (ECM) interactions by cleaving laminin receptors (LR), resulting in apoptosis of larva-proper epithelial cells (LE). In addition, T3 upregulates the expression of MMP-9 in macrophages (Mφ), which migrate from the connective tissue (CT) into the degenerating larval epithelium and engulf epithelial apoptotic bodies (3; purple). If the larva-proper cells survive, adult lymphocytes may immunologically remove them (4; green). These multiple pathways act in concert to enable massive apoptosis in the appropriate place at the appropriate time. On the other hand, the adult progenitor cells (AE), which are possibly predetermined before metamorphic climax (pre-AE), do not undergo apoptosis but proliferate via T3-induced pathways (grey).

As for the other MMPs identified as TH response genes, there are the duplicated MMP-9 genes, MMP-9 and MMP-9TH (Fujimoto et al. 2006). Their ISH analyses showed that MMP9-TH mRNA is highly expressed in fibroblasts just beneath the epithelium undergoing apoptosis, similar to ST3 mRNA (Hasebe et al. 2007). Considering that mammalian MMP-9 is known to degrade type IV collagen, another major component of the basal lamina ECM, amphibian MMP-9TH may also promote the epithelial apoptosis by altering cell-ECM interactions. On the other hand, cells expressing MMP-9 mRNA spatiotemporally coincide with the macrophages that migrate into the larval epithelium and then engulf the apoptotic bodies (Hasebe et al. 2007), suggesting its involvement in functions of the macrophages. It is worth analyzing how ST3 cooperate with these MMPs to enable massive and well-organized apoptosis during natural metamorphosis.

Immunological pathway

It is well recognized that the immune system also undergoes the larval-to-adult remodeling with a transition of major histocompatibility complex (MHC) antigens during X. laevis metamorphosis (Du Pasquier & Flajnik 1990; Rollins-Smith 1998). Apoptosis of larval lymphocytes occurs in two major immunological organs, the spleen and the thymus during metamorphosis (Grant et al. 1998). Although it still remains unknown whether the larval lymphocytes are replaced by adult ones in the peripheral organs such as the intestine, previous studies reported that lymphocytes are frequently located in the intestinal epithelium both before and after metamorphosis (Marshall & Dixon 1978; McAvoy & Dixon 1978) and during metamorphosis (Ishizuya-Oka, unpubl. data, 1996). Then, whenever adult lymphocytes coexist with larval epithelial cells, they must recognize the epithelial cells as nonself. In fact, in the X. laevis tail, another peripheral organ, adult T lymphocytes have been shown to recognize the larval epidermal cells in vitro (Izutsu et al. 2000). Considered together, the immunological pathway may be biologically significant as an assurance program in the intestine to remove the larval cells, if other apoptotic pathways are blocked.

Lessons from apoptosis in other amphibian organs

Apoptosis occurs in many remodeling and larva-specific organs other than the intestine during metamorphosis. Its comparisons among different organs are useful for gaining better understanding of apoptosis during amphibian metamorphosis. In the X. laevis tail, a typical larva-specific organ extensively studied (Yoshizato 1989, 2007), the number of apoptotic cells peaks at stages 63/64 in muscles (Nishikawa & Hayashi 1995; Nakajima & Yaoita 2003) and at stage 58 in the caudal spinal cord (Estabel et al. 2003), whereas it peaks at stages 60/61 in the intestinal epithelium as mentioned above. This raises a question why apoptosis in different organs/tissues occurs at different stages, although all of them are exposed to the same plasma TH level. One proposed explanation for this is that deiodinases regulate TH activity locally in the peripheral tissues/organs (Becker et al. 1997; Cai & Brown 2004). The amphibian thyroid gland secrets two TH hormones, thyroxine (T4) and 3,5,3′-triiodothyronines (T3), a biologically more active hormone. Type I and II deiodinase (D1, D2) are known to convert T4 to T3, while type III deiodinase (D3) inactivates T3 (Huang et al. 1999; Gereben et al. 2008). It is possible that differences in activity of these enzymes among different organs/tissues may spatiotemporally regulate apoptosis by altering the activity of TH.

Caspases and regulatory mitochondrial factors such as BID, Bax, and xR11, a homologue of mammalian Bcl-XL, have been shown to be involved in TH-induced apoptosis both in the tail (Nakajima et al. 2000; Sachs et al. 2004; Rowe et al. 2005; Du Pasquier et al. 2006) and the brain (Coen et al. 2001, 2007). In addition, in the X. laevis tail muscles, similar to the intestinal epithelium, it has been demonstrated that TH can induce apoptosis cell-autonomously both in vitro (Yaoita & Nakajima 1997) and in vivo (Das et al. 2002). On the other hand, previous culture studies indicated that the epidermal-dermal interactions are necessary for the tail regression of Rana tadpoles (Niki et al. 1982; Kinoshita et al. 1989; Yoshizato 2007), suggesting the involvement of cell–cell or cell–ECM interactions in the tail apoptosis. Although its molecular bases remain mostly unknown, the involvement of ST3 in the tail apoptosis has been recently shown by using Tg X. laevis tadpoles. Noteworthy, in the tail muscles, overexpression of ST3 causes apoptosis without LR cleavage (Mathew et al. 2009). This implies that ST3 induces apoptosis in the muscles via a LR-independent pathway. In contrast, in the tail epidermis expressing LR, ST3 has been proposed to induce apoptosis via a LR-dependent pathway as proposed in the intestinal epithelium (Mathew et al. 2010).

Furthermore, a recent study using Tg tadpoles indicated that Ouro proteins, which encode keratin-related proteins, function as larva-specific immune antigens (Mukaigasa et al. 2009), suggesting a key role of the immune system in the tail regression. Thus, also in the tail, the multiple apoptotic pathways seem to work together to ensure massive apoptosis. Although each pathway has been only fragmentarily elucidated at the molecular level so far, comparison of genome-wide microarray data across the intestine and the tail (Das et al. 2006; Buchholz et al. 2007; Heimeier et al. 2010) and functional analyses of common apoptosis-related genes should pave a way to clarify the similarity and diversity of apoptotic pathways among amphibian organs.

Evolutionary conservation of apoptosis in mammalian organs

Amphibian metamorphosis is considered to mimic the evolutionary process from the aquatic fish to terrestrial tetrapods in the Devonian period of the Paleozoic era and is often compared with mammalian postnatal development when the endogenous TH level transiently increases (Tata 1993; Hodin et al. 1994; Shi 1999). In the mammalian intestine, high levels of TH during this period are required for maturation of the intestine. Thereafter, although the TH level becomes lower, TH continues to function for maintaining a cell-renewal system of the intestinal epithelium throughout the mammalian adulthood. Interestingly, in the mouse adult intestine, TH has been shown to promote not only apoptosis of damaged epithelial cells but also proliferation of surviving epithelial progenitor cells during intestinal regeneration after irradiation-caused DNA damage (Kress et al. 2008). These similarities between amphibian and mammalian intestines predict that functions of TH during the amphibian intestinal remodeling are at least partly conserved in the mammalian postnatal and adult intestines. Currently, a growing number of TH response genes have been identified by genome-wide microarrays in the mouse intestine, and they include genes common to both the mouse and Xenopus intestines (Kress et al. 2009). Although their functions in apoptosis remain unknown, precise comparison of more numerous TH response genes between the two species in future will be interesting from the viewpoint of evolutionary biology.

At the other extreme, it should be pointed out that apoptosis-related genes identified as amphibian TH response genes may have a conserved function in mammalian apoptosis, even if they are not identified as TH response genes in mammals. For example, ST3 was initially cloned as a MMP associated with human breast cancer (Basset et al. 1990), and its dependence on TH remains unproven in the mammalian organs. Although ST3 functions have not yet been demonstrated in the mammalian model, possibly due to redundancy in MMPs activity, their expression is strictly localized in fibroblasts surrounding (i) the epithelium that undergoes apoptosis during development (Lefebvre et al. 1992) and (ii) various growing cancers including the colorectal cancer (Basset et al. 1997; Egeblad & Werb 2002), which highly express LR (Sanjuan et al. 1996; Menard et al. 1997). These expression profiles of ST3 and LR strongly suggest conserved functions of ST3 from frog to human (Fiorentino et al. 2009).

Conclusions and future perspectives

During amphibian metamorphosis, most organs undergo apoptosis in a spatiotemporally-regulated manner to survive as an individual. Because of its absolute dependence of TH, amphibian metamorphosis provides a unique opportunity to study the entire mechanisms underlying apoptosis at the molecular level (Tata 1994). In the X. laevis intestine, functional analyses of TH response genes both in vivo and in vitro (Fu et al. 2002; Buchholz et al. 2004; Ikuzawa et al. 2006) provide an increasing body of evidence for the existence of multiple apoptotic pathways (Fig. 3).

One notion obtained from this model is that TH simultaneously initiates not only a cell-autonomous pathway but also extracellular pathways leading to apoptosis. That is, TH directly affects larval epithelial cells and cell-autonomously causes their apoptosis and, at the same time, induces fibroblasts to express ST3, which in turn cleavages LR, resulting in apoptosis of the epithelial cells. Previous experimental studies have shown that either one of the pathways is sufficient to induce apoptosis by itself, but acts synergistically with each other to cause massive apoptosis. In addition, if the larval cells survive by a blockage of these pathways, the immunological pathway may be available for their removal. Although we have not yet obtained the entire picture of the apoptotic pathways, the progress of functional analysis of TH response genes by using modern genetic methodologies (Rankin et al. 2009) may shed light on the molecular understanding of complicated cell–cell and cell–ECM interactions.

Another notion is that cellular responses to TH are diverse and depend on the cell-types. The intestinal epithelium of the X. laevis tadpole is divided into two types of cells, i.e., larva-proper cells and adult progenitor/stem cells, by the onset of metamorphic climax at the latest (Ishizuya-Oka & Shi 2007). TH induces the former cells to undergo apoptosis and the latter cells to proliferate. This raises a question why the adult progenitor cells escape from the apoptotic pathways and proliferate. One possible explanation is that some anti-apoptotic signals protect the adult cells, as proposed in human colorectal tumor cells, where hedgehog signals are active and may have a protective effect against apoptosis (Qualtrough et al. 2004). To identify such anti-apoptotic signals, it is worth studying functions of TH response genes whose expression is specifically localized in the adult progenitor cells. Furthermore, by using Tg tadpoles expressing green fluorescent protein (GFP), we have recently shown that the adult progenitor cells originate from some differentiated epithelial cells during the larval period (Ishizuya-Oka et al. 2009), when precursor cells of the adult progenitor cells are not morphologically distinguished from the other epithelial cells destined to undergo apoptosis. This raises the fundamental question of when and how the precursor cells have different fates and are determined to become the adult progenitor cells. One possible mechanism is that molecular cues from the neighboring tissues/cells may be necessary for the epithelial cells to dedifferentiate into the adult progenitor cells, as proposed during organogenesis of the embryonic gut, where mesodermal/mesenchymal cues exert various effects on differentiation of the endoderm (Yasugi & Mizuno 2008). Considering that the postembryonic organs consist of a greater diversity of cells than that of embryonic ones, to address this question, future studies should be directed towards more precise identification of each cell-type at the genetic level and their cell lineage tracing until the onset of metamorphic climax.

Acknowledgments

I would like to thank Dr Y.-B. Shi and Dr T. Hasebe for collaborations. I am also grateful to Professor T. Mizuno for his continuous encouragement. This research was supported in part by JSPS Grants-in-Aid for Scientific Research (C).

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