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Dual functions of thyroid hormone receptors during Xenopus development
Comparative Biochemistry and Physiology Part B 126 (2000) 199 – 211 www.elsevier.com/locate/cbpb
Metamorphosis
Dual functions of thyroid hormone receptors during Xenopus development Laurent M. Sachs a, Sashko Damjanovski a, Peter L. Jones a, Qing Li a, Tosikazu Amano a, Shuichi Ueda b, Yun-Bo Shi a,*, Atsuko Ishizuya-Oka b a
Laboratory of Molecular Embryology, National Institute of Child Health and Human De6elopment, National Institutes of Health, Bethesda, MD 20892 -5431, USA b Department of Histology and Neurobiology, Dokkyo Uni6ersity School of Medicine, Mibu, Tochigi 321 -02, Japan Received 18 August 1999; received in revised form 16 November 1999; accepted 18 November 1999
Abstract Thyroid hormone (TH) plays a causative role in anuran metamorphosis. This effect is presumed to be manifested through the regulation of gene expression by TH receptors (TRs). TRs can act as both activators and repressors of a TH-inducible gene depending upon the presence and absence of TH, respectively. We have been investigating the roles of TRs during Xenopus lae6is development, including premetamorphic and metamorphosing stages. In this review, we summarize some of the studies on the TRs by others and us. These studies reveal that TRs have dual functions in frog development as reflected in the following two aspects. First, TRs function initially as repressors of TH-inducible genes in premetamorphic tadpoles to prevent precocious metamorphosis, thus ensuring a proper period of tadpole growth, and later as activators of these genes to activate the metamorphic process. Second, TRs can promote both cell proliferation and apoptosis during metamorphosis, depending upon the cell type in which they are expressed. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Apoptosis; Cell death; Cell proliferation; Metamorphosis; Transcriptional regulation; Xenopus lae6is; Amphibian; Thyroid hormone receptor
1. Introduction Thyroid hormone (TH) influences diverse biological processes (Hetzel, 1989; Mandel et al., 1993). In adult mammals, TH regulates the physiological function of many organs (Guernsey and Edelman, 1983; Freake and Oppenheimer, 1995; Silva, 1995). In humans, inappropriate levels of Presented at the International Symposium ‘‘The Frontiers of the Biology of Amphibia’’, Hiroshima, Japan, March 22– 24, 1999. * Corresponding author. Tel.: + 1-301-4021004; fax: + 1301-4021323. E-mail address: [email protected] (Y.-B. Shi)
TH lead to various diseases. In addition, TH also plays important roles during embryogenesis and organogenesis. Since early part of this century, hypothyroidism has been known to cause severe developmental defects in humans, most notably human cretinism, a form of defect characterized by mental retardation and short stature due to deficiencies in brain and skeletal development (Dussault and Ruel, 1987; Hetzel, 1989; Mandel et al., 1993; Porterfield and Hendrich, 1993). Compared to other vertebrates, anuran development is perhaps the most dependent on TH. Anuran development takes place in two phases. Its embryogenesis occurs prior to the formation of
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a functional thyroid gland to form a free-living tadpole. However, metamorphosis, i.e. the transformation of the tadpole to a frog, is absolutely dependent upon the presence of sufficient levels of TH in the plasma (Dodd and Dodd, 1976; Gilbert and Frieden, 1981). Blocking the synthesis of endogenous TH inhibits metamorphosis, while simply adding physiological concentrations of TH to rearing water of premetamorphic tadpoles induces precocious transformations. Furthermore, TH can induce metamorphosis in organ cultures, suggesting that it act directly within target tissues/ organs (Ishizuya-Oka and Shimozawa, 1991; Oofusa and Yoshizato, 1991; Tata et al., 1991). TH functions by regulating gene transcription through its nuclear receptor or thyroid hormone receptors (TRs) (Sap et al., 1986; Weinberger et al., 1986). TRs belong to the superfamily of nuclear receptors that also include steroid receptors, retinoic acid receptors, and 9-cis retinoic acid receptors (RXRs) (Mangelsdorf et al., 1995). Like most receptors of this family, TR has a DNA binding domain in the N-terminal half (Lazar, 1993; Tsai and O’Malley, 1994; Mangelsdorf et al., 1995) that recognizes thyroid hormone response elements (TREs), and a TH-binding domain in the C-terminal half. Although TRs can bind to TREs as monomers or homodimers, in vivo they most likely function as heterodimers formed with RXRs. Upon binding to TREs, TR/ RXR heterodimers can regulate the transcription of the target genes in a TH-dependent manner, thus mediating the biological effects of TH. The consequences of this regulation depend upon target cell/tissue types. In this paper, we discuss some possible biological functions of TR/RXR heterodimers during frog development, focusing mostly on our own studies in X. lae6is.
2. Transcriptional activation and repression by TR Thyroid hormone can both activate and repress gene transcription through TRs (Tsai and O’Malley, 1994; Yen and Chin, 1994; Shi et al., 1996a; Wolffe et al., 1997). Only a few genes are known to be repressed by TH, and currently relatively little is known about how TH represses these genes through TRs. These genes contain negative TREs, which differ in sequence from the TREs found in TH-inducible genes. Whether and how
such differences in TRE sequences influence the positive or negative transcriptional regulation by TRs remains to be determined. The vast majority of the genes that are regulated by thyroid hormones at the transcriptional level are upregulated by TH. These genes contain one or more TREs, which often consist of a direct repeat of the consensus sequence AGGTCA separated by 4 bp (Naar et al., 1991; Umesono et al., 1991). Interestingly, these class of genes can be both activated and repressed by TR/RXR heterodimers, depending upon the presence and absence of TH, respectively (Fig. 1, Tsai and O’Malley, 1994; Shi et al., 1996a; Wolffe et al., 1997). Both the activation and repression require the presence of a TRE in the target promoter. On the other hand, distinct cofactors are recruited by TRs, depending upon the presence or absence of TH, to effect the outcome of the transcriptional regulation by TR/RXR (Fig. 1, Shi et al., 1996a; Chen and Li, 1998; Koenig, 1998). Several TR-interacting transcriptional corepressors have been identified. Among them, the beststudied are N-CoR and SMRT (Chen and Evans, 1995; Horlein et al., 1995; Chen and Li, 1998). Both of them can interact with unliganded TRs and the binding of TH to TRs inhibits this interaction. How these corepressors mediate transcriptional repression by unliganded TRs is unclear. However, both N-CoR and SMRT can interact with the corepressor Sin-3A, and thus, can be recruited into large complexes that also contain histone deacetylases such as RPD3 (Fig. 1, Heinzel et al., 1997; Nagy et al., 1997). Thus, one possible mechanism of transcriptional repression by unliganded TR/RXR is through histonedeacetylation which is turn facilitates the formation of a more repressive chromatin structure (for more details, see below). Numerous proteins have been isolated based on their interactions with TH-bound TRs (Lee et al., 1995; Shi et al., 1996a; Chen and Li, 1998). In contrast to the corepressors, these coactivators interact strongly with liganded TR but not with unliganded TR. Interestingly, many of these coactivators, such as CBP/p300, SRC-1, and pCAF, are themselves histone acetylases (Fig. 1, Ogryzko et al., 1996; Spencer et al., 1997; Blanco et al., 1998). Thus, their recruitment by TRs may bring about the opposite changes in chromatin structure compared to the recruitment of the corepressors by unliganded TRs. The resulting increase in
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Fig. 1. A proposed mechanism for transcriptional regulation by TRs. TR functions as a heterodimer with RXR. The heterodimer binds to the TRE in a target gene. In the absence of TH, the heterodimer represses gene transcription likely through the recruitment of corepressor complex containing the corepressor N-CoR (Horlein et al., 1995) or SMRT (Chen and Evans, 1995). The corepressor interacts with Sin3A, which in turn recruits a histone deacetylase such as Rpd3 to deacetylate histones (Heinzel et al., 1997; Nagy et al., 1997), thus affecting transcription. Upon binding by TH, a conformational change takes place in the heterodimer, which may be responsible for the release of the corepressor complex. Liganded TR also recruits a coactivator complex containing coactivators such as SRC-1 or CBP/p300, and P/CAF, and/or the so-called DRIP/TRAP coactivator complex (Freedman, 1999). The DRIP/TRAP complex may contact RNA polymerase directly to activate gene transcription. On the other hand, the SRC-1, CBP/p300, and P/CAF complexes may function through chromatin modification as they possess histone acetylase activity. In addition, transcriptional activation is associated with chromatin disruption, which may be due to the recruitment of chromatin remodeling factors by TR/RXR. This chromatin disruption may be necessary for transcriptional activation by TR/RXR. In addition to TBP, the TATA box binding protein, and RNA polymerase, some other basal transcription factors are also depicted in the figure.
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acetylation levels of histones or other proteins may in part contribute to the transcriptional activation of the target genes as discussed below. Histone acetylation has long been shown to affect transcription (Wolffe, 1996). Histone acetylation occurs in the basic tail domains of the histones. Increases in histone acetylation levels are expected to reduce the charge – charge interactions between the positively charged histone tails and the negatively charged DNA within the nucleosome. This may lead to a less compact chromatin structure, allowing better access of the DNA to transcription factors to activate transcription. A possible mechanism by which TR/ RXR regulates transcription is, therefore, through the regulation of histone acetylation levels. In the absence of TH, TR/RXR binds to TRE and recruits to the target gene a deacetylase-containing complex through its interaction with a corepressor, leading to localized histone deacetylation and transcriptional repression. Upon the binding of TH to TR/RXR, the corepressor complex is relieved and a coactivator complex is recruited, leading to increased levels of histone acetylation and transcriptional activation. Consistently, by using an in vivo reconstituted transcription system in frog oocytes, we have shown that inhibiting histone deacetylases with trichostatin A blocks TR/RXR mediated repression of the TH-dependent X. lae6is TRbA promoter (Wong et al., 1998). Conversely, over-expression of a histone deacetylase, the Xenopus RPD3, leads to transcriptional repression of the promoter and this repression can be eliminated by introducing TR/ RXR in the presence of TH (Wong et al., 1998). In addition to histone acetylation, TR/RXR may regulate gene transcription through alterations in chromatin structure at another level, i.e. chromatin disruption. Normal chromatin consists of a regularly placed nucleosome every 180 –200 bp of DNA (Wolffe, 1995). Alteration of this nucleosome array can be detected by micrococcal nuclease digestion, which occurs preferentially in the inter-nucleosomal region, generating a ladder of DNA fragments differing in sizes by multiples of 180–200 bp. In addition, if the template chromatin is a circular plasmid DNA, such changes can also be detected by measuring the superhelical density of the DNA on an agarose gel. Using these two methods, Wong et al. (1995, 1998) have demonstrated that liganded TR/RXR drastically alters the chromatin structure of a TRE-contain-
ing template in the reconstituted frog oocyte transcription system. The change by one TRE-bound TR/RXR was found to be equivalent to the loss of two to three nucleosomes. Furthermore, this chromatin disruption is tightly associated with transcriptional activation by TH-bound TR/ RXR, both requiring the same domains of TR. On the other hand, it is not sufficient for transcriptional activation. Interestingly, this disruption appears to be unrelated to changes in histone acetylation because overexpression of the histone deacetylase RPD3 or blocking deacetylases with trichostatin A fails to produce such disruptions (Wong et al., 1998). Thus, chromatin disruption is likely to be necessary for transcriptional activation by TH-bound TR/RXR and multiple levels of changes in chromatin are involved in transcriptional repression and activation by TR/RXR.
3. Genes regulated by TH during metamorphosis TRs have been shown to be present in X. lae6is and Rana catesbeiana (Yaoita et al., 1990; Schneider and Galton, 1991; Helbing et al., 1992). This is consistent with the view that TH regulates amphibian metamorphosis by controlling target gene transcription through nuclear receptors. In addition, many genes have been found to be regulated by TH during metamorphosis (Gilbert et al., 1996). However, many of these TH response genes are unlikely to be regulated by TRs at the transcriptional level (Shi, 1996). A systematic isolation of early TH response genes, i.e. those whose mRNA levels are altered within 24 h of TH treatment of premetamorphic tadpoles, was carried out in the tail, hindlimb, and intestine of X. lae6is by Buckbinder and Brown (1992), Shi and Brown (1993), Wang and Brown (1993) and subsequently in the brain by Denver et al. (1997). The characterization of these genes has yielded both expected and surprising findings. Many of the genes are indeed directly regulated by TRs (direct response genes) as their regulation by TH can occur in the presence of inhibitors of protein synthesis (Table 1). In addition, many genes encoding transcription factors, including TRb genes themselves, are found to be among these direct response genes. This supports the notion that TH induces a cascade of gene regulation to effect the metamorphic transformations of individual tissues/organs.
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matrix, and proteins involved in signal transduction, etc. Such diversity is also reflected in the direct TH-response genes identified through other means, e.g. the R. catesbeiana matrix metalloproteinase collagenase 1, and a transcription factor aC/EBP (Chen et al., 1994; Oofusa et al., 1994). These results argue against a simple, but possible, model where TH induces a cascade of gene regulation through sequential activation of a series of transcription factors, with the last step being the regulation of genes that are responsible for tissue specific changes during metamorphosis. Instead, it appears that TH simultaneously in-
Most of the direct response genes are ubiquitously regulated by TH, even though different organs undergo vastly different transformations. For example, TRb genes are upregulated by TH in all of the organs analyzed, such as the tail, limb, intestine, and brain, even though the tail resorbs, limb undergoes de novo development, and the intestine and brain partially but drastically remodel during metamorphosis. Another surprising finding is that the early response genes belong to diverse groups (Table 1). They include genes encoding transcription factors, matrix metelloproteinases that degrade the extracellular
Table 1 Some of the early TH upregulated genes isolated by subtraction from Xenopus tadpole tissues Gene name
Homologous genes
Response to THa
Possible function
Tail 1/3 Tail 7b IU 16/33 Tail 8/9TH/b TRb
Zinc finger (BTEB) Zip (Fra-2) NFI Zip (E4BP4)
Direct ND Direct Direct Direct
Transcription factorsb,c,d,f,g,m As above As above As above As above
Tail 11 Tail 14
Collagenase-3 Stromelysin-3
ND Direct
Matrix metalloproteinasesb,d,f,h As above
Tail 13 Tail D
FAPa N-Aspartyl dipeptidase
ND ND
Proteasesf
Tail C Tail 14 Tail 15 Xh20
Fibronectin Integrin a-1 Type III deiodinase Protein disulfide isomerase
ND Direct Direct Direct
ECM proteinf ECM receptorf TH inactivationb,c,i Protein isomerizationc
Xh1
Cytochrome c oxidase subunit
ND
Oxidative phosphorylationc
IU22 IU24 IU12 IU27 P
Nonhepatic arginase Na+/PO− 4 cotransporter Transmembrane protein Sonic hedgehog Rat clathrin B chain
ND Direct ND Direct ND
Proline biosynthesis etc.d,f,j d,k PO− 4 transport Amino acid transportl Morphogend,n Vesicular intracellular transporte
B H J
Heat-shock protein Yeast MCM3, mouse P1 Mouse eIF-4A
Direct ND ND
Rapid cell growthe As above As above
a
A direct response indicates that the regulation is resistant to protein synthesis inhibition; ND, not determinable. Wang and Brown (1993) c Denver et al. (1997) d Shi and Brown (1993) e Buckbinder and Brown (1992) f Brown et al. (1996) g Ishizuya-Oka et al. (1997a) h Patterton et al. (1995) i St. Germain et al. (1994) j Patterton and Shi (1994) k Ishizuya-Oka et al. (1997b) l Liang et al. (1997), Torrents et al. (1998) m Puzianowska-Kuznicka and Shi (1996) n Stolow and Shi (1995) b
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Fig. 2. Developmental expression of TR and RXR genes suggests dual functions for TR/RXR in frog development. The TH-inducible genes stromelysin-3 (ST3) and sonic hedgehog (HH) are expressed during late embryogenesis when little TR/RXR mRNAs are present. As the TRa and RXRa genes are activated, ST3 and HH genes are repressed. When endogenous TH levels rise after stage 54, both ST3 and TRb genes are activated. HH is activated only in selective organs, thus not observable when whole animals are use for analysis as shown here. The TR and RXR mRNA levels are based on Yaoita and Brown (1990)and Wong and Shi (1995). The HH and ST3 mRNA levels are based on Stolow and Shi (1995) and Patterton et al. (1995), respectively. Thyroid hormone T4 levels are from Leloup and Buscaglia (1977).
duces both intra- and extra-cellular pathways to initiate metamorphosis. Furthermore, these findings argue that the ubiquitous TH-induced early genes function together with pre-existing, tissuespecific factors to activate the downstream organspecific metamorphic pathways.
4. Involvement of TR in gene repression in premetamorphic tadpoles and activation in metamorphosing tadpole To investigate the role of TRs in regulating the TH response genes during metamorphosis, we and others have analyzed the expression of TR genes in various organs at different developmental stages in R. catesbeiana and X. lae6is (Yaoita and Brown, 1990; Kawahara et al., 1991; Schneider and Galton, 1991; Helbing et al., 1992; Wang and Brown, 1993; Eliceiri and Brown, 1994; Shi et al., 1994; Wong and Shi, 1995; Fairclough and Tata, 1997). In general, TRa and b genes are highly expressed in an organ when metamorphosis occurs although different organs undergo metamorphosis at different stages. For example, in the hindlimb of X. lae6is, the receptor mRNAs are high when limb morphogenesis, i.e. digit formation, takes place around stages 52 – 54. On the other hand, the receptor genes are upregulated
after stage 62 in the tail because tail resorption occurs mainly after stage 62 (Nieuwkoop and Faber, 1956). Moreover, we have found that RXR genes (RXRa and g) are coordinately expressed with the TR genes in various organs (Wong and Shi, 1995), supporting a role of RXR in the formation of TR/RXR heterodimers to mediate the effects of TH during metamorphosis. These studies have also revealed that the TR and RXR genes are expressed in premetamorphic Xenopus tadpoles (Yaoita and Brown, 1990; Kawahara et al., 1991; Shi et al., 1994; Wong and Shi, 1995). In particular, the TRa and RXRa genes are upregulated around stage 40–45 (Fig. 2), when tadpole feeding begins (Nieuwkoop and Faber, 1956). Interestingly, this upregulation of the receptor gene expression coincides with the downregulation of several TH response genes, e.g. stromelysin-3 (Patterton et al., 1995) and sonic hedgehog (Stolow and Shi, 1995), which are upregulated by TH during metamorphosis and are also expressed during embryogenesis (Fig. 2). Since TH is not yet available during this period of tadpole development (Fig. 2, Leloup and Buscaglia, 1977), TR/RXR heterodimers likely function as unliganded transcription repressors for these genes. As embryogenesis is completed and tadpole feeding begins, those TH response genes, which are important for organogenesis of
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the tadpole and frog but not required for tadpole growth, need to be repressed. The unliganded TR/RXR heterodimers thus may function to repress these genes to ensure a proper period of tadpole growth. The maturation of thyroid gland later during development then reactivates these genes through the same receptors to initiate the metamorphic transformation. A direct proof is yet lacking for the dual functions of TR/RXR in frog development, i.e. to repress the TH response genes in premetamorphic tadpoles and reactivate them during metamorphosis. On the other hand, by microinjecting mRNAs encoding TR and/or RXR into fertilized X. lae6is eggs, we have overexpressed these receptors in embryos prior to the activation of endogenous receptor genes (Puzianowska-Kuznicka et al., 1997). Such overexpression leads to distinct developmental defects, such as axis truncation and deformed/missing eyes, depending upon the presence or absence of TH. Furthermore, in the absence of TH, TR overexpression results in the downregulation of at least one TH response gene, the Xenopus sonic hedgehog gene. When TH is added to the embryo culture medium, several TH response genes, including stromelysin-3, are strongly activated. For the sonic hedgehog gene, the downregulation is relieved but little further activation is observed, consistent with the fact that sonic hedgehog is activated by TH only in certain organs during metamorphosis (Stolow and Shi, 1995). Thus, the regulation of gene expression by the overexpressed receptors is specific and reflective of the regulatory roles that they play during natural metamorphosis. Furthermore, RXRs are required for both the effects of TR on embryogenesis and regulation of TH response genes by TRs in the absence or presence of TH, demonstrating that TR/RXR heterodimers are indeed the functional complexes during development. That is, TR/RXR heterodimers function as transcriptional repressors of TH-inducible genes in premetamorphic tadpoles when TH is absent, and as transcriptional activators during metamorphosis when TH is present.
5. Roles of TR in apoptosis and cell proliferation during metamorphosis Anuran metamorphosis involves three major types of transformations. Some organs, such as
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the limb, undergo de novo development, involving extensive cell proliferation followed by cell differentiation and organ morphogenesis. Others, such as the tail and gills, which are tadpole specific, completely degenerate through mostly, if not entirely, programmed cell death or apoptosis (Dodd and Dodd, 1976; Gilbert and Frieden, 1981). The majority of the organs, such as the intestine and liver, are present in both tadpoles and frogs. However, they are drastically remodeled into often morphologically different adult structures with distinct functional properties during metamorphosis. This remodeling process often involves apoptotic removal of some larval tissues with the organ and development of adult tissues. Despite such diversity in tissue transformations, all are controlled by TH. Furthermore, individual organs are genetically programmed to undergo their predetermined changes as demonstrated by organ transplantation experiments. For example, a tail transplanted to the body region undergoes degeneration together with the host tail while an eye transplanted to the tail will not resorb even though the host tail resorbs completely (Weber, 1964). In addition, most, if not all, of these changes are organ-autonomous as they can also occur in organ cultures in vitro when TH is added to the culture media (Ishizuya-Oka and Shimozawa, 1991; Oofusa and Yoshizato, 1991; Tata et al., 1991). Finally, the expression studies described above have shown that TR and RXR gene expression is temporally correlated with metamorphosis in individual organs. These argue that TRs can function to promote cell death (tissue resorption) or cell proliferation and differentiation (organ development) during metamorphosis depending upon the organs in which they are expressed. By using in situ hybridization, we have carried out a detailed analysis of TRb gene expression during intestinal metamorphosis (Shi and Ishizuya-Oka, 1997). As expected from Northern blot analysis, little or no TRb mRNA is present in any tissues within the premetamorphic intestine. Around stage 57, TRb mRNA becomes detectable in some larval epithelial cells and by stage 59, just prior to the onset of larval epithelial apoptosis, all larval epithelial cells express high levels of TRb mRNA (Fig. 3). Subsequently, as apoptosis takes place in the larval epithelium, the TRb mRNA levels are downregulated (stage 61, Fig. 3D). In contrast, TRb mRNA levels are high in the prolif-
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erating adult epithelial cells as soon as they can be identified as cell islets in between the larval epithelium and the connective tissue. The TRb expression in the adult epithelial cells remains high until stage 62 when adult epithelial cell differentiation begins. Likewise, in the connective tissue and muscles, TRb mRNA levels are high when cells proliferate but are downregulated as these cells
differentiate to form the adult connective tissue and muscles, respectively (Fig. 3C, E). Thus, even with a single organ, TRs (at least TRb) appear to be involved in promoting both cell proliferation and apoptosis, depending upon the cell types in which they are expressed. In particular, at least in the intestine during metamorphosis, high levels of TRb mRNA appear to be incompatible with high
Fig. 3. In situ hybridization reveals that TRb gene expression correlates with larval epithelial cell death and adult cell proliferation during intestinal metamorphosis. (There are two TRb genes, TRbA and TRbB, in Xenopus lae6is. The in situ hybridization here detects both). (A) Stage 55, little TRb mRNA can be detected in premetamorphic intestine. (B) Stage 57, TRb genes are activated in the larval epithelium (arrows, E) facing the lumen (L) but not yet in the connective tissue (CT) or muscle (M). (C) Stage 60, the adult epithelial cells can now be recognized as small islets (arrowheads) between the larval epithelium (LE) and the connective tissue and are expressing TRb. TRb genes are now also expressed strongly in the connective tissue and circular muscle (CM) but only very weakly in the longitudinal muscle (LM). (D) Stage 61, the proliferating adult epithelium (AE) continues to express TRb while the degenerating larval epithelium ceases to express TRb. (E) Stage 62, TRb mRNA signals in the longitudinal muscle is now as strong as in the circular muscle. The connective tissue and adult epithelium continue to express TRb. (F) Stage 63, multiple folds (Fo) are formed as adult epithelial cells differentiate. The TRb genes are expressed in the connective tissue and muscle (very weakly in the circular muscle) but not in the differentiating adult epithelial cells. (G) Stage 66, TRb genes are repressed in all intestinal tissues by the end of metamorphosis. For details, see Shi and Ishizuya-Oka (1997).
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Fig. 4. A model for the dual functions of TR/RXR and for at lease some TH-induced genes during frog development. During embryogenesis, the TH-inducible genes are expressed to facilitate embryonic organ development. At the end of larval development, tadpole feeding begins and these genes need to be repressed. The upregulation of TRa and RXRa genes in the absence of TH serves this repressor role, thus allowing tadpole growth. As thyroid gland matures toward the end of this premetamorphic period, TH is synthesized and secreted into the plasma. TH then relieves the repression of TH-inducible genes by the unliganded TR/RXR and further activates them, thus initiating adult organ development (metamorphosis). TH-bound TR/RXR may also downregulate the expression of some genes. Currently, little is known about such genes. Thus, the possible involvement of such genes is indicated by dashed lines.
degrees of differentiation. Thus, in the differentiated larval epithelial cells, high levels of TRb expression are associated with apoptosis. On the other hand, as the cells of the adult epithelium, connective tissue, and muscles begin to differentiate, they need to shut down their TRb expression to prevent likely deleterious (apoptotic) consequences associated with high levels of TRb mRNA. Direct evidence for a role of TRa and/or TRb in promoting cell proliferation and apoptosis comes from in vitro studies of primary intestinal cells (Su et al., 1997). Partially purified fibroblasts and larval epithelial cells from tadpole intestine can be cultured in vitro, where they respond to TH in a similar fashion as that which occurs during natural metamorphosis. In particular, the fibroblasts increase in number and this proliferation is stimulated by including TH in culture medium. In contrast, the epithelial cells gradually decrease in number and this reduction is greatly enhanced by TH. Interestingly, analysis of DNA synthesis reveals that TH stimulated cell proliferation in both cell types. On the other hand, apoptosis is induced by TH in the larval epithelial cells but not in the fibroblasts. These results argue that TRs indeed function directly to mediate the effects of TH in stimulating the proliferation of the intestinal fibroblasts while causing the epithelial cells to undergo programmed cell death.
6. Conclusion Thyroid hormone regulates a wide range of biological processes across most animal species. Although it may affect some of these processes through non-genomic actions such as interacting with cytosolic TH binding proteins (Davis and Davis, 1996), it is now clear that thyroid hormone receptors are the major players in mediating the biological effects of TH. So far only two types of TRs, TRa and TRb have been identified and little evidence supports the existence of additional TRs. In addition, human patients with mutated TRb gene share similar developmental defects as hypothyroid patients. Likewise, gene knockout studies in mice suggest that TRa and TRb are responsible for the major known biological effects of TH (Forrest et al., 1996a,b; Fraichard et al., 1997; Wikstrom et al., 1998; Gautheir et al., 1999). The roles of TRa and TRb in regulating anuran metamorphosis are also supported by their temporal and spatial expression profiles in both R. catesbeiana and X. lae6is. Furthermore, these receptors appear to have dual functions depending upon the cell types and developmental stages when they are expressed (Fig. 4). In premetamorphic tadpoles, they are likely to function as unliganded transcriptional repressors to block the expression of TH response genes that are involved in metamorphosis, thus ensuring a proper period of tadpole growth. When TH is
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synthesized and secreted into the circulating plasma, it binds to the receptors and converts them into activators to activate the TH-inducible genes to initiate metamorphosis. During metamorphosis, these receptors can promote apoptosis in larval specific cell types while enhancing the proliferation of adult cells. Our studies involving overexpression of TR/ RXR in embryos have provided some in vivo evidence that supports the involvement of TR/ RXR heterodimers in repressing TH-inducible genes in the absence of TH and in activating them these genes when TH is present. However, conclusive evidence for the dual functions of TR/RXR is yet to be obtained. Furthermore, most of the studies on TR/RXR expression are on mRNA levels, although limited Western blot and immunohistochemical analyses support the conclusions (Eliceiri and Brown, 1994; Fairclough and Tata, 1997). Thus, more detailed analysis of TR and RXR protein levels in individual cell types during metamorphosis are important to support the proposed dual roles of the heterodimers. Such analysis will further provide evidence whether there are functional difference among the RXR isoforms, i.e. RXRa, b, g (little information is available on RXRb expression during metamorphosis), and between TRa and TRb. Functional studies of the receptors during metamorphosis will benefit from the use of primary cell cultures or cell lines established from tadpole tissues, such as the one from Xenopus tail muscles cells (Yaoita and Nakajima, 1997). Finally, studies using the recently developed transgenic technology in X. lae6is (Kroll and Amaya, 1996) will likely provide direct evidence for receptor function during metamorphosis, as was done for the type III deiodinase (Huang et al., 1999).
Acknowledgements We thank Kieu Pham for preparing the manuscript.
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Metamorphosis
Dual functions of thyroid hormone receptors during Xenopus development Laurent M. Sachs a, Sashko Damjanovski a, Peter L. Jones a, Qing Li a, Tosikazu Amano a, Shuichi Ueda b, Yun-Bo Shi a,*, Atsuko Ishizuya-Oka b a
Laboratory of Molecular Embryology, National Institute of Child Health and Human De6elopment, National Institutes of Health, Bethesda, MD 20892 -5431, USA b Department of Histology and Neurobiology, Dokkyo Uni6ersity School of Medicine, Mibu, Tochigi 321 -02, Japan Received 18 August 1999; received in revised form 16 November 1999; accepted 18 November 1999
Abstract Thyroid hormone (TH) plays a causative role in anuran metamorphosis. This effect is presumed to be manifested through the regulation of gene expression by TH receptors (TRs). TRs can act as both activators and repressors of a TH-inducible gene depending upon the presence and absence of TH, respectively. We have been investigating the roles of TRs during Xenopus lae6is development, including premetamorphic and metamorphosing stages. In this review, we summarize some of the studies on the TRs by others and us. These studies reveal that TRs have dual functions in frog development as reflected in the following two aspects. First, TRs function initially as repressors of TH-inducible genes in premetamorphic tadpoles to prevent precocious metamorphosis, thus ensuring a proper period of tadpole growth, and later as activators of these genes to activate the metamorphic process. Second, TRs can promote both cell proliferation and apoptosis during metamorphosis, depending upon the cell type in which they are expressed. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Apoptosis; Cell death; Cell proliferation; Metamorphosis; Transcriptional regulation; Xenopus lae6is; Amphibian; Thyroid hormone receptor
1. Introduction Thyroid hormone (TH) influences diverse biological processes (Hetzel, 1989; Mandel et al., 1993). In adult mammals, TH regulates the physiological function of many organs (Guernsey and Edelman, 1983; Freake and Oppenheimer, 1995; Silva, 1995). In humans, inappropriate levels of Presented at the International Symposium ‘‘The Frontiers of the Biology of Amphibia’’, Hiroshima, Japan, March 22– 24, 1999. * Corresponding author. Tel.: + 1-301-4021004; fax: + 1301-4021323. E-mail address: [email protected] (Y.-B. Shi)
TH lead to various diseases. In addition, TH also plays important roles during embryogenesis and organogenesis. Since early part of this century, hypothyroidism has been known to cause severe developmental defects in humans, most notably human cretinism, a form of defect characterized by mental retardation and short stature due to deficiencies in brain and skeletal development (Dussault and Ruel, 1987; Hetzel, 1989; Mandel et al., 1993; Porterfield and Hendrich, 1993). Compared to other vertebrates, anuran development is perhaps the most dependent on TH. Anuran development takes place in two phases. Its embryogenesis occurs prior to the formation of
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a functional thyroid gland to form a free-living tadpole. However, metamorphosis, i.e. the transformation of the tadpole to a frog, is absolutely dependent upon the presence of sufficient levels of TH in the plasma (Dodd and Dodd, 1976; Gilbert and Frieden, 1981). Blocking the synthesis of endogenous TH inhibits metamorphosis, while simply adding physiological concentrations of TH to rearing water of premetamorphic tadpoles induces precocious transformations. Furthermore, TH can induce metamorphosis in organ cultures, suggesting that it act directly within target tissues/ organs (Ishizuya-Oka and Shimozawa, 1991; Oofusa and Yoshizato, 1991; Tata et al., 1991). TH functions by regulating gene transcription through its nuclear receptor or thyroid hormone receptors (TRs) (Sap et al., 1986; Weinberger et al., 1986). TRs belong to the superfamily of nuclear receptors that also include steroid receptors, retinoic acid receptors, and 9-cis retinoic acid receptors (RXRs) (Mangelsdorf et al., 1995). Like most receptors of this family, TR has a DNA binding domain in the N-terminal half (Lazar, 1993; Tsai and O’Malley, 1994; Mangelsdorf et al., 1995) that recognizes thyroid hormone response elements (TREs), and a TH-binding domain in the C-terminal half. Although TRs can bind to TREs as monomers or homodimers, in vivo they most likely function as heterodimers formed with RXRs. Upon binding to TREs, TR/ RXR heterodimers can regulate the transcription of the target genes in a TH-dependent manner, thus mediating the biological effects of TH. The consequences of this regulation depend upon target cell/tissue types. In this paper, we discuss some possible biological functions of TR/RXR heterodimers during frog development, focusing mostly on our own studies in X. lae6is.
2. Transcriptional activation and repression by TR Thyroid hormone can both activate and repress gene transcription through TRs (Tsai and O’Malley, 1994; Yen and Chin, 1994; Shi et al., 1996a; Wolffe et al., 1997). Only a few genes are known to be repressed by TH, and currently relatively little is known about how TH represses these genes through TRs. These genes contain negative TREs, which differ in sequence from the TREs found in TH-inducible genes. Whether and how
such differences in TRE sequences influence the positive or negative transcriptional regulation by TRs remains to be determined. The vast majority of the genes that are regulated by thyroid hormones at the transcriptional level are upregulated by TH. These genes contain one or more TREs, which often consist of a direct repeat of the consensus sequence AGGTCA separated by 4 bp (Naar et al., 1991; Umesono et al., 1991). Interestingly, these class of genes can be both activated and repressed by TR/RXR heterodimers, depending upon the presence and absence of TH, respectively (Fig. 1, Tsai and O’Malley, 1994; Shi et al., 1996a; Wolffe et al., 1997). Both the activation and repression require the presence of a TRE in the target promoter. On the other hand, distinct cofactors are recruited by TRs, depending upon the presence or absence of TH, to effect the outcome of the transcriptional regulation by TR/RXR (Fig. 1, Shi et al., 1996a; Chen and Li, 1998; Koenig, 1998). Several TR-interacting transcriptional corepressors have been identified. Among them, the beststudied are N-CoR and SMRT (Chen and Evans, 1995; Horlein et al., 1995; Chen and Li, 1998). Both of them can interact with unliganded TRs and the binding of TH to TRs inhibits this interaction. How these corepressors mediate transcriptional repression by unliganded TRs is unclear. However, both N-CoR and SMRT can interact with the corepressor Sin-3A, and thus, can be recruited into large complexes that also contain histone deacetylases such as RPD3 (Fig. 1, Heinzel et al., 1997; Nagy et al., 1997). Thus, one possible mechanism of transcriptional repression by unliganded TR/RXR is through histonedeacetylation which is turn facilitates the formation of a more repressive chromatin structure (for more details, see below). Numerous proteins have been isolated based on their interactions with TH-bound TRs (Lee et al., 1995; Shi et al., 1996a; Chen and Li, 1998). In contrast to the corepressors, these coactivators interact strongly with liganded TR but not with unliganded TR. Interestingly, many of these coactivators, such as CBP/p300, SRC-1, and pCAF, are themselves histone acetylases (Fig. 1, Ogryzko et al., 1996; Spencer et al., 1997; Blanco et al., 1998). Thus, their recruitment by TRs may bring about the opposite changes in chromatin structure compared to the recruitment of the corepressors by unliganded TRs. The resulting increase in
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Fig. 1. A proposed mechanism for transcriptional regulation by TRs. TR functions as a heterodimer with RXR. The heterodimer binds to the TRE in a target gene. In the absence of TH, the heterodimer represses gene transcription likely through the recruitment of corepressor complex containing the corepressor N-CoR (Horlein et al., 1995) or SMRT (Chen and Evans, 1995). The corepressor interacts with Sin3A, which in turn recruits a histone deacetylase such as Rpd3 to deacetylate histones (Heinzel et al., 1997; Nagy et al., 1997), thus affecting transcription. Upon binding by TH, a conformational change takes place in the heterodimer, which may be responsible for the release of the corepressor complex. Liganded TR also recruits a coactivator complex containing coactivators such as SRC-1 or CBP/p300, and P/CAF, and/or the so-called DRIP/TRAP coactivator complex (Freedman, 1999). The DRIP/TRAP complex may contact RNA polymerase directly to activate gene transcription. On the other hand, the SRC-1, CBP/p300, and P/CAF complexes may function through chromatin modification as they possess histone acetylase activity. In addition, transcriptional activation is associated with chromatin disruption, which may be due to the recruitment of chromatin remodeling factors by TR/RXR. This chromatin disruption may be necessary for transcriptional activation by TR/RXR. In addition to TBP, the TATA box binding protein, and RNA polymerase, some other basal transcription factors are also depicted in the figure.
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acetylation levels of histones or other proteins may in part contribute to the transcriptional activation of the target genes as discussed below. Histone acetylation has long been shown to affect transcription (Wolffe, 1996). Histone acetylation occurs in the basic tail domains of the histones. Increases in histone acetylation levels are expected to reduce the charge – charge interactions between the positively charged histone tails and the negatively charged DNA within the nucleosome. This may lead to a less compact chromatin structure, allowing better access of the DNA to transcription factors to activate transcription. A possible mechanism by which TR/ RXR regulates transcription is, therefore, through the regulation of histone acetylation levels. In the absence of TH, TR/RXR binds to TRE and recruits to the target gene a deacetylase-containing complex through its interaction with a corepressor, leading to localized histone deacetylation and transcriptional repression. Upon the binding of TH to TR/RXR, the corepressor complex is relieved and a coactivator complex is recruited, leading to increased levels of histone acetylation and transcriptional activation. Consistently, by using an in vivo reconstituted transcription system in frog oocytes, we have shown that inhibiting histone deacetylases with trichostatin A blocks TR/RXR mediated repression of the TH-dependent X. lae6is TRbA promoter (Wong et al., 1998). Conversely, over-expression of a histone deacetylase, the Xenopus RPD3, leads to transcriptional repression of the promoter and this repression can be eliminated by introducing TR/ RXR in the presence of TH (Wong et al., 1998). In addition to histone acetylation, TR/RXR may regulate gene transcription through alterations in chromatin structure at another level, i.e. chromatin disruption. Normal chromatin consists of a regularly placed nucleosome every 180 –200 bp of DNA (Wolffe, 1995). Alteration of this nucleosome array can be detected by micrococcal nuclease digestion, which occurs preferentially in the inter-nucleosomal region, generating a ladder of DNA fragments differing in sizes by multiples of 180–200 bp. In addition, if the template chromatin is a circular plasmid DNA, such changes can also be detected by measuring the superhelical density of the DNA on an agarose gel. Using these two methods, Wong et al. (1995, 1998) have demonstrated that liganded TR/RXR drastically alters the chromatin structure of a TRE-contain-
ing template in the reconstituted frog oocyte transcription system. The change by one TRE-bound TR/RXR was found to be equivalent to the loss of two to three nucleosomes. Furthermore, this chromatin disruption is tightly associated with transcriptional activation by TH-bound TR/ RXR, both requiring the same domains of TR. On the other hand, it is not sufficient for transcriptional activation. Interestingly, this disruption appears to be unrelated to changes in histone acetylation because overexpression of the histone deacetylase RPD3 or blocking deacetylases with trichostatin A fails to produce such disruptions (Wong et al., 1998). Thus, chromatin disruption is likely to be necessary for transcriptional activation by TH-bound TR/RXR and multiple levels of changes in chromatin are involved in transcriptional repression and activation by TR/RXR.
3. Genes regulated by TH during metamorphosis TRs have been shown to be present in X. lae6is and Rana catesbeiana (Yaoita et al., 1990; Schneider and Galton, 1991; Helbing et al., 1992). This is consistent with the view that TH regulates amphibian metamorphosis by controlling target gene transcription through nuclear receptors. In addition, many genes have been found to be regulated by TH during metamorphosis (Gilbert et al., 1996). However, many of these TH response genes are unlikely to be regulated by TRs at the transcriptional level (Shi, 1996). A systematic isolation of early TH response genes, i.e. those whose mRNA levels are altered within 24 h of TH treatment of premetamorphic tadpoles, was carried out in the tail, hindlimb, and intestine of X. lae6is by Buckbinder and Brown (1992), Shi and Brown (1993), Wang and Brown (1993) and subsequently in the brain by Denver et al. (1997). The characterization of these genes has yielded both expected and surprising findings. Many of the genes are indeed directly regulated by TRs (direct response genes) as their regulation by TH can occur in the presence of inhibitors of protein synthesis (Table 1). In addition, many genes encoding transcription factors, including TRb genes themselves, are found to be among these direct response genes. This supports the notion that TH induces a cascade of gene regulation to effect the metamorphic transformations of individual tissues/organs.
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matrix, and proteins involved in signal transduction, etc. Such diversity is also reflected in the direct TH-response genes identified through other means, e.g. the R. catesbeiana matrix metalloproteinase collagenase 1, and a transcription factor aC/EBP (Chen et al., 1994; Oofusa et al., 1994). These results argue against a simple, but possible, model where TH induces a cascade of gene regulation through sequential activation of a series of transcription factors, with the last step being the regulation of genes that are responsible for tissue specific changes during metamorphosis. Instead, it appears that TH simultaneously in-
Most of the direct response genes are ubiquitously regulated by TH, even though different organs undergo vastly different transformations. For example, TRb genes are upregulated by TH in all of the organs analyzed, such as the tail, limb, intestine, and brain, even though the tail resorbs, limb undergoes de novo development, and the intestine and brain partially but drastically remodel during metamorphosis. Another surprising finding is that the early response genes belong to diverse groups (Table 1). They include genes encoding transcription factors, matrix metelloproteinases that degrade the extracellular
Table 1 Some of the early TH upregulated genes isolated by subtraction from Xenopus tadpole tissues Gene name
Homologous genes
Response to THa
Possible function
Tail 1/3 Tail 7b IU 16/33 Tail 8/9TH/b TRb
Zinc finger (BTEB) Zip (Fra-2) NFI Zip (E4BP4)
Direct ND Direct Direct Direct
Transcription factorsb,c,d,f,g,m As above As above As above As above
Tail 11 Tail 14
Collagenase-3 Stromelysin-3
ND Direct
Matrix metalloproteinasesb,d,f,h As above
Tail 13 Tail D
FAPa N-Aspartyl dipeptidase
ND ND
Proteasesf
Tail C Tail 14 Tail 15 Xh20
Fibronectin Integrin a-1 Type III deiodinase Protein disulfide isomerase
ND Direct Direct Direct
ECM proteinf ECM receptorf TH inactivationb,c,i Protein isomerizationc
Xh1
Cytochrome c oxidase subunit
ND
Oxidative phosphorylationc
IU22 IU24 IU12 IU27 P
Nonhepatic arginase Na+/PO− 4 cotransporter Transmembrane protein Sonic hedgehog Rat clathrin B chain
ND Direct ND Direct ND
Proline biosynthesis etc.d,f,j d,k PO− 4 transport Amino acid transportl Morphogend,n Vesicular intracellular transporte
B H J
Heat-shock protein Yeast MCM3, mouse P1 Mouse eIF-4A
Direct ND ND
Rapid cell growthe As above As above
a
A direct response indicates that the regulation is resistant to protein synthesis inhibition; ND, not determinable. Wang and Brown (1993) c Denver et al. (1997) d Shi and Brown (1993) e Buckbinder and Brown (1992) f Brown et al. (1996) g Ishizuya-Oka et al. (1997a) h Patterton et al. (1995) i St. Germain et al. (1994) j Patterton and Shi (1994) k Ishizuya-Oka et al. (1997b) l Liang et al. (1997), Torrents et al. (1998) m Puzianowska-Kuznicka and Shi (1996) n Stolow and Shi (1995) b
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Fig. 2. Developmental expression of TR and RXR genes suggests dual functions for TR/RXR in frog development. The TH-inducible genes stromelysin-3 (ST3) and sonic hedgehog (HH) are expressed during late embryogenesis when little TR/RXR mRNAs are present. As the TRa and RXRa genes are activated, ST3 and HH genes are repressed. When endogenous TH levels rise after stage 54, both ST3 and TRb genes are activated. HH is activated only in selective organs, thus not observable when whole animals are use for analysis as shown here. The TR and RXR mRNA levels are based on Yaoita and Brown (1990)and Wong and Shi (1995). The HH and ST3 mRNA levels are based on Stolow and Shi (1995) and Patterton et al. (1995), respectively. Thyroid hormone T4 levels are from Leloup and Buscaglia (1977).
duces both intra- and extra-cellular pathways to initiate metamorphosis. Furthermore, these findings argue that the ubiquitous TH-induced early genes function together with pre-existing, tissuespecific factors to activate the downstream organspecific metamorphic pathways.
4. Involvement of TR in gene repression in premetamorphic tadpoles and activation in metamorphosing tadpole To investigate the role of TRs in regulating the TH response genes during metamorphosis, we and others have analyzed the expression of TR genes in various organs at different developmental stages in R. catesbeiana and X. lae6is (Yaoita and Brown, 1990; Kawahara et al., 1991; Schneider and Galton, 1991; Helbing et al., 1992; Wang and Brown, 1993; Eliceiri and Brown, 1994; Shi et al., 1994; Wong and Shi, 1995; Fairclough and Tata, 1997). In general, TRa and b genes are highly expressed in an organ when metamorphosis occurs although different organs undergo metamorphosis at different stages. For example, in the hindlimb of X. lae6is, the receptor mRNAs are high when limb morphogenesis, i.e. digit formation, takes place around stages 52 – 54. On the other hand, the receptor genes are upregulated
after stage 62 in the tail because tail resorption occurs mainly after stage 62 (Nieuwkoop and Faber, 1956). Moreover, we have found that RXR genes (RXRa and g) are coordinately expressed with the TR genes in various organs (Wong and Shi, 1995), supporting a role of RXR in the formation of TR/RXR heterodimers to mediate the effects of TH during metamorphosis. These studies have also revealed that the TR and RXR genes are expressed in premetamorphic Xenopus tadpoles (Yaoita and Brown, 1990; Kawahara et al., 1991; Shi et al., 1994; Wong and Shi, 1995). In particular, the TRa and RXRa genes are upregulated around stage 40–45 (Fig. 2), when tadpole feeding begins (Nieuwkoop and Faber, 1956). Interestingly, this upregulation of the receptor gene expression coincides with the downregulation of several TH response genes, e.g. stromelysin-3 (Patterton et al., 1995) and sonic hedgehog (Stolow and Shi, 1995), which are upregulated by TH during metamorphosis and are also expressed during embryogenesis (Fig. 2). Since TH is not yet available during this period of tadpole development (Fig. 2, Leloup and Buscaglia, 1977), TR/RXR heterodimers likely function as unliganded transcription repressors for these genes. As embryogenesis is completed and tadpole feeding begins, those TH response genes, which are important for organogenesis of
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the tadpole and frog but not required for tadpole growth, need to be repressed. The unliganded TR/RXR heterodimers thus may function to repress these genes to ensure a proper period of tadpole growth. The maturation of thyroid gland later during development then reactivates these genes through the same receptors to initiate the metamorphic transformation. A direct proof is yet lacking for the dual functions of TR/RXR in frog development, i.e. to repress the TH response genes in premetamorphic tadpoles and reactivate them during metamorphosis. On the other hand, by microinjecting mRNAs encoding TR and/or RXR into fertilized X. lae6is eggs, we have overexpressed these receptors in embryos prior to the activation of endogenous receptor genes (Puzianowska-Kuznicka et al., 1997). Such overexpression leads to distinct developmental defects, such as axis truncation and deformed/missing eyes, depending upon the presence or absence of TH. Furthermore, in the absence of TH, TR overexpression results in the downregulation of at least one TH response gene, the Xenopus sonic hedgehog gene. When TH is added to the embryo culture medium, several TH response genes, including stromelysin-3, are strongly activated. For the sonic hedgehog gene, the downregulation is relieved but little further activation is observed, consistent with the fact that sonic hedgehog is activated by TH only in certain organs during metamorphosis (Stolow and Shi, 1995). Thus, the regulation of gene expression by the overexpressed receptors is specific and reflective of the regulatory roles that they play during natural metamorphosis. Furthermore, RXRs are required for both the effects of TR on embryogenesis and regulation of TH response genes by TRs in the absence or presence of TH, demonstrating that TR/RXR heterodimers are indeed the functional complexes during development. That is, TR/RXR heterodimers function as transcriptional repressors of TH-inducible genes in premetamorphic tadpoles when TH is absent, and as transcriptional activators during metamorphosis when TH is present.
5. Roles of TR in apoptosis and cell proliferation during metamorphosis Anuran metamorphosis involves three major types of transformations. Some organs, such as
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the limb, undergo de novo development, involving extensive cell proliferation followed by cell differentiation and organ morphogenesis. Others, such as the tail and gills, which are tadpole specific, completely degenerate through mostly, if not entirely, programmed cell death or apoptosis (Dodd and Dodd, 1976; Gilbert and Frieden, 1981). The majority of the organs, such as the intestine and liver, are present in both tadpoles and frogs. However, they are drastically remodeled into often morphologically different adult structures with distinct functional properties during metamorphosis. This remodeling process often involves apoptotic removal of some larval tissues with the organ and development of adult tissues. Despite such diversity in tissue transformations, all are controlled by TH. Furthermore, individual organs are genetically programmed to undergo their predetermined changes as demonstrated by organ transplantation experiments. For example, a tail transplanted to the body region undergoes degeneration together with the host tail while an eye transplanted to the tail will not resorb even though the host tail resorbs completely (Weber, 1964). In addition, most, if not all, of these changes are organ-autonomous as they can also occur in organ cultures in vitro when TH is added to the culture media (Ishizuya-Oka and Shimozawa, 1991; Oofusa and Yoshizato, 1991; Tata et al., 1991). Finally, the expression studies described above have shown that TR and RXR gene expression is temporally correlated with metamorphosis in individual organs. These argue that TRs can function to promote cell death (tissue resorption) or cell proliferation and differentiation (organ development) during metamorphosis depending upon the organs in which they are expressed. By using in situ hybridization, we have carried out a detailed analysis of TRb gene expression during intestinal metamorphosis (Shi and Ishizuya-Oka, 1997). As expected from Northern blot analysis, little or no TRb mRNA is present in any tissues within the premetamorphic intestine. Around stage 57, TRb mRNA becomes detectable in some larval epithelial cells and by stage 59, just prior to the onset of larval epithelial apoptosis, all larval epithelial cells express high levels of TRb mRNA (Fig. 3). Subsequently, as apoptosis takes place in the larval epithelium, the TRb mRNA levels are downregulated (stage 61, Fig. 3D). In contrast, TRb mRNA levels are high in the prolif-
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erating adult epithelial cells as soon as they can be identified as cell islets in between the larval epithelium and the connective tissue. The TRb expression in the adult epithelial cells remains high until stage 62 when adult epithelial cell differentiation begins. Likewise, in the connective tissue and muscles, TRb mRNA levels are high when cells proliferate but are downregulated as these cells
differentiate to form the adult connective tissue and muscles, respectively (Fig. 3C, E). Thus, even with a single organ, TRs (at least TRb) appear to be involved in promoting both cell proliferation and apoptosis, depending upon the cell types in which they are expressed. In particular, at least in the intestine during metamorphosis, high levels of TRb mRNA appear to be incompatible with high
Fig. 3. In situ hybridization reveals that TRb gene expression correlates with larval epithelial cell death and adult cell proliferation during intestinal metamorphosis. (There are two TRb genes, TRbA and TRbB, in Xenopus lae6is. The in situ hybridization here detects both). (A) Stage 55, little TRb mRNA can be detected in premetamorphic intestine. (B) Stage 57, TRb genes are activated in the larval epithelium (arrows, E) facing the lumen (L) but not yet in the connective tissue (CT) or muscle (M). (C) Stage 60, the adult epithelial cells can now be recognized as small islets (arrowheads) between the larval epithelium (LE) and the connective tissue and are expressing TRb. TRb genes are now also expressed strongly in the connective tissue and circular muscle (CM) but only very weakly in the longitudinal muscle (LM). (D) Stage 61, the proliferating adult epithelium (AE) continues to express TRb while the degenerating larval epithelium ceases to express TRb. (E) Stage 62, TRb mRNA signals in the longitudinal muscle is now as strong as in the circular muscle. The connective tissue and adult epithelium continue to express TRb. (F) Stage 63, multiple folds (Fo) are formed as adult epithelial cells differentiate. The TRb genes are expressed in the connective tissue and muscle (very weakly in the circular muscle) but not in the differentiating adult epithelial cells. (G) Stage 66, TRb genes are repressed in all intestinal tissues by the end of metamorphosis. For details, see Shi and Ishizuya-Oka (1997).
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Fig. 4. A model for the dual functions of TR/RXR and for at lease some TH-induced genes during frog development. During embryogenesis, the TH-inducible genes are expressed to facilitate embryonic organ development. At the end of larval development, tadpole feeding begins and these genes need to be repressed. The upregulation of TRa and RXRa genes in the absence of TH serves this repressor role, thus allowing tadpole growth. As thyroid gland matures toward the end of this premetamorphic period, TH is synthesized and secreted into the plasma. TH then relieves the repression of TH-inducible genes by the unliganded TR/RXR and further activates them, thus initiating adult organ development (metamorphosis). TH-bound TR/RXR may also downregulate the expression of some genes. Currently, little is known about such genes. Thus, the possible involvement of such genes is indicated by dashed lines.
degrees of differentiation. Thus, in the differentiated larval epithelial cells, high levels of TRb expression are associated with apoptosis. On the other hand, as the cells of the adult epithelium, connective tissue, and muscles begin to differentiate, they need to shut down their TRb expression to prevent likely deleterious (apoptotic) consequences associated with high levels of TRb mRNA. Direct evidence for a role of TRa and/or TRb in promoting cell proliferation and apoptosis comes from in vitro studies of primary intestinal cells (Su et al., 1997). Partially purified fibroblasts and larval epithelial cells from tadpole intestine can be cultured in vitro, where they respond to TH in a similar fashion as that which occurs during natural metamorphosis. In particular, the fibroblasts increase in number and this proliferation is stimulated by including TH in culture medium. In contrast, the epithelial cells gradually decrease in number and this reduction is greatly enhanced by TH. Interestingly, analysis of DNA synthesis reveals that TH stimulated cell proliferation in both cell types. On the other hand, apoptosis is induced by TH in the larval epithelial cells but not in the fibroblasts. These results argue that TRs indeed function directly to mediate the effects of TH in stimulating the proliferation of the intestinal fibroblasts while causing the epithelial cells to undergo programmed cell death.
6. Conclusion Thyroid hormone regulates a wide range of biological processes across most animal species. Although it may affect some of these processes through non-genomic actions such as interacting with cytosolic TH binding proteins (Davis and Davis, 1996), it is now clear that thyroid hormone receptors are the major players in mediating the biological effects of TH. So far only two types of TRs, TRa and TRb have been identified and little evidence supports the existence of additional TRs. In addition, human patients with mutated TRb gene share similar developmental defects as hypothyroid patients. Likewise, gene knockout studies in mice suggest that TRa and TRb are responsible for the major known biological effects of TH (Forrest et al., 1996a,b; Fraichard et al., 1997; Wikstrom et al., 1998; Gautheir et al., 1999). The roles of TRa and TRb in regulating anuran metamorphosis are also supported by their temporal and spatial expression profiles in both R. catesbeiana and X. lae6is. Furthermore, these receptors appear to have dual functions depending upon the cell types and developmental stages when they are expressed (Fig. 4). In premetamorphic tadpoles, they are likely to function as unliganded transcriptional repressors to block the expression of TH response genes that are involved in metamorphosis, thus ensuring a proper period of tadpole growth. When TH is
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synthesized and secreted into the circulating plasma, it binds to the receptors and converts them into activators to activate the TH-inducible genes to initiate metamorphosis. During metamorphosis, these receptors can promote apoptosis in larval specific cell types while enhancing the proliferation of adult cells. Our studies involving overexpression of TR/ RXR in embryos have provided some in vivo evidence that supports the involvement of TR/ RXR heterodimers in repressing TH-inducible genes in the absence of TH and in activating them these genes when TH is present. However, conclusive evidence for the dual functions of TR/RXR is yet to be obtained. Furthermore, most of the studies on TR/RXR expression are on mRNA levels, although limited Western blot and immunohistochemical analyses support the conclusions (Eliceiri and Brown, 1994; Fairclough and Tata, 1997). Thus, more detailed analysis of TR and RXR protein levels in individual cell types during metamorphosis are important to support the proposed dual roles of the heterodimers. Such analysis will further provide evidence whether there are functional difference among the RXR isoforms, i.e. RXRa, b, g (little information is available on RXRb expression during metamorphosis), and between TRa and TRb. Functional studies of the receptors during metamorphosis will benefit from the use of primary cell cultures or cell lines established from tadpole tissues, such as the one from Xenopus tail muscles cells (Yaoita and Nakajima, 1997). Finally, studies using the recently developed transgenic technology in X. lae6is (Kroll and Amaya, 1996) will likely provide direct evidence for receptor function during metamorphosis, as was done for the type III deiodinase (Huang et al., 1999).
Acknowledgements We thank Kieu Pham for preparing the manuscript.
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