Identification of Direct Thyroid Hormone Response Genes Reveals the Earliest Gene Regulation Programs during Frog Metamorphosis

Thyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis. Thyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis. IntroductionThyroid hormone (T3) 2The abbreviations used are: T3thyroid hormoneTRthyroid receptorTREthyroid response elementRXR9-cis-retinoic acid receptorGOGene ontologyCHXcycloheximideqRTquantitative reverse transcriptionChIPchromatin immunoprecipitation. is critical for adult organ homeostasis and function and also essential for vertebrate development (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 15: 263-291Crossref PubMed Scopus (155) Google Scholar, 7.Franklyn J.A. Gammage M.D. Trends Endocrinol. Metab. 1996; 7: 50-54Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 8.Silva J.E. Thyroid. 1995; 5: 481-492Crossref PubMed Scopus (308) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar). T3 deficiency during development leads to severe developmental defects in mammals, including cretinism in human, which is characterized by severe short stature and mental retardation (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar). During early mammalian development, there is little T3 in the fetus, although some maternal T3 reaches the embryo. High levels of T3 are present only during the so-called postembryonic period, which spans from several months prior to birth to several months after birth in human (3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 10.Howdeshell K.L. Environ. Health Perspect. 2002; 110: 337-348Crossref PubMed Scopus (306) Google Scholar). This is a critical period of organ growth and maturation, and thus, not surprisingly, T3 deficiency during this period causes severe developmental defects (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar, 11.Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar, 12.Hsu J.H. Brent G.A. TEM. 1998; 9: 103-112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Interestingly, appropriate levels of maternal T3 are also important to ensure proper mammalian development (13.de Escobar G.M. Obregón M.J. del Rey F.E. Best Pract. Res. Clin. Endocrinol. Metab. 2004; 18: 225-248Crossref PubMed Scopus (431) Google Scholar, 14.de Escobar G.M. Obregón M.J. del Rey F.E. Public Health Nutrition. 2007; 10: 1554-1570Crossref PubMed Scopus (238) Google Scholar, 15.Anselmo J. Cao D. Karrison T. Weiss R.E. Refetoff S. JAMA. 2004; 292: 691-695Crossref PubMed Scopus (211) Google Scholar). These observations suggest that proper levels of T3 in both the mother and fetus are critical for mammalian development, which makes it difficult to separate the direct effects of T3 on the development of the fetus versus indirect effects through maternal influence. Furthermore, there are only a limited number of known direct T3 response genes in different model systems, and no systematic analysis has been carried out to isolate such genes in development. All these have hampered our understanding of how T3 regulates development in vivo.Amphibian metamorphosis is a postembryonic process that is also dependent on T3 (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 18.Buchholz D.R. Tomita A. Fu L. Paul B.D. Shi Y.B. Mol. Cell. Biol. 2004; 24: 9026-9037Crossref PubMed Scopus (112) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). During metamorphosis, different tissues have different fates (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 306: 20-33Crossref PubMed Scopus (305) Google Scholar). For example, the tail and gills undergo resorption, the brain, skin, intestine, and other visceral organs undergo remodeling while the limbs are generated de novo. All these changes are controlled by T3 in mostly organ-autonomous manner.T3 action is primarily mediated through thyroid receptors (TRs), which are transcription factors and are members of nuclear receptor superfamily (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar, 20.Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 21.Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). TRs bind to chromosomal sites in the promoter regions (known as thyroid response elements (TRE)) of direct response genes of T3. For T3-inducible genes, these binding sites are often composed of two direct repeats of the consensus sequence AGGTCA with a 4-bp spacer sequence. TR functions mainly as a heterodimer with 9-cis-retinoic acid receptor (RXR) at these TREs and represses and activates these genes by recruiting corepressor and in the absence of have a function model for TR function during metamorphosis Y.B. J. M.A. BioEssays. 1996; 18: PubMed Scopus Google Scholar, S. T. S. Shi Y.B. A. Comp. Biochem. Physiol. Biochem. Mol. Biol. PubMed Scopus Google Scholar). to this in the absence of direct target genes to ensure proper growth of the tadpoles and organ the of these target genes to metamorphosis. the by us and have shown that TRs are both necessary and sufficient to the effects of T3 in laevis (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). the and developmental changes in the different tissues are through gene by Furthermore, TRs have functions during metamorphosis. corepressor in tadpoles to and to metamorphosis B.D. Fu L. D.R. Shi Y.B. Mol. Cell. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, D.R. Paul B.D. Shi Y.B. Dev. 2007; PubMed Scopus Google Scholar, B.D. D.R. Fu L. Shi Y.B. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, B.D. D.R. Fu L. Shi Y.B. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the levels of also the of Paul B.D. T. Shi Y.B. Mol. Cell. Biol. 2009; PubMed Scopus Google and we and have the genes in different organs during both and metamorphosis (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 306: 20-33Crossref PubMed Scopus (305) Google Scholar, S. Shi Y.B. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. T. Shi Y.B. Dev. Biol. 2007; PubMed Scopus Google Scholar, Cai L. D.D. Dev. Biol. 2006; PubMed Scopus Google Scholar, L. D.D. Dev. Biol. 2007; PubMed Scopus Google Scholar). These have us with understanding of the gene changes and signaling pathways in metamorphosis. these genes are regulated although a have been shown to direct target genes of TRs J. Shi Y.B. J. Biol. 1994; Full Text PDF PubMed Google Scholar, A. 2002; PubMed Scopus Google Scholar, L. Tomita A. D.R. Shi Y.B. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Shi Y.B. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Y.B. Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect L.I. Tata J.R. Atkinson B.G. Academic Press, New York1996Google Scholar). Here we have of the in microarray analysis of gene in laevis and in the Xenopus to identify direct target genes of which are in the effects of T3 in metamorphosis. This is in by the that laevis and undergo metamorphosis. TR and genes are regulated and function in both during development Shi Y.B. PubMed Scopus Google Scholar). the regulation of immediate early, direct TR target genes by T3 of new protein to identify these direct we laevis tadpoles with T3 to induce metamorphosis and protein synthesis to the synthesis of new We the T3 response genes by laevis Gene ontology analysis revealed that these genes are enriched in categories important for the earliest of during metamorphosis. these genes are regulated by TR at the transcriptional we carried out analysis of the genes in by in DNA binding These showed that of the genes functional TREs in and their More importantly, we that TR bound to these TREs in tadpoles and regulated their promoters in vivo. our not only direct TR target genes in but also revealed a number of signaling pathways that are regulated by T3 as the step regulates developmental processes and organ function and in different vertebrate of T3 to some of the effects of T3 Thyroid. 1996; PubMed Scopus Google TR is to the of T3 action through transcriptional The immediate early, direct target genes of TR critical to the effects of T3. a number of have been carried out to identify T3 response genes in different the genes from such both immediate early, direct target genes of TR as as T3 response Amphibian metamorphosis is model to study T3 action in development of on T3 and the ability to easily this process of maternal effects as in (3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). we have the of two microarray analysis in laevis and sequence in to not only identify immediate early, direct target genes of T3 but also provide evidence to their regulation by TR in tadpoles in vivo. Furthermore, our gene ontology analysis has revealed interesting pathways induced by T3 at the earliest of the microarray analysis in laevis 188 genes up-regulated and 249 genes down-regulated by T3 in the absence of new protein Interestingly, of these genes not as T3 response genes in the absence of during the the to some the some This have the of the changes for some genes with the of T3 regulation the from a regulated gene to a gene and in the of some of the genes at levels and thus not with on the microarray in the absence of by the by with as some of the genes regulated by T3 response genes and thus not regulated by T3 in the of some genes with a of regulation by T3 the in the T3 T3 to some it is that of the genes regulated by T3 in the of are T3 response genes during is also by our in a of laevis the of We have shown that TR and genes are regulated and function in and laevis Shi Y.B. PubMed Scopus Google Scholar). of the a direct TR target genes in laevis that have been in are regulated by T3 in Shi Y.B. PubMed Scopus Google Scholar). A. D. and Our also that the genes of the T3 response genes in laevis are regulated in the that the gene regulation is and our showed that of the genes with laevis and have TREs of their transcription showed of the genes has at of binding to the heterodimer in More importantly, our in has shown that TR is bound to the TREs of target genes in tadpoles and that the binding to some of the TREs T3 that in the transcription we have further that TR regulates the promoters of the genes in in the of not of the genes are regulated by TR during Xenopus development. These findings allowed us for the to a Xenopus consensus on a number of The consensus and the to for TREs in other TR target genes by of the International on for Molecular Biology. Press, Scholar). in this is the identification of direct target genes of T3 in different which are important to that we are microarray analysis showed that only a of genes regulated by T3 in the absence of is that the genes regulated by T3 both immediate early, direct T3 response genes, which are T3 response genes, and T3 response that the functions of the genes regulated by T3 in the absence of and in the of also only our categories analysis revealed that there are a number of categories the genes induced by T3 and induced by T3 in the of the other there are categories the down-regulated genes by T3 and by T3 in the of with the down-regulated genes the two direct T3 response genes by T3 in the of are enriched in categories to transcriptional such as transcription factors and of these transcription factors have been as direct of such as and J. Shi Y.B. J. Biol. 1994; Full Text PDF PubMed Google Scholar, A. 2002; PubMed Scopus Google Scholar, A. S. Shi Y.B. Dev. PubMed Scopus Google Scholar). Interestingly, categories to transcriptional regulation are not enriched in the genes regulated by T3 of the of response genes in this These the that a transcription gene regulation important in metamorphosis and suggest that the earliest members of the gene pathways to changes a of transcription enriched in the direct T3 target genes is and This that the remodeling of the and other protein are important early signaling induced by T3 during metamorphosis. of the T3 direct response genes during metamorphosis is L. Tomita A. D.R. Shi Y.B. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Y.B. D.D. J. Biol. 1993; Full Text PDF PubMed Google Scholar, D.D. J. Biol. 1993; Full Text PDF PubMed Google Scholar, D. Shi Y.B. Dev. Biol. 1995; PubMed Scopus Google which has been shown to essential for during metamorphosis L. A. D.R. T. Shi Y.B. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Fu L. Shi Y.B. J. Biol. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. S. S. Shi Y.B. J. Biol. PubMed Scopus Google Scholar, L. D. Shi Y.B. Mol. Dev. 2002; PubMed Scopus Google Scholar). signaling processes through the action of are important early and during our analysis revealed the of categories to and DNA in the genes induced by T3 by T3 in the of Interestingly, categories enriched in the direct target genes, DNA categories enriched in the genes induced by T3 These suggest in the absence of protein T3 induce The direct target genes by are not sufficient for DNA but the to the This is by the of genes but the absence of DNA genes, the genes in S. 2006; 5: PubMed Scopus Google in the direct T3 target genes and the absence of the to and in the T3 response genes, and their in the genes induced by T3 suggest the that genes are to it of importance in the to the function of the direct target genes to these IntroductionThyroid hormone (T3) 2The abbreviations used are: T3thyroid hormoneTRthyroid receptorTREthyroid response elementRXR9-cis-retinoic acid receptorGOGene ontologyCHXcycloheximideqRTquantitative reverse transcriptionChIPchromatin immunoprecipitation. is critical for adult organ homeostasis and function and also essential for vertebrate development (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 15: 263-291Crossref PubMed Scopus (155) Google Scholar, 7.Franklyn J.A. Gammage M.D. Trends Endocrinol. Metab. 1996; 7: 50-54Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 8.Silva J.E. Thyroid. 1995; 5: 481-492Crossref PubMed Scopus (308) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar). T3 deficiency during development leads to severe developmental defects in mammals, including cretinism in human, which is characterized by severe short stature and mental retardation (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar). During early mammalian development, there is little T3 in the fetus, although some maternal T3 reaches the embryo. High levels of T3 are present only during the so-called postembryonic period, which spans from several months prior to birth to several months after birth in human (3.Tata J.R. BioEssays. 1993; 15: 239-248Crossref PubMed Scopus (209) Google Scholar, 10.Howdeshell K.L. Environ. Health Perspect. 2002; 110: 337-348Crossref PubMed Scopus (306) Google Scholar). This is a critical period of organ growth and maturation, and thus, not surprisingly, T3 deficiency during this period causes severe developmental defects (5.Hetzel B.S. The Story of Iodine Deficiency: An International Challenge in Nutrition. Oxford University Press, Oxford1989Google Scholar, 11.Porterfield S.P. Hendrich C.E. Endocr. Rev. 1993; 14: 94-106PubMed Google Scholar, 12.Hsu J.H. Brent G.A. TEM. 1998; 9: 103-112Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Interestingly, appropriate levels of maternal T3 are also important to ensure proper mammalian development (13.de Escobar G.M. Obregón M.J. del Rey F.E. Best Pract. Res. Clin. Endocrinol. Metab. 2004; 18: 225-248Crossref PubMed Scopus (431) Google Scholar, 14.de Escobar G.M. Obregón M.J. del Rey F.E. Public Health Nutrition. 2007; 10: 1554-1570Crossref PubMed Scopus (238) Google Scholar, 15.Anselmo J. Cao D. Karrison T. Weiss R.E. Refetoff S. JAMA. 2004; 292: 691-695Crossref PubMed Scopus (211) Google Scholar). These observations suggest that proper levels of T3 in both the mother and fetus are critical for mammalian development, which makes it difficult to separate the direct effects of T3 on the development of the fetus versus indirect effects through maternal influence. Furthermore, there are only a limited number of known direct T3 response genes in different model systems, and no systematic analysis has been carried out to isolate such genes in development. All these have hampered our understanding of how T3 regulates development in vivo.Amphibian metamorphosis is a postembryonic process that is also dependent on T3 (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 17.Brown D.D. Cai L. Dev. Biol. 2007; 306: 20-33Crossref PubMed Scopus (305) Google Scholar, 18.Buchholz D.R. Tomita A. Fu L. Paul B.D. Shi Y.B. Mol. Cell. Biol. 2004; 24: 9026-9037Crossref PubMed Scopus (112) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). During metamorphosis, different tissues have different fates (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 306: 20-33Crossref PubMed Scopus (305) Google Scholar). For example, the tail and gills undergo resorption, the brain, skin, intestine, and other visceral organs undergo remodeling while the limbs are generated de novo. All these changes are controlled by T3 in mostly organ-autonomous manner.T3 action is primarily mediated through thyroid receptors (TRs), which are transcription factors and are members of nuclear receptor superfamily (1.Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 2.Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1501) Google Scholar, 9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar, 20.Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 21.Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). TRs bind to chromosomal sites in the promoter regions (known as thyroid response elements (TRE)) of direct response genes of T3. For T3-inducible genes, these binding sites are often composed of two direct repeats of the consensus sequence AGGTCA with a 4-bp spacer sequence. TR functions mainly as a heterodimer with 9-cis-retinoic acid receptor (RXR) at these TREs and represses and activates these genes by recruiting corepressor and in the absence of have a function model for TR function during metamorphosis Y.B. J. M.A. BioEssays. 1996; 18: PubMed Scopus Google Scholar, S. T. S. Shi Y.B. A. Comp. Biochem. Physiol. Biochem. Mol. Biol. PubMed Scopus Google Scholar). to this in the absence of direct target genes to ensure proper growth of the tadpoles and organ the of these target genes to metamorphosis. the by us and have shown that TRs are both necessary and sufficient to the effects of T3 in laevis (9.Shi Y.B. Thyroid. 2009; 19: 987-999Crossref PubMed Scopus (73) Google Scholar, 19.Buchholz D.R. Paul B.D. Fu L. Shi Y.B. Gen. Comp. Endocrinol. 2006; 145: 1-19Crossref PubMed Scopus (171) Google Scholar). the and developmental changes in the different tissues are through gene by Furthermore, TRs have functions during metamorphosis. corepressor in tadpoles to and to metamorphosis B.D. Fu L. D.R. Shi Y.B. Mol. Cell. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, D.R. Paul B.D. Shi Y.B. Dev. 2007; PubMed Scopus Google Scholar, B.D. D.R. Fu L. Shi Y.B. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, B.D. D.R. Fu L. Shi Y.B. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the levels of also the of Paul B.D. T. Shi Y.B. Mol. Cell. Biol. 2009; PubMed Scopus Google and we and have the genes in different organs during both and metamorphosis (4.Shi Y.B. Amphibian Metamorphosis: From Morphology to Molecular Biology. John Wiley 306: 20-33Crossref PubMed Scopus (305) Google Scholar, S. Shi Y.B. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. T. Shi Y.B. Dev. Biol. 2007; PubMed Scopus Google Scholar, Cai L. D.D. Dev. Biol. 2006; PubMed Scopus Google Scholar, L. D.D. Dev. Biol. 2007; PubMed Scopus Google Scholar). These have us with understanding of the gene changes and signaling pathways in metamorphosis. these genes are regulated although a have been shown to direct target genes of TRs J. Shi Y.B. J. Biol. 1994; Full Text PDF PubMed Google Scholar, A. 2002; PubMed Scopus Google Scholar, L. Tomita A. D.R. Shi Y.B. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Shi Y.B. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Y.B. Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect L.I. Tata J.R. Atkinson B.G. Academic Press, New York1996Google Scholar). Here we have of the in microarray analysis of gene in laevis and in the Xenopus to identify direct target genes of which are in the effects of T3 in metamorphosis. This is in by the that laevis and undergo metamorphosis. TR and genes are regulated and function in both during development Shi Y.B. PubMed Scopus Google Scholar). the regulation of immediate early, direct TR target genes by T3 of new protein to identify these direct we laevis tadpoles with T3 to induce metamorphosis and protein synthesis to the synthesis of new We the T3 response genes by laevis Gene ontology analysis revealed that these genes are enriched in categories important for the earliest of during metamorphosis. these genes are regulated by TR at the transcriptional we carried out analysis of the genes in by in DNA binding These showed that of the genes functional TREs in and their More importantly, we that TR bound to these TREs in tadpoles and regulated their promoters in vivo. our not only direct TR target genes in but also revealed a number of signaling pathways that are regulated by T3 as the step metamorphosis.