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

Abstract

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)2 is critical for adult organ homeostasis and function and also essential for vertebrate development (1-9). 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). 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, 10). This is a critical period of organ growth and maturation, and thus, not surprisingly, T3 deficiency during this period causes severe developmental defects (5, 11, 12). Interestingly, appropriate levels of maternal T3 are also important to ensure proper mammalian development (13-15). 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, 16). Studies in anurans such as Xenopus laevis in the past century have shown t...

Xenopus tropicalis: An Ideal Experimental Animal in Amphibia

“…Information on X. tropicalis in the bioinformatic search for transposable elements [17] and binding sites of transcriptional factors such as thyroid hormone receptor [5,8], and in the genome-wide comparisons with mammalian sequences to predict the cis-regulatory elements, for example, conserved noncoding elements [29].…”

Adult-type myogenesis of the frog Xenopus laevis specifically suppressed by notochord cells but promoted by spinal cord cells in vitro

“…One is the deletion of larval organs such as tadpole tail (Kerr et al 1974), the second is the formation of new organs such as fore-and hindlimbs (Muntz 1975), and the third is the larval-to-adult conversion of organs such as dorsal muscles (Nishikawa and Hayashi 1994;Shimizu-Nishikawa et al 2002) and intestinal epithelium (Ishizuya-Oka and Ueda 1996;Ishizuya-Oka et al 2001). These events are triggered by the action of thyroid hormone, triiodothyronine (T 3 ; Das et al 2009;Mukhi et al 2010) and modulated by cell-to-cell (Schreiber et al 2009) and cell-to-extracellular matrix interactions (Ishizuya-Oka et al 2000). Amphibian metamorphosis thus serves as an attractive system for studying organ remodeling during development.…”

Thyroid Hormone Activates Protein Arginine Methyltransferase 1 Expression by Directly Inducing c-Myc Transcription during Xenopus Intestinal Stem Cell Development

“…41 and data not shown), suggesting conserved transcriptional regulation mechanisms in X. laevis and X. tropicalis, just like all other genes that we and others have studied during metamorphosis (58,59,61,66,67). Alignment of PRMT1 genomic sequences of X. laevis and X. tropicalis PRMT1 revealed several conserved segments with over 75% identity in the upstream regions and the first intron (Fig.…”

“…The KF330 and KF331 primers for PRMT1 were used for SYBR Green PCR. The primers for EF1␣ and c-Myc were as published (61,62).…”

“…To investigate this possibility, we took advantage of the conservation in gene regulation during metamorphosis between X. laevis and X. tropicalis, as the regulation of gene expression during metamorphosis is conserved between the two species for all genes analyzed so far (58,59,61,66,67). Thus, we searched for the putative TRE in the X. tropicalis c-Myc gene.…”