UNLIGANDED THYROID HORMONE RECEPTOR ΑCONTROLS DEVELOPMENTAL TIMING IN XENOPUS TROPICALIS. Earlier studies have shown that during development, of the two TR genes (TRαand TRβ), TRαexpression appears earlier than TH synthesis and secretion into the plasma. This and the ability of TRs to regulate gene expression both in the presence and absence of TH have implicated a role of unliganded TR during vertebrate development. On the other hand, it has been difficult to study the role of unliganded TR during development in mammals due to the difficulty to manipulate the uterus-enclosed, late stage embryos. Amphibian development offers an excellent opportunity to address this question. We have designed TALENs (transcriptional activator like effector nucleases) to mutate the TRαgene in Xenopus tropicalis. We show that knockdown of TRαenhances tadpole growth in premetamorphic tadpoles, in part because of increased growth hormone gene expression. More importantly, the knockdown also accelerates animal development, with the knockdown animals initiating metamorphosis at a younger age and with a smaller body size. On the other hand, such tadpoles are resistant to exogenous TH and have delayed natural metamorphosis. Thus, our studies have not only directly demonstrated a critical role of endogenous TRαin mediating the metamorphic effect of TH but also revealed novel functions for unliganded TRαduring early development, that is, regulating both the tadpole growth rate and the timing of metamorphosis. TH-INDUCED HISTONE METHYLTRANSFERASE DOT1L IS REQUIRED FOR POSTEMBRYONIC DEVELOPMENT BUT DISPENSABLE FOR XENOPUS EMBRYOGENESIS. In our study of gene regulation by TR during metamorphosis, we discovered that the levels of the methylation of histone H3K79 at TH target genes were upregulated during natural and TH-induced metamorphosis. Interestingly, the histone methyltransferase Dot1L is the only known enzyme capable of methylating histone H3K79, suggesting that Dot1L may function as a TR coactivator. In addition, we have shown that Dot1L is directly activated by TH via TR at the transcription level. To investigate the role of Dot1L during metamorphosis, we have generated a Dot1L-specific TALEN nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We have shown that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis since normal feeding tadpoles were formed. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings and the lack of maternal Dot1L expression suggest that Dot1L and H3K79 methylation are dispensable for embryogenesis but essential for tadpole growth and development prior to metamorphosing into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of two separate phases of vertebrate development with distinct requirements for epigenetic modifications. GLOBAL EXPRESSION PROFILING REVEALS GENETIC PROGRAMS UNDERLYING THE DEVELOPMENTAL DIVERGENCE BETWEEN MOUSE AND HUMAN EMBRYOGENESIS. Through a collaborative effort to study global gene regulation during vertebrate development, we have analyzed the developmental gene expression profiles in mouse. Mouse has served as an excellent model for studying human development and diseases. Advances in transgenic and knockout studies in mouse have dramatically strengthened the use of this model and significantly improved our understanding of gene function during development in the past few decades. On the other hand, little information is known about the gene regulatory networks governing the mouse organogenesis. Importantly, mouse and human development diverges during organogenesis. For instance, the mouse embryo is born around the end of organogenesis while in human the subsequent fetal period of ongoing growth and maturation of most organs spans more than 2/3 of human embryogenesis. To investigate the underlying molecular basis, we carried out a detailed analysis of the global gene expression profiles from egg to the end of organogenesis in mouse (5). Our studies revealed distinct temporal regulation patterns for genes belonging to different functional (Gene Ontology or GO) categories that support their roles during organogenesis. More importantly, comparative analyses identified both conserved and divergent gene regulation programs in mouse and human organogenesis, with the latter likely responsible for the developmental divergence between the two species, and further suggest a novel developmental strategy during vertebrate evolution. Given our earlier observation that genes function in a given process tends to be developmentally co-regulated during organogenesis, our microarray data should help to identify genes associated with mouse development and/or infer the developmental functions of unknown genes. In addition, our study might be useful for investigating the molecular basis of vertebrate evolution.
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