ADAPTING TALEN AND CRISPR TECHNOLOGIES FOR STUDYING GENE FUNCTION DURING METAMORPHOSIS. Gene knockout and knockdown had been very difficult if not impossible in Xenopus tadpoles. We have now successfully adapted the TALEN and CRISPR technologies to knockdown endogenous genes in Xenopus tadpoles by microinjecting TALEN and CRISPR RNAs into fertilized eggs. Importantly, we showed that we could achieve very high efficiencies of mutations in the target genes in the resulting premetamorphic tadpoles, making it possible to carry out functional studies directly on these tadpoles, the so called F0 genetic studies. This eliminates the need for F1 or F2 generation animals, thus allowing much faster determination of gene function. This has enabled us to investigate the roles of TRα and a histone methyltransferase, a likely TR-coactivator as described below. DISCOVERED A NOVEL FUNCTION FOR TRα DURING DEVELOPMENT. We developed a TALEN that could mutate TRα gene in X. tropicalis with over 90% efficiency by injecting TALEN mRNAs into fertilized eggs, making it possible to analyze the role of TRα in the resulting F0 animals. Consistent with our dual function model for TR, we observed that knocking down TRα accelerated animal development, with the knockdown animals reaching the onset of metamorphosis earlier. On the other hand, they were resistant to exogenous T3 treatment and had delayed natural metamorphosis. Thus, our studies directly demonstrated a critical role of endogenous TRα both in mediating the metamorphic effect of T3 during metamorphosis and in preventing precocious initiation of metamorphosis when T3 is absent. Surprisingly, we also found that TRα knockdown enhanced tadpole growth in premetamorphic tadpoles. This novel function of unliganded TRα (as little T3 is present during premetamorphosis) appears to be due to increased growth hormone gene expression. Further analyses suggest that the two functions of unliganded TRα during metamorphosis, i.e., regulating tadpole growth rate and the time to reach the onset of metamorphosis, are independent of each other. While this is the first direct evidence for a critical role of unliganded TR in vertebrate development, TRα knockout studies suggest that unliganded TRα is important for postembryonic regulation of heart rate and gene expression. Similarly, deleting the gene encoding the T3-inactivating enzyme, type 3 deiodinase, causes auditory defects, arguing for the importance of maintaining a very low level of T3, which would lead to more unliganded TR, for cochlear development. Thus unliganded TR may be important for postembryonic development in mammals as well. IDENTIFIED THE HISTONE METHYLTRANSFERASE DOT1L AS A DIRECT TARGET GENE OF TR AND HAVING AN ESSENTIAL ROLE IN PREMETAMORPHIC TADPOLE GROWTH. We have earlier identified Dot1L, the only HMT capable of methylating histone H3K79 in vitro, as a direct target gene of T3. Interestingly, the level of H3K79 methylation is strongly increased by T3 at TR target genes, suggesting that Dot1L is upregulated by TR and in turn functions as a TR coactivator. To investigate Dot1L function, we generated a Dot1L-specific TALEN that was extremely efficient in mutating Dot1L when expressed in X. tropicalis fertilized eggs, creating animals with almost no Dot1L and little H3K79 methylation. We observed that Dot1L knockdown had no apparent effect on embryogenesis. On the other hand, it severely retarded tadpole growth and led to tadpole lethality before metamorphosis. These findings suggest that Dot1L and H3K79 methylation plays an important role for tadpole growth and development prior to metamorphosis. Our analyses further revealed interesting similarities between Xenopus and mouse development and suggest the existence of two separate phases of vertebrate development with distinct requirements for epigenetic modifications.
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