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The regions of expression of Ripply1 and Ripply2, presumptive transcriptional corepressors, overlap at the presomitic mesoderm during somitogenesis in mouse and zebrafish. Ripply1 is required for somite segmentation in zebrafish, but the developmental role of Ripply2 remains unclear in both species. Here, we generated Ripply2 knock-out mice to investigate the role of Ripply2. Defects in segmentation of the axial skeleton were observed in the homozygous mutant mice. Moreover, somite segmentation and expression of Notch2 and Uncx4.1 were disrupted. These findings indicate that Ripply2 is involved in somite segmentation and establishment of rostrocaudal polarity.

Amphibians have been used for over a century as experimental animals. In the field of developmental biology in particular, much knowledge has been accumulated from studies on amphibians, mainly because they are easy to observe and handle. Xenopus laevis is one of the most intensely investigated amphibians in developmental biology at the molecular level. Thus, Xenopus is highly suitable for studies on the mechanisms of organ differentiation from not only a single fertilized egg, as in normal development, but also from undifferentiated cells, as in the case of in vitro organogenesis. Based on the established in vitro organogenesis methods, we have identified many genes that are indispensable for normal development in various organs. These experimental systems are useful for investigations of embryonic development and for advancing regenerative medicine. Developmental Dynamics 238:1309 -1320, 2009.

Non-universal codons are known to be used in the mitochondrial (mt) 3 genetic code (1-6). DNA sequencing of proteincoding genes in various metazoan mtDNAs followed by comparison of the deduced amino acid sequences with those of other species revealed amino acid assignment deviations for the codons UGA, AUA, AGR, and AAA. The termination codon UGA in the universal genetic code specifies Trp in the mitochondria of all species investigated so far (1, 2). The AUA codon for Ile changes to specify Met in most metazoa except echinoderms, planaria, and coelenterates (2-4). AGR codons are used for Ser in most invertebrates, for Gly in urochordates, and for termination in vertebrates (7-10). The AAA codon specifies Asn in echinoderms (7) and platyhelminths (11).Our group previously reported that the mitochondrial decoding system of ascidian Halocynthia roretzi employs four non-universal genetic codes: UGA for Trp, AUA for Met and AGR for Gly (Table 1) (9, 12). Sequence analysis of H. roretzi mtDNA identified three tDNAs responsible for these non-universal codons. Each of these tDNAs contains thymidine (T) at the first (wobble) position of their anticodons (Table 1). Because an unmodified U at the wobble position can decode any of the four bases at the third letter of the codon, according to the mitochondrial four-way wobble rule (5, 13), the uridines of these tRNAs at the wobble position must be modified in order to decipher their respective non-universal genetic codes accurately. To elucidate the molecular mechanism of the decoding system, it is necessary to determine the chemical structure of the anticodon wobble base in H. roretzi mt tRNAs. We previously reported 5-carboxymethylaminomethyluridine at the wobble position of mt tRNA Met (AUR) from H. roretzi (14). However, the nucleotide identification was inaccurate because it was analyzed by conventional two-dimensional TLC, and the modification was determined based on a comparison of its rate of flow value with that of known modified nucleotides (15). In addition, we also reported that H. roretzi mt tRNA Gly -(AGR) has an unidentified modified U at the wobble position (16). We describe here the chemical structures of the wobble bases in mt tRNAs for non-universal genetic codes in the H. roretzi mitochondrial decoding system and discuss evolutionary insights into the role of wobble modification in genetic code alteration. EXPERIMENTAL PROCEDURESIsolation of mt tRNAs-Two hundred grams of the commercially available edible muscle of an ascidian, H. roretzi, was minced, and the tRNA fraction was obtained by phenol extraction as described previously (17). Two thousand A 260 units of crude tRNA were separated by the DEAE-cellulose column * This work was supported by grants-in-aid for scientific research (C) from The Japan Society for the Promotion of Science (to K. W.) and by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan (to Takeo Suzuki and Tsutomu Suzuki). □ S The on-line version of this article...

T-box factor, Tbx6, is a prerequisite for somite segmentation in vertebrates. We recently identified a negative regulator of Tbx6, Bowline, which represses the expression of genes involved in somite segmentation by suppressing the transcriptional activity of Tbx6. According to this function, bowline gene expression is restricted to the most anterior presomitic mesoderm where the somite segmentation program terminates, although it remains unclear how bowline expression is activated. To address this, we investigated the cis-regulatory region of bowline. Measuring luciferase activity driven by the bowline promoter, we found that Tbx6, Thylacine1, and E47 synergistically activate bowline expression in vitro. We also found that Tbx6, Thylacine1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. Our findings indicated that besides being a negative regulator of Tbx6, bowline itself is also regulated by Tbx6, suggesting the negative feedback loop of Tbx6-Bowline in the termination step of somite segmentation.