The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses. Received: 14 July 1999 / Accepted: 4 September 1999     

Neuronal function of Tbx20 conserved from nematodes to vertebrates

The Tbx20 orthologue, mab-9, is required for development of the Caenorhabditis elegans hindgut, whereas several vertebrate Tbx20 genes promote heart development. Here we show that Tbx20 orthologues also have a role in motor neuron development that is conserved between invertebrates and vertebrates. mab-9 mutants exhibit guidance defects in dorsally projecting axons from motor neurons located in the ventral nerve cord. Danio rerio (Zebrafish) tbx20 morphants show defects in the migration patterns of motor neuron soma of the facial and trigeminal motor neuron groups. Human TBX20 is expressed in motor neurons in the developing hindbrain of human embryos and we show that human TBX20 can substitute for zebrafish tbx20 in promoting cranial motor neuron migration. mab-9 is also partially able to rescue the zebrafish migration defect, whereas other vertebrate T-box genes cannot. Conversely we show that the human TBX20 T-box domain can rescue motor neuron defects in C. elegans. These data suggest the functional equivalence of Tbx20 orthologues in regulating the development of specific motor neuron groups. We also demonstrate the functional equivalence of human and C. elegans Tbx20 T-box domains for regulating male tail development in the nematode even though these genes play highly diverged roles in organogenesis.     

Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis

Members of the T-box family of proteins play a fundamental role in patterning the developing vertebrate heart; however, the precise cellular requirements for any one family member and the mechanism by which individual T-box genes function remains largely unknown. In this study, we have investigated the cellular and molecular relationship between two T-box genes, Tbx5 and Tbx20. We demonstrate that blocking Tbx5 or Tbx20 produces phenotypes that display a high degree of similarity, as judged by overall gross morphology, molecular marker analysis and cardiac physiology, implying that the two genes are required for and have non-redundant functions in early heart development. In addition, we demonstrate that although co-expressed, Tbx5 and Tbx20 are not dependent on the expression of one another, but rather have a synergistic role during early heart development. Consistent with this proposal, we show that TBX5 and TBX20 can physically interact and map the interaction domains, and we show a cellular interaction for the two proteins in cardiac development, thus providing the first evidence for direct interaction between members of the T-box gene family.     

UV-mediated regulation of the anti-senescence factor Tbx2

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.     

Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family

 We report on a new zebrafish T-box-containing gene, tbx16. It encodes a message that is first detected throughout the blastoderm soon after the initiation of zygotic gene expression. Following gastrulation, expression becomes restricted to paraxial mesoderm and later primarily to the developing tail bud. To gain an evolutionary prospective on the potential function of this gene, we have analyzed its phylogenetic relationships to known T-box genes from other species. Zebrafish tbx16 is likely orthologous to the chicken Tbx6L and Xenopus Xombi/Antipodean/Brat/VegT genes. Our analysis also shows that zebrafish tbx6 and mouse Tbx6 genes are paralogous to zebrafish tbx16. We present evidence which argues, that despite the same name and similar expression, zebrafish tbx6 and mouse Tbx6 genes are not orthologous to each other but instead represent relatively distant paralogs. The expression patterns of all genes are discussed in the light of their evolutionary relationships. Received: 27 November 1997 / Accepted: 27 January 1998