The Role of T-box Proteins in Vertebrate Germ Layer Formation and Patterning

All of the tissues in triploblastic organisms, with the exception of the germ cells, arise from the three germ layers, ectoderm, mesoderm and the endoderm. The identification of the genes that underlie the differentiation of these layers is crucial to our understanding of development. T-box family proteins are DNA-binding transcriptional regulators that play important roles during germ layer formation in the early vertebrate embryo. Well-characterized members of this family, including the transcriptional activators Brachyury and VegT, are essential for the proper formation of mesoderm and endoderm, respectively. To date, T-box proteins have not been shown to play a role in the promotion of the third primary germ layer, ectoderm. In this study, we have identified two T-box proteins, Tbx2 and Tbx3, as important players in the development of the vertebrate embryo with majority of our work focused on Tbx2. Our studies indicate that the T-box factor Tbx2 is both sufficient and necessary for ectodermal differentiation in the frog Xenopus laevis. Tbx2 is expressed zygotically in the presumptive ectoderm, during blastula and gastrula stages. Ectopic expression of Tbx2 represses mesoderm and endoderm, while loss of Tbx2 leads to inappropriate expression of mesoderm- and endoderm-specific genes in the region fated to give rise to ectoderm. Misexpression of Tbx2 also promotes neural tissue in animal cap explants, suggesting that Tbx2 plays a role in both the establishment of ectodermal fate and its dorsoventral patterning. Our studies demonstrate that Tbx2 functions as a transcriptional repressor during germ layer formation, and suggest that this activity is mediated in part through repression of target genes that are stimulated, in the mesendoderm, by activating T-box proteins. In addition to Tbx2, we also identified Tbx3, another T-box transcription factor, whose expression begins early in development. Our data indicate that Tbx3 is expressed in the animal pole of Xenopus laevis embryos, where it functions to repress mesodermal and endodermal genes. Taken together, our results point to a critical role for T-box proteins in limiting the potency of blastula-stage progenitor cells during vertebrate germ layer differentiation

Measuring the Rate of Conjugal Plasmid Transfer in a Bacterial Population Using Quantitative PCR

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F+) to a recipient (F−) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F+ transconjugants and the recovery time between successive conjugation events of the F+ donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10−10 (cells/mL · min)−1. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F+ populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome