RNA interference approach to dissect roles of notochord regulators

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We have recently developed a novel method to control the production of the transcription factor Brachyury/T in cells by using a lentivirus that produces an shRNA directed against Brachyury and, when introduced into cells (or animals), causes the loss of Brachyury/T. This novel tool will enable us to alter Brachyury function during mouse development to understand how it regulates cell fate and behavior, and then to determine whether its function is necessary for notochord cell survival. It has been hypothesized that Brachyury plays a central role in the formation of cancers that arise from notochord (chordomas), and this tool may also provide the impetus for devising new therapies for these very difficult to treat cancers. We have introduced this shRNA construct into transgenic mice and employed Cre-lox technology to enable us to selectively activate its expression in different embryonic tissues at specific times. We have shown that early activation of this knock-down construct in mouse embryos that are heterozygous for the Brachyury null mutation (T+/-) reproduces the null embryonic lethal phenotype with loss of the body axis caudal to the forelimb level. The knock-down on a T wild-type background produces a weaker phenotype with loss of the body axis at the hindlimb bud level. Now that we have shown that Brachyury can be effectively removed from cells and animals using this approach, we are using it to study the normal function of Brachyury in regulating growth and cell fate during notochord formation, primitive streak migration to produce the main body mesoderm, and in limb development. The activation of Brachyury expression has been proposed to be essential for the genesis of chordomas, cancers that arise in notochord remnants. The gene knock-down tools we have developed to study Brachyury function will help to test this hypothesis and unravel the possible mechanisms by which this gene may promote tumor formation. We are also using genetic strategies in chick to analyze the function of Gnot (noto) and Brachyury and have discovered that these two factors may play complementary roles in regulating cell proliferation in the embryo. Unlike mouse, in chick Gnot (Noto) is expressed in limb, as well as in the notochord. Interestingly, we previously found that Brachyury is expressed in limb in chick and in mouse embryos, and based on genetic studies, we proposed that Brachyury plays a positive role in limb bud outgrowth be regulating signaling from the apical ectodermal ridge to promote limb bud outgrowth. Using a mis-expression approach in chick embryos, we have also gained new insights on the function of another transcription factor that regulates notochord formation, Gnot/noto. In contrast to Brachyury/T, which positively regulates limb growth, Gnot/noto inhibits growth. This appears to also occur by modulating the apical ectodermal ridge function to lower the amount of FGF signaling from this specialized ectoderm. We are now analyzing Gnot (Noto) effects on global gene expression using cDNA microarrays to identify other downstream targets of Gnot/noto (such as direct targets in mesoderm). These studies will uncover the functional mechanism of Gnot/noto effects on limb development, and may also give new insights on its role in notochord development, which could help develop new potential therapeutic interventions for chordoma.