Interactions of T-box genes in the zebrafish mesoderm: TBx6 and No Tail

The T-box genes encode a family of transcription factors important for tissue differentiation and morphogenesis. Like the Hox genes, T-box genes are often expressed in overlapping patterns within developmental precursor populations. T-box gene mutations often affect only a subset of the cells that normally express the genes. I propose that, similar to the Hox genes, the co-expression of T-box genes allows for genetic interactions that may alter a T-box gene's effect within a portion of its expression domain. Support of this hypothesis comes from the analysis of interactions between no tail and tbx6, T-box genes that are co-expressed in the ventral mesoderm. These experiments are presented in this thesis. Three T-box genes are expressed in a nested array in the mesoderm of the zebrafish early gastrula. Their combined expression defines two domains of mesoderm with distinct developmental fates. no tail, tbx6 and spadetail are expressed in ventral mesoderm precursors that give rise to nonaxial tissues; no tail is the only known T-box gene expressed in the dorsal mesoderm precursors that give rise to notochord, an axial structure. During gastrulation, no tail expression is extinguished in the involuting ventral mesoderm but is maintained in the presumptive notochord in a manner that depends on continuous wildtype no tail function. I tested whether tbx6 might be responsible for inhibiting no tail function in the ventral mesoderm, leading to its expression being down-regulated in these cells. I found that one function of tbx6 appears to be to antagonize the function and potentially the expression of no tail in this cell population. Ectopic expression of tbx6 appears to mimic the effects of no tail loss of function mutations or ectopic expression of a known No Tail antagonist, a No Tail-Engrailed repressor chimera in several different assays. Ectopic expression of tbx6causes a deficit in notochord formation by 24 hours of development and suppresses MyoD expression in paraxial mesoderm. Both of these have been shown to be dependent on functional No Tail protein. Expression of a repressor form of the Tbx6 protein, a Thx6-Engrailed chimera, has similar effects on no tail function. In vitro binding studies and studies of competition between Tbx6 and No Tail indicate that the Tbx6 protein can compete with No Tail binding at specific sites and that Tbx6 fails to activate transcription at many of these sites. Importantly, I was able to show that Tbx6 and No Tail have different specificities for certain arrangements of binding site, opening the possibility that Tbx6 does more than simply antagonize No Tail. Theses studies clearly indicate that the prevailing view of T-domain transcription factors as simple activation factors needs to be rethought. Coexpression of T-box genes can potentially lead to complex interactions that control tissue differentiation.