Control of precisely timed gene expression during notochord development by Brachyury

PROJECT SUMMARY/ABSTRACT Precise temporal regulation of stage-specific gene expression is fundamental for the proper development of all organisms. Different molecular mechanisms that ensure the regionalized activation of gene expression, such as activator gradients or localized transcriptional repressors, have been elucidated in

remarkable detail; however, the structural features of cis-regulatory sequences that enable transcription factors to achieve the timely deployment of their downstream genes remain mostly unclear. The evolutionarily conserved transcription factor Brachyury provides an example of a regulator that controls the spatial and

temporal expression of a large number of genes, many of which are required for the development of the notochord. The notochord is the main feature shared by all chordate embryos, from tunicates to humans. During the early embryogenesis of all chordates, the notochord provides support and patterning signals to the

developing body and is indispensable for patterning neural tube, endoderm, paraxial mesoderm, and the structures that they will form. In the tunicate Ciona, an invertebrate chordate, Brachyury is exclusively expressed in notochord cells, where it controls the expression of hundreds of genes. Even though Ciona Brachyury (Ci-Bra) is steadily

transcribed and transported to the nuclei of the notochord cells throughout development, the genes downstream of Ci-Bra exhibit a distinctive, temporally staggered onset. In an effort to identify the cis-regulatory strategies responsible for the precise temporal regulation of notochord gene expression by Ci-Bra, we have

characterized several cis-regulatory regions (aka enhancers) that control the notochord expression of Ci-Bra- downstream genes characterized by different temporal onsets. These studies have led us to formulate a working hypothesis that explains how Brachyury controls its early-, middle- and late-onset target genes. This

hypothesis will be tested through the following experimental approaches: the elucidation of the cis-regulatory principles underlying the sequential activation of early-onset and middle-onset notochord genes directly controlled by Ci-Bra (Aim 1); the functional analysis of two transcription factors that activate expression of late-

onset notochord genes (Aim 2); the exploration of the role of transcriptional repressors in notochord development (Aim 3). These studies will shed light on the modalities employed by a pivotal transcription factor to set the temporal context for developmental processes of widespread relevance, such as convergent extension and

extracellular matrix secretion, and will provide the mechanistic framework that is needed to diagnose and prevent malformations and tumorigenesis associated with the multifaceted transcription factor Brachyury.