Brachyury, Foxa2 and the cis-Regulatory Origins of the Notochord

Abstract

A main challenge of modern biology is to understand how specific constellations of genes are activated to differentiate cells and give rise to distinct tissues. This study focuses on elucidating how gene expression is initiated in the notochord, an axial structure that provides support and patterning signals to embryos of humans and all other chordates. Although numerous notochord genes have been identified, the regulatory DNAs that orchestrate development and propel evolution of this structure by eliciting notochord gene expression remain mostly uncharted, and the information on their configuration and recurrence is still quite fragmentary. Here we used the simple chordate Ciona for a systematic analysis of notochord cis-regulatory modules (CRMs), and investigated their composition, architectural constraints, predictive ability and evolutionary conservation. We found that most Ciona notochord CRMs relied upon variable combinations of binding sites for the transcription factors Brachyury and/or Foxa2, which can act either synergistically or independently from one another. Notably, one of these CRMs contains a Brachyury binding site juxtaposed to an (AC) microsatellite, an unusual arrangement also found in Brachyury-bound regulatory regions in mouse. In contrast, different subsets of CRMs relied upon binding sites for transcription factors of widely diverse families. Surprisingly, we found that neither intra-genomic nor interspecific conservation of binding sites were reliably predictive hallmarks of notochord CRMs. We propose that rather than obeying a rigid sequence-based cis-regulatory code, most notochord CRMs are rather unique. Yet, this study uncovered essential elements recurrently used by divergent chordates as basic building blocks for notochord CRMs.

Brachyury controlsCionanotochord fate as part of a feedforward network and not as a unitary master regulator

“…If Foxa.a is an important co-regulator of notochord gene expression acting not just upstream of Bra but also in parallel, then FoxA binding motifs should be overrepresented in the enhancers of notochord enriched genes. (José-Edwards et al, 2015) previously found that several CRMs capable of driving notochord expression of a reporter at mid-tailbud frequently contained functionally important Bra and Fox binding sites, but did not address whether these or other binding motifs are statistically enriched across notochord genes overall. Transcription factor binding site (TFBS) enrichment analysis also provides an alternate means of predicting regulators that may have been overlooked based on complex or imperfectly annotated expression patterns.…”

“…Mnx's role is not clear, but it is less important than Foxa.a in misexpression assays. FoxA is known to be a key regulator of notochord fate in both vertebrates (Ang and Rossant, 1994;Weinstein et al, 1994) and tunicates (Imai et al, 2006;José-Edwards et al, 2015;Kumano et al, 2006), but the details of the regulatory networks connecting FoxA, Bra and downstream targets are not well understood in any model. We pursued several parallel strategies to clarify these relationships.…”

“…Brachyury misexpression in the endodermal, A-line neural and notochord lineages under the control of the Foxa.a enhancer does not typically give rise to a larger notochord with additional intercalated cells, but instead to a mass of cells with little morphological differentiation . Several notochord enriched genes have been identified that are not induced by ectopic Bra expression (José-Edwards et al, 2011;Kugler et al, 2008), and several notochord enhancers have been identified that drive early notochord expression via essential Fox binding sites but with no recognizable Bra sites (José-Edwards et al, 2015).…”

Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos

“…Yet, their embryos are virtually identical. This paradoxical situation represents an excellent case to study the evolution and diversification of developmental mechanisms, CRMs and DSD (12,14,(26)(27)(28)(29)(30)(31)(32)(33)(34).…”

Structural and functional significance of microsatellites

“…The (AT)8(N)12GT(AT)7 configuration of microsatellite found in the Hypersensitive site of the structure is associated with a special form of sickle cells -Tunisian βs chromosomes [118]. In some gene clusters the changes in microsatellite structure drastically modify the transcription factor binding and gene switching [119]. The marker D12S96 is localized 5.653 cM downstream the vitamin D receptor (VDR) gene.…”