Brachyury is necessary for mesoderm formation but not posterior neural identity.

<p>(A) Schematic of the conditions used for mesoderm differentiation. (B) qRT-PCR analysis of the expression of <i>Tbx6</i>, <i>Cdx2</i> and <i>Hoxb1</i> relative to b-actin at D3 of differentiation in wild-type (wt) and Brachyury null cells (Bra^−/−) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while <i>Cdx2</i> and <i>Hoxb1</i> continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra^−/− ESCs exposed to CHIR from D2–D5 (Meso conditions). Posterior Hox genes <i>Hoxc8</i> and <i>Hoxc9</i> are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers <i>Sox1</i> and <i>Sox2</i> are expressed only in Bra^−/− cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra^−/− and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra^−/− cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not β-Tubulin (Tuj1) whereas Bra^−/− cells fail to produce Desmin/MyoD and differentiate into neurons expressing β-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N_P) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log₂₊ scale). All data used for the plots can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s014" target="_blank">Data S6</a>. (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling.