Sox transcription factors play widespread roles during development; however, their versatile funtions have a relatively simple basis: the binding of a Sox protein alone to DNA does not elicit transcriptional activation or repression, but requires binding of a partner transcription factor to an adjacent site on the DNA. Thus, the activity of a Sox protein is dependent upon the identity of its partner factor and the context of the DNA sequence to which it binds. In this Primer, we provide an mechanistic overview of how Sox family proteins function, as a paradigm for transcriptional regulation of development involving multi-transcription factor complexes, and we discuss how Sox factors can thus regulate diverse processes during development.

A population of axial stem cells present in the caudal lateral epiblast of the mouse embryo is known to produce both neural plate and mesoderm cells, but the factors regulating this choice are unknown. Takemoto et al. now show that Tbx6-dependent modulation of the developmental transcription factor Sox2 drives the fate of these axial stem cells. In the absence of Tbx6, cells aberrantly upregulated Sox2 activity, meaning that cells originally destined to be mesoderm turned into ectopic neural tubes. This work and recent cell-lineage analyses challenge the textbook view that segregation of the three germ layers (ectoderm, mesoderm, endoderm) is the process specifying cell lineages in early embryos. Rather, the paraxial mesoderm (producing muscle and bone) and neural plate (producing the brain and spinal cord) seem to be derived from common stem-cell-like precursors. A common stem cell is known to produce both neural plate and mesoderm, but the factors regulating this choice are unknown. This study determines that Tbx6-dependent modulation of the developmental transcription factor Sox2 drives the fate of axial stem cells. In the absence of Tbx6, cells aberrantly upregulated Sox2 activity, with the result that cells originally destined to be mesoderm turned into ectopic neural tubes. In the absence of the N1 enhancer, this aberrant regulation is corrected and cells follow the appropriate fate, even in the absence of Tbx6. Therefore, active repression of the neural fate program is essential for mesoderm tissue to develop from axial stem cells. The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast1,2. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm3 must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate4,5,6. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.

Somatic development initiates from the epiblast in post-implantation mammalian embryos. Recent establishment of epiblast stem cell (EpiSC) lines has opened up new avenues of investigation of the mechanisms that regulate the epiblast state and initiate lineage-specific somatic development. Here, we investigated the role of cell-intrinsic core transcriptional regulation in the epiblast and during derivation of the anterior neural plate (ANP) using a mouse EpiSC model. Cells that developed from EpiSCs in one day in the absence of extrinsic signals were found to represent the ANP of ~E7.5 embryos. We focused on transcription factors that are uniformly expressed in the E6.5 epiblast but in a localized fashion within or external to the ANP at E7.5, as these are likely to regulate the epiblast state and ANP development depending on their balance. Analyses of the effects of knockdown and overexpression of these factors in EpiSCs on the levels of downstream transcription factors identified the following regulatory functions: cross-regulation among Zic, Otx2, Sox2 and Pou factors stabilizes the epiblastic state; Zic, Otx2 and Pou factors in combination repress mesodermal development; Zic and Sox2 factors repress endodermal development; and Otx2 represses posterior neural plate development. All of these factors variably activate genes responsible for neural plate development. The direct interaction of these factors with enhancers of Otx2, Hesx1 and Sox2 genes was demonstrated. Thus, a combination of regulatory processes that suppresses non-ANP lineages and promotes neural plate development determines the ANP.

Sox2 is universally expressed in the neural and placodal primordia in early stage embryos, and this expression depends on various phylogenetically conserved enhancers having different regional and temporal specificities. The enhancer N‐3 was identified as a regulator of the Sox2 gene active in the diencephalon, optic vesicle, and after the contact of the vesicle with the ectoderm, in the lens placodal surface area, suggesting its involvement in embryonic visual system development. A 36‐bp minimal essential core sequence was defined in the 568‐bp‐long enhancer N‐3, which in a tetrameric form emulates the original enhancer activity. The core sequence comprises a SOX‐binding sequence and a non‐canonical PAX6 (Paired domain) binding sequence, and is activated by the synergistic action of SOX2 and PAX6 in transfected cells. The SOX and PAX6 binding sequences of the N‐3 core are arranged with the same orientation and spacing as the DC5 sequence of the δ‐crystallin enhancer previously demonstrated to be cooperatively bound by SOX2 and PAX6. The N‐3 core sequence was also bound by these factors in a cooperative fashion, but with a higher threshold of these factors’ levels than DC5, and the enhancer effect of the tetrameric sequence activated by exogenous SOX2 and PAX6 was less pronounced than that of DC5. The observations suggest that gene activation mechanisms that depend on the cooperative interaction of SOX2 and PAX6 but with different thresholds of the factor levels are crucial for the regulation of visual system development.