Sox2 gene regulation via the D1 enhancer in embryonic neural tube and neural crest by the combined action of SOX2 and ZIC2.

The transcription factor (TF) SOX2 regulates various stem cells and tissue progenitors via functional interactions with cell type-specific partner TFs that co-bind to enhancer sequences. Neural progenitors are the major embryonic tissues where SOX2 assumes central regulatory roles. In order to characterize the partner TFs of SOX2 in neural progenitors, we investigated the regulation of the D1 enhancer of the Sox2 gene, which is activated in the embryonic neural tube (NT) and neural crest (NC), using chicken embryo electroporation. We identified essential TF binding sites for a SOX, and two ZIC TFs in the activation of the D1 enhancer. By comparison of dorso-ventral and antero-posterior patterns of D1 enhancer activation, and the effect of mutations on the enhancer activation patterns with TF expression patterns, we determined SOX2 and ZIC2 as the major D1 enhancer-activating TFs. Binding of these TFs to the D1 enhancer sequence was confirmed by chromatin immunoprecipitation analysis. The combination of SOX2 and ZIC2 TFs activated the enhancer in both the NT and NC. These results indicate that SOX2 and ZIC2, which have been known to play major regulatory roles in neural progenitors, do functionally cooperate. In addition, the recently demonstrated SOX2 expression during the NC development is accounted for at least partly by the D1 enhancer activity. Deletion of the D1 enhancer sequence from the mouse genome, however, did not affect the mouse development, indicating functional redundancies of other enhancers.

The many faces of Sox2 function in neural crest development.

Neural crest (NC) cells give rise to a wide variety of cell types and tissues, such as neurons and glial cells in the peripheral nervous system. Sox2, which encodes an HMG-box transcription factor, is known to mediate pluripotency of primordial germ cells and embryonic stem (ES)/induced pluripotent stem (iPS) cells, and to regulate central nervous system development. Previous studies have revealed that Sox2 is also an important regulator of NC development. This review summarizes the well-established inhibitory roles of Sox2 in NC formation and subsequent neuronal differentiation of NC-derived cells. This review also covers recent studies suggesting additional roles for Sox2 in early NC development, neurogenesis, and glial differentiation of NC-derived cells.

Nasal and otic placode specific regulation of Sox2 involves both activation by Sox-Sall4 synergism and multiple repression mechanisms.

Transcription factor gene Sox2 is expressed throughout sensory development, but the enhancers that regulate the gene vary depending on the developmental stages and tissues. To gain new insights into the gene regulatory network in sensory placode specification, regulation of the nasal-otic bispecific NOP1 enhancer of Sox2 was investigated in chicken embryos. Deletion and mutational analyses using electroporation showed that transcriptional repression mechanisms in combination with activation mechanisms determine placodal specificity. Activation of the NOP1 enhancer involves synergistic action by Sall4 and SoxB1/SoxE factors that bind to the adjacent sites. Deletion of repressive elements resulted in widening of the tissue area for enhancer activity to a region where the expression of Sall4 and SoxB1/E overlaps, e.g., the CNS and neural crest. Among multiple repressive elements that contribute to the placodal confinement of the NOP1 enhancer activity, CACCT/CACCTG motifs bound by Zeb/Snail family repressors play important roles. Overexpression of δEF1 (Zeb1) or Snail2 (Slug) strongly inhibited NOP1 activity. These data indicate that both activation by Sall4-Sox synergism and multiple repression mechanisms involving Zeb/Snail factors are essential for Sox2 regulation to be confined to the nasal and otic placodes.

Cooperation of Sall4 and Sox8 transcription factors in the regulation of the chicken Sox3 gene during otic placode development.

To elucidate the transcriptional regulation that underlies specification of the otic placode, we investigated the Sox3 downstream enhancer Otic1 of the chicken, the activity of which is restricted to and distributed across the entire otic placode. The 181-bp Otic1 enhancer sequence was dissected into a 68-bp minimal activating sequence, which exhibited dimer enhancer activity in the otic placode and cephalic neural crest, and this was further reduced to a 25-bp Otic1 core sequence, which also showed octamer enhancer activity in the same regions. The Otic1 core octamer was activated by the combined action of Sall4 and the SoxE transcription factors (TFs) Sox8 or Sox9. Binding of Sall4, Sox8 and Sox9 to the Otic1 sequence in embryonic tissues was confirmed by ChIP-qPCR analysis. The core-adjoining 3' side sequences of Otic1 augmented its enhancer activity, while inclusion of the CAGGTG sequence in the immediate 3' end of the 68-bp sequence repressed its enhancer activity outside the otic placode. The CAGGTG sequence likely serves as the binding sites of the repressor TFs δEF1 (Zeb1), Sip1 (Zeb2), and Snail2, all of which are expressed in the cephalic neural crest but not in the otic placode. Therefore, the combination of Sall4-Sox8-dependent activation and CAGGTG sequence-dependent repression determines otic placode development. Although the Otic1 sequence is not conserved in mammals or fishes, the activation mechanism is, as Otic1 was also activated in otic placode tissues developed from mouse embryonic stem cells and transient transgenic zebrafish embryos.

Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in 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 epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm 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 plate. 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.

Regulation of trunk neural crest delamination by δEF1 and Sip1 in the chicken embryo.

The vertebrate Zfhx1 transcription factor family comprises δEF1 and Sip1, which bind to CACCT-containing sequences and act as transcriptional repressors. It has been a longstanding question whether these transcription factors share the same regulatory functions in vivo. It has been shown that neural crest (NC) delamination depends on the Sip1 activity at the cranial level in mouse and chicken embryos, and it remained unclear how NC delamination is regulated at the trunk level. We observed that the expression of δEF1 and Sip1 overlaps in many tissues in chicken embryos, including NC cells at the trunk level. To clarify the above questions, we separately knocked down δEF1 and Sip1 or in combination in NC cells by electroporation of vectors expressing short hairpin RNAs (shRNAs) against respective mRNAs on the dorsal side of neural tubes that generate NC cells. In all cases, the migrating NC cell population was significantly reduced, paralleled by the decreased expression of δEF1 or Sip1 targeted by shRNAs. Expression of Sox10, the major transcription factor that regulates NC development, was also decreased by the shRNAs against δEF1 or Sip1. We conclude that the trunk NC delamination is regulated by both δEF1 and Sip1 in an analogous manner, and that these transcription factors can share equivalent regulatory functions in embryonic tissues.

Sixteen additional enhancers associated with the chicken Sox2 locus outside the central 50-kb region.

The transcription factor Sox2 plays a central role in the regulation of neuro-sensory development, and many other developmental processes. To gain an in depth understanding of the Sox2 gene regulation, we previously investigated the Sox2-proximal 50-kb region of the chicken genome to determine enhancers based on functional assays using chicken embryo electroporation. We identified 11 enhancers with specificity for neuro-sensory tissues. In this study, we extended the analysis of Sox2 locus-associated enhancers to a 200-kb region and identified 16 additional enhancers with functions in neuro-sensory development. These enhancers roughly correspond to a fraction of the sequence blocks that are highly conserved between chicken and mammalian genomes. The neural enhancers were activated in sequence, thereby creating a complex pattern of functional overlaps in the developing central nervous system (CNS). The variations in the specificities of the sensory enhancers also reflected the intermediate steps of sensory tissue development. This study provides an example where a single transcription factor gene has numerous regulatory elements that allow it to fulfill many functional roles in different biological contexts.

Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1.

Sox2 is required for proper neuronal formation in the CNS, but the molecular mechanisms involved are not well characterized. Here, we addressed the role of Sox2 in neurogenesis of the developing chicken inner ear. Overexpressing Sox2 from a constitutive (ß-actin) promoter induces the expression of the proneural gene, Neurogenin1 (Ngn1); however, the expression of a downstream target of Ngn1, Neurod1, is unchanged. As a result, there is a reduction of neural precursors to delaminate and populate the developing cochleo-vestibular ganglion. In contrast, overexpression of either Ngn1 or Neurod1 is sufficient to promote the neural fate in this system. These results suggest that high levels of Sox2 inhibit progression of neurogenesis in the developing inner ear. Furthermore, we provide evidence that Ngn1 and Neurod1 inhibit Sox2 transcription through a phylogenetically conserved Sox2 enhancer to mediate neurogenesis. We propose that Sox2 confers neural competency by promoting Ngn1 expression, and that negative feedback inhibition of Sox2 by Ngn1 is an essential step in the progression from neural precursor to nascent neuron.

The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates.

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.

B1 and B2 Sox gene expression during neural plate development in chicken and mouse embryos: universal versus species-dependent features.

Cumulative evidence now indicates pivotal roles for the group B1 Sox genes, Sox1, Sox2 and Sox3 in the genesis and development of neural primordia. Shared functions for the Sox1, Sox2 and Sox3 protein products have also been indicated. This emphasizes the importance and integral role of the group B1 Sox genes in regulating the neural primordia. We here review what is currently known about the expression patterns of both the group B1 Sox genes and the related group B2 Sox21 gene during the embryonic stages when the neural plate develops. These expression profiles are compared between the chicken and mouse embryos, both representatives of amniote species. This comparison indicates a gross conservation of the regulation of individual Sox genes, yet also demonstrates the existence of species-dependent variations, which should be taken into account when data from different species are being compared. To link the expression patterns and transcriptional regulation of these genes, contribution of gene-specific enhancers are discussed. The regulation of B1 Sox genes in the axial stem cells, the common precursors to the posterior neural plate and paraxial mesoderm and located at the posterior end of developing neural plate, is also highlighted in this review. This article thus provides a guide to performing readouts of B1/B2 Sox expression data during neural plate development in amniotes.

Regulation of Sox2 in the pre-placodal cephalic ectoderm and central nervous system by enhancer N-4.

The development of various tissues originating from the cephalic placodes is accompanied by the expression of the Sox2 gene. This Sox2 expression initiates in the pre-placodal cephalic ectoderm, and is regulated by enhancer N-4, which also regulates Sox2 in the embryonic central nervous system (CNS) posterior to the diencephalon. As the regulation of enhancer N-4 in the ectoderm likely reflects that of the pre-placodal cell state, its regulatory elements were characterized. A 110-bp minimal and essential sequence of N-4 (mini-N-4) was determined. By mutational and deletion analyses, nine regulatory elements were determined in the mini-N-4 sequence: three elements involved in activation in both the cephalic ectoderm and CNS, three elements specifically involved in activation in the cephalic ectoderm, three elements individually involved in activation in the mesencephalon, repression in the prosencephalon, and retinoic acid response in the rhombomeric region. The cephalic ectoderm-specific elements include two potential sites for the binding of nuclear receptors, suggestive of a nuclear receptor-dependent regulation. Multimers of the 3' half of the mini-N-4 sequence, including all of the cephalic ectodermal elements, show strong and selective activity in the cephalic ectoderm, providing a powerful genetic tool for the manipulation of gene activities in the placodal lineages.

Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs.

The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3.

The absence of SOX2 in the anterior foregut alters the esophagus into trachea and bronchi in both epithelial and mesenchymal components.

In the anterior foregut (AFG) of mouse embryos, the transcription factor SOX2 is expressed in the epithelia of the esophagus and proximal branches of respiratory organs comprising the trachea and bronchi, whereas NKX2.1 is expressed only in the epithelia of respiratory organs. Previous studies using hypomorphic Sox2 alleles have indicated that reduced SOX2 expression causes the esophageal epithelium to display some respiratory organ characteristics. In the present study, we produced mouse embryos with AFG-specific SOX2 deficiency. In the absence of SOX2 expression, a single NKX2.1-expressing epithelial tube connected the pharynx and the stomach, and a pair of bronchi developed in the middle of the tube. Expression patterns of NKX2.1 and SOX9 revealed that the anterior and posterior halves of SOX2-deficient AFG epithelial tubes assumed the characteristics of the trachea and bronchus, respectively. In addition, we found that mesenchymal tissues surrounding the SOX2-deficient NKX2.1-expressing epithelial tube changed to those surrounding the trachea and bronchi in the anterior and posterior halves, as indicated by the arrangement of smooth muscle cells and SOX9-expressing cells and by the expression of Wnt4 (esophagus specific), Tbx4 (respiratory organ specific), and Hoxb6 (distal bronchus specific). The impact of mesenchyme-derived signaling on the early stage of AFG epithelial specification has been indicated. Our study demonstrated an opposite trend where epithelial tissue specification causes concordant changes in mesenchymal tissues, indicating a reciprocity of epithelial-mesenchymal interactions.

Nodal paralogues underlie distinct mechanisms for visceral left-right asymmetry in reptiles and mammals.

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.

Anteroventrally localized activity in the optic vesicle plays a crucial role in the optic development.

The vertebrate eye develops from the optic vesicle (OV), a laterally protrusive structure of the forebrain, by a coordinated interaction with surrounding tissues. The OV then invaginates to form an optic cup, and the lens placode develops to the lens vesicle at the same time. These aspects in the early stage characterize vertebrate eye formation and are controlled by appropriate dorsal-ventral coordination. In the present study, we performed surgical manipulation in the chick OV to remove either the dorsal or ventral half and examined the development of the remaining OV. The results show that the dorsal and ventral halves of the OV have a clearly different developmental pattern. When the dorsal half was removed, the remaining ventral OV developed into an entire eye, while the dorsal OV developed to a pigmented vesicle consisting of retinal pigmented epithelium alone. These results indicate that the ventral part of the OV retains the potency to develop the entire eye structure and plays an essential role in proper eye development. In subsequent manipulations of early chick embryos, it was found that only the anterior ventral quadrant of the OV has the potential to develop the entire eye and that no other part of the OV has a similar activity. Fgf8 expression was localized in this portion and no Fgf8 expression was observed within the OV when the ventral OV was removed. These results suggest that the anterior ventral portion of the OV plays a crucial role in the proper development of the eye, possibly generating the dorsal-ventral gradients of signal proteins within the eye primordium.

Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals.

Sox2 expression marks neural and sensory primordia at various stages of development. A 50 kb genomic region of chicken Sox2 was isolated and scanned for enhancer activity utilizing embryo electroporation, resulting in identification of a battery of enhancers. Although Sox2 expression in the early embryonic CNS appears uniform, it is actually pieced together by five separate enhancers with distinct spatio-temporal specificities, including the one activated by the neural induction signals emanating from Hensen's node. Enhancers for Sox2 expression in the lens and nasal/otic placodes and in the neural crest were also determined. These functionally identified Sox2 enhancers exactly correspond to the extragenic sequence blocks conspicuously conserved between chicken and mammals, which are not discernible by sequence comparison among mammals.

Evolution of non-coding regulatory sequences involved in the developmental process: reflection of differential employment of paralogous genes as highlighted by Sox2 and group B1 Sox genes.

In higher vertebrates, the expression of Sox2, a group B1 Sox gene, is the hallmark of neural primordial cell state during the developmental processes from embryo to adult. Sox2 is regulated by the combined action of many enhancers with distinct spatio-temporal specificities. DNA sequences for these enhancers are conserved in a wide range of vertebrate species, corresponding to a majority of highly conserved non-coding sequences surrounding the Sox2 gene, corroborating the notion that the conservation of non-coding sequences mirrors their functional importance. Among the Sox2 enhancers, N-1 and N-2 are activated the earliest in embryogenesis and regulate Sox2 in posterior and anterior neural plates, respectively. These enhancers differ in their evolutionary history: the sequence and activity of enhancer N-2 is conserved in all vertebrate species, while enhancer N-1 is fully conserved only in amniotes. In teleost embryos, Sox19a/b play the major pan-neural role among the group B1 Sox paralogues, while strong Sox2 expression is limited to the anterior neural plate, reflecting the absence of posterior CNS-dedicated enhancers, including N-1. In Xenopus, neurally expressed SoxD is the orthologue of Sox19, but Sox3 appears to dominate other B1 paralogues. In amniotes, however, Sox19 has lost its group B1 Sox function and transforms into group G Sox15 (neofunctionalization), and Sox2 assumes the dominant position by gaining enhancer N-1 and other enhancers for posterior CNS. Thus, the gain and loss of specific enhancer elements during the evolutionary process reflects the change in functional assignment of particular paralogous genes, while overall regulatory functions attributed to the gene family are maintained.

Enhancer analysis by chicken embryo electroporation with aid of genome comparison.

The identification of the enhancers associated with each developmentally regulated gene is a first step to clarify the regulatory mechanisms underlying embryogenesis. The electroporation technique using chicken embryo is a powerful tool to identify such enhancers. The technique enables us to survey a large genomic region and to analyze the enhancers in great detail. Comparison of the genomic sequences of the chicken and other vertebrate species identifies conserved non-coding sequence blocks to which the functionally identified enhancers often correspond. In this review, I describe in detail the methods to analyze the enhancers using the chicken embryo electroporation and genome comparison.

Enhancer Analyses Using Chicken Embryo Electroporation.

Chicken embryo electroporation is a powerful tool used to identify and analyze enhancers involved in developmental gene regulation. In this chapter, the basic procedures and underlying principles of enhancer analysis using chicken embryo electroporation are described in the following steps: (1) identification of enhancers in a wide genomic region, (2) determination of the full enhancer region, (3) definition of the core enhancer regions, and (4) analysis of a functional transcription factor binding sequences in the core region.

Efficient identification of regulatory sequences in the chicken genome by a powerful combination of embryo electroporation and genome comparison.

Recently expanded knowledge of gene regulation clearly indicates that the regulatory sequences of a gene, usually identified as enhancers, are widely distributed in the gene locus, revising the classical view that they are clustered in the vicinity of genes. To identify regulatory sequences for Sox2 expression governing early neurogenesis, we scanned the 50-kb region of the chicken Sox2 locus for enhancer activity utilizing embryo electroporation, resulting in identification of a number of enhancers scattered throughout the analyzed genomic span. The 'pan-neural' Sox2 expression in early embryos is actually brought about by the composite activities of five separate enhancers with distinct spatio-temporal specificities. These and other functionally defined enhancers exactly correspond to extragenic sequence blocks that are conspicuously conserved between the chicken and mammalian genomes and that are embedded in sequences with a wide range of sequence conservation between humans and mice. The sequences conserved between amniotes and teleosts correspond to subregions of the enhancer subsets which presumably represent core motifs of the enhancers, and the limited conservation partly reflects divergent expression patterns of the gene. The phylogenic distance between the chicken and mammals appears optimal for identifying a battery of genetic regulatory elements as conserved sequence blocks, and chicken embryo electroporation facilitates functional characterization of these elements.