Summary Naïve and primed pluripotency is characterized by distinct signaling requirements, transcriptomes and developmental properties, but both cellular states share key transcriptional regulators, Oct4, Sox2 and Nanog. Here we demonstrate that transition between these two pluripotent states is associated with widespread Oct4 relocalization, mirrored by global rearrangement of enhancer chromatin landscapes. Our genomic and biochemical analyses identified candidate mediators of primed state-specific Oct4 binding, including Otx2 and Zic2/3. Even when differentiation cues are blocked, premature Otx2 overexpression is sufficient to exit the naïve state, induce transcription of a substantial subset of primed pluripotency-associated genes and redirect Oct4 to previously inaccessible enhancer sites. However, ability of Otx2 to engage new enhancer regions is determined by its levels, cis-encoded properties of the sites and signaling environment. Our results illuminate regulatory mechanisms underlying pluripotency and suggest that capacity of transcription factors such as Otx2 and Oct4 to pioneer new enhancer sites is highly context-dependent.

We studied the expression of two vertebrate homeobox genes, Otx1 and Otx2, related to orthodenticle, a gene expressed in the developing head of Drosophila. Both genes are expressed in restricted regions of the developing rostral brain including the presumptive cerebral cortex and olfactory bulbs. The expression patterns of the two genes in diencephalon suggest that they both have a role in establishing the boundary between presumptive dorsal and ventral thalamus. They are also expressed in regions of the developing olfactory, auricolar and ocular system, including the covering of the optic nerve. Otx1 expression is detectable from day 8 of gestation in telencephalic, diencephalic and mesencephalic regions. From day 10.5 of gestation its expression extends to some metencephalic areas. Otx2 appears to be already expressed in the epiblast of prestreak embryos. It persists in the entire embryonic ectoderm for some time after the onset of gastrulation. In midstreak embryos its expression appears progressively restricted to the anterior embryonic ectoderm corresponding to presumptive fore‐ and mid‐brain. In early midgestation embryos it is expressed in telencephalic, diencephalic and mesencephalic regions but from day 11.75 of gestation its expression disappears from dorsal telencephalon and is confined to diencephalic and mesencephalic regions. Otx2 is one of the earliest genes expressed in the epiblast and immediately afterwards is expressed in anterior neuroectoderm, demarcating rostral brain regions even before headfold formation. Its gene product contains a homeodomain of the bicoid class and is able to recognize and transactivate a bicoid target sequence.

Insight into the genetic control of the identity of specific regions along the body axis of vertebrates has resulted primarily from the study of vertebrate homologues of regulatory genes operating in the Drosophila trunk, but little is known about the development of most anterior regions of the body either in flies or vertebrates. Three Drosophila genes have been identified that are important in controlling the development of the head, two of which, empty spiracles and orthodenticle, have been cloned and shown to contain a homeobox. We previously cloned and characterized Emx1 and Emx2, two mouse genes related to empty spiracles that are expressed in restricted regions of the developing forebrain, including the presumptive cerebral cortex and olfactory bulbs. Here we report the identification of Otx1 and Otx2, which are related to orthodenticle. We have compared the expression domains of the four genes in the developing rostral brain of mouse embryos at a developmental stage, day 10 post coitum, when they are all expressed. Otx2 is expressed in every dorsal and most ventral regions of telencephalon, diencephalon and mesencephalon. The Otx1 expression domain is similar to that of Otx2, but contained within it. The Emx2 expression domain is comprised of dorsal telencephalon and small diencephalic regions, both dorsally and ventrally. Finally, Emx1 expression is exclusively confined to the dorsal telencephalon. Thus at the time when regional specification of major brain regions takes place, the expression domains of the four genes seem to be continuous regions contained within each other in the sequence Emx1 less than Emx2 less than Otx1 less than Otx2.

We cloned two homeobox genes, Emx1 and Emx2, related to empty spiracles, a gene expressed in very anterior body regions during early Drosophila embryogenesis, and studied their expression in mouse embryos. Emx1 expression is detectable from day 9.5 of gestation whereas Emx2 appears to be already expressed in 8.5 day embryos. Both genes are expressed in the presumptive cerebral cortex and olfactory bulbs. Emx1 is expressed exclusively there, whereas Emx2 is also expressed in some neuroectodermal areas in embryonic head including olfactory placodes in earlier stages and olfactory epithelia later in development.

RETINOIC acid had been implicated as a natural morphogen in chicken and frog embryogenesis, and is presumed to act through the gene regulatory activity of a family of nuclear receptors. Homeobox genes, which specify positional information in Drosophila and possibly in vertebrate embryogenesis, are among the candidate responsive genes. We previously reported that retinoic acid specifically induces human homeobox gene (HOX) expression in the embryonal carcinoma cell line NT2/D1. We now show that the nine genes of the HOX2 cluster are differentially activated in NT2/D1 cells exposed to retinoic acid concentrations ranging from 10(-8) to 10(-5) M. Genes located in the 3' half of the cluster are induced at peak levels by 10(-8) M retinoic acid, whereas a concentration of 10(-6) to 10(-5) M is required to fully activate 5' genes. At both high and low retinoic acid concentrations, HOX2 genes are sequentially activated in embryonal carcinoma cells in the 3' to 5' direction.

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The specification of distinct neuronal cell-types is controlled by inducing signals whose interpretation in distinct areas along the central nervous system provides neuronal progenitors with a precise and typical expression code of transcription factors. To gain insights into this process, we investigated the role of Otx2 in the specification of identity and fate of neuronal progenitors in the ventral midbrain. To achieve this, Otx2 was inactivated by Cre recombinase under the transcriptional control of En1. Lack of Otx2 in the ventrolateral and posterior midbrain results in a dorsal expansion of Shh expression and in a dorsal and anterior rotation of the midbrain-hindbrain boundary and Fgf8 expression. Indeed, in this mutant correct positioning of the ventral site of midbrain-hindbrain boundary and Fgf8 expression are efficiently controlled by Otx1 function, thus allowing the study of the identity and fate of neuronal progenitors of the ventral midbrain in the absence of Otx2. Our results suggest that Otx2 acts in two ways: by repressing Nkx2.2 in the ventral midbrain and maintaining the Nkx6.1-expressing domain through dorsal antagonism on Shh. Failure of this control affects the identity code and fate of midbrain progenitors, which exhibit features in common with neuronal precursors of the rostral hindbrain even though the midbrain retains its regional identity and these neuronal precursors are rostral to Fgf8 expression. Dopaminergic neurons are greatly reduced in number, red nucleus precursors disappear from the ventral midbrain where a relevant number of serotonergic neurons are generated. These results indicate that Otx2 is an essential regulator of the identity, extent and fate of neuronal progenitor domains in the ventral midbrain and provide novel insights into the mechanisms by which neuronal diversity is generated in the central nervous system.

A number of vertebrate genes of the Dlx gene family have been cloned in mouse, frog, and zebrafish. These genes contain a homeobox related to that of Distalless, a gene expressed in the developing head and limbs of Drosophila embryos. We cloned and studied the expression of two members of this family, which we amaed DlxS and Dax6, in human and mouse. The two human genes, DLXS and DLX6, are closely linked in an inverted convergent configuration in a region of chromosome 7, at 7q22. Similarly, the two human genes DLXI and DLK2 are closely linked in a convergent configuration at 2q32, near the HOXD (previously HOX4) locus. In situ hybridization experiments in mouse embryos revealed expression of DlxS and Dlx6 mRNA in restricted regions of ventral diencephalon and basal teencephalon, with a distribution very similar to that reported forDMl and Dx2 mRNA. A surprising feature of DlxS and Dlx6 is that they are also expressed in all skeletal sutures of dtion embryos after the first cartilae formation. The expreion pattern of these genes, together with their chromosome calation, may provide useftl cues for the study of congenital disorders in which there is a combination of cranlofacial and limb defects.Many vertebrate genes have been identified by virtue of their nucleotide sequence similarity with Drosophila developmental genes. Many homeobox-containing genes (1) have been identified on this basis. The Dlx gene family (2-7) has been identified because these genes contain a homeobox related to that of Distalless (Dli, also known as Ba) a gene expressed in the head and limbs of the developing fruit fly (8-9).Cloned Dlx sequences in the mouse (2-4), frog (6, 7), and zebrafish (5) have been shown to correspond to at least four different genes, Dlxi-Dlx4. A detailed expression analysis has been carried out for murine Dlxi (2, 10, 11) and Dlx2 (3, 4, 12) genes. They appear to be expressed within the central nervous system of midgestation mouse embryos in specific regions ofthe forebrain, but not in more posterior parts of the neural tube. In early embryos they are also expressed in branchial arches, in the otic vesicle, and in facial and limb primordia. Expression in the developing inner ear has been also reported (5) for the zebrafish cognate of Dlx3. With the notable exception of Xdli2 (7), several frog genes (Xenopus) of the Dlx family have been identified (6, 7) that are similarly expressed in the anterior portion of the embryonic neural tube. In many instances, a correlation of their expression domain with forebrain regionalization (13,14) has been suggested (2)(3)(4)(5)(6)(7)(10)(11)(12) MATERIALS AND METHODSExpression Analysis. A cDNA library prepared from 8-week human embryos (15) was screened at low-stringency conditions with a short Dli genomic sequence including the homeobox (8). Four classes of homologous cDNA clones, corresponding to DLX), DLX2, DLX5, and DLX6, were found. Using these cDNA clones as probes, we screened in turn a human genomic library constructed in cosmids (15) to study the transcrip...