Silvia K. Nicolis

Hippocampal development and neural stem cell maintenance require Sox2 -dependent regulation of Shh

Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.

Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations

In multicellular organisms, transcription regulation is one of the central mechanisms modelling lineage differentiation and cell-fate determination. Transcription requires dynamic chromatin configurations between promoters and their corresponding distal regulatory elements. It is believed that their communication occurs within large discrete foci of aggregated RNA polymerases termed transcription factories in three-dimensional nuclear space. However, the dynamic nature of chromatin connectivity has not been characterized at the genome-wide level. Here, through a chromatin interaction analysis with paired-end tagging approach using an antibody that primarily recognizes the pre-initiation complexes of RNA polymerase II, we explore the transcriptional interactomes of three mouse cells of progressive lineage commitment, including pluripotent embryonic stem cells, neural stem cells and neurosphere stem/progenitor cells. Our global chromatin connectivity maps reveal approximately 40,000 long-range interactions, suggest precise enhancer–promoter associations and delineate cell-type-specific chromatin structures. Analysis of the complex regulatory repertoire shows that there are extensive colocalizations among promoters and distal-acting enhancers. Most of the enhancers associate with promoters located beyond their nearest active genes, indicating that the linear juxtaposition is not the only guiding principle driving enhancer target selection. Although promoter–enhancer interactions exhibit high cell-type specificity, promoters involved in interactions are found to be generally common and mostly active among different cells. Chromatin connectivity networks reveal that the pivotal genes of reprogramming functions are transcribed within physical proximity to each other in embryonic stem cells, linking chromatin architecture to coordinated gene expression. Our study sets the stage for the full-scale dissection of spatial and temporal genome structures and their roles in orchestrating development.

Sox2 roles in neural stem cells

Throughout vertebrate evolution, Sox2 marks the developing nervous system from its earliest developmental stages and, therein, the most undifferentiated precursor cells, including stem cells. Recent gene targeting studies investigated the function of Sox2 in two neuronal systems: the developing eye and brain. These studies uncovered a requirement for Sox2 in the maintenance of neural stem cells, as well as a downstream role in the differentiation of specific neuron sub-types. In both systems, Sox2 action is markedly dose-dependent, and downstream-target gene studies are beginning to reveal the mechanisms of Sox2 function.

Signaling through BMP receptors promotes respiratory identity in the foregut via repression of SOX2

The mammalian foregut gives rise to the dorsally located esophagus and stomach and the ventrally located trachea and lung. Proper patterning and morphogenesis of the common foregut tube and its derived organs is essential for viability of the organism at birth. Here, we show that conditional inactivation of BMP type I receptor genes Bmpr1a and Bmpr1b (Bmpr1a;b) in the ventral endoderm leads to tracheal agenesis and ectopic primary bronchi. Molecular analyses of these mutants reveal a reduction of ventral endoderm marker NKX2-1 and an expansion of dorsal markers SOX2 and P63 into the prospective trachea and primary bronchi. Subsequent genetic experiments show that activation of canonical WNT signaling, previously shown to induce ectopic respiratory fate in otherwise wild-type mice, is incapable of promoting respiratory fate in the absence of Bmpr1a;b. Furthermore, we find that inactivation of Sox2 in Bmpr1a;b mutants does not suppress ectopic lung budding but does rescue trachea formation and NKX2-1 expression. Together, our data suggest that signaling through BMPR1A;B performs at least two roles in early respiratory development: first, it promotes tracheal formation through repression of Sox2; and second, it restricts the site of lung bud initiation.

Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest

The cellular origin and molecular mechanisms regulating pigmentation of head and neck are largely unknown. Melanocyte specification is controlled by the transcriptional activity of Mitf, but no general logic has emerged to explain how Mitf and progenitor transcriptional activities consolidate melanocyte and progenitor cell fates. We show that cranial melanocytes arise from at least two different cellular sources: initially from nerve-associated Schwann cell precursors (SCPs) and later from a cellular source that is independent of nerves. Unlike the midbrain-hindbrain cluster from which melanoblasts arise independently of nerves, a large center of melanocytes in and around cranial nerves IX-X is derived from SCPs, as shown by genetic cell-lineage tracing and analysis of ErbB3-null mutant mice. Conditional gain- and loss-of-function experiments show genetically that cell fates in the neural crest involve both the SRY transcription factor Sox2 and Mitf, which consolidate an SCP progenitor or melanocyte fate by cross-regulatory interactions. A gradual downregulation of Sox2 in progenitors during development permits the differentiation of both neural crest- and SCP-derived progenitors into melanocytes, and an initial small pool of nerve-associated melanoblasts expands in number and disperses under the control of endothelin receptor B (Ednrb) and Wnt5a signaling.

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self‐renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self‐renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP‐Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple‐repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog‐Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self‐renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self‐renewal.

The transcription factor Sox2 is required for osteoblast self-renewal

The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2–3 passages failed to yield any Sox2-null cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2-null cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming ‘osteospheres’ are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells

While the Polycomb complex is known to regulate cell identity in ES cells, its role in controlling tissue‐specific stem cells is not well understood. Here we show that removal of Ezh1 and Ezh2, key Polycomb subunits, from mouse skin results in a marked change in fate determination in epidermal progenitor cells, leading to an increase in the number of lineage‐committed Merkel cells, a specialized subtype of skin cells involved in mechanotransduction. By dissecting the genetic mechanism, we showed that the Polycomb complex restricts differentiation of epidermal progenitor cells by repressing the transcription factor Sox2. Ablation of Sox2 results in a dramatic loss of Merkel cells, indicating that Sox2 is a critical regulator of Merkel cell specification. We show that Sox2 directly activates Atoh1, the obligate regulator of Merkel cell differentiation. Concordantly, ablation of Sox2 attenuated the Ezh1/2‐null phenotype, confirming the importance of Polycomb‐mediated repression of Sox2 in maintaining the epidermal progenitor cell state. Together, these findings define a novel regulatory network by which the Polycomb complex maintains the progenitor cell state and governs differentiation in vivo.

Sox2 Is Required to Maintain Cancer Stem Cells in a Mouse Model of High-Grade Oligodendroglioma

The stem cell-determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor-induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells.

Essential Role of Sox2 for the Establishment and Maintenance of the Germ Cell Line

Sox2 is a pluripotency‐conferring gene expressed in primordial germ cells (PGCs) and postnatal oocytes, but the role it plays during germ cell development and early embryogenesis is unknown. Since Sox2 ablation causes early embryonic lethality shortly after blastocyst implantation, we generated mice bearing Sox2‐conditional deletion in germ cells at different stages of their development through the Cre/loxP recombination system. Embryos lacking Sox2 in PGCs show a dramatic decrease of germ cell numbers at the time of their specification. At later stages, we found that Sox2 is strictly required for PGC proliferation. On the contrary, Sox2 deletion in meiotic oocytes does not impair postnatal oogenesis and early embryogenesis, indicating that it is not essential for oocyte maturation or for zygotic development. We also show that Sox2 regulates Kit expression in PGCs and binds to discrete transcriptional regulatory sequences of this gene, which is known to be important for PGCs survival and proliferation. Sox2 also stimulates the expression of Zfp148, which is required for normal development of fetal germ cells, and Rif1, a potential regulator of PGC pluripotency. STEM Cells2013;31:1408–1421