Biomedical subjects

Ian Chambers

Publications and source records attributed to Ian Chambers.

10 recordsLinked to original sources

PubMed2025

NANOG is repurposed after implantation to repress Sox2 and begin pluripotency extinction.

Loss of pluripotency is an essential step in post-implantation development that facilitates the emergence of somatic cell identities essential for gastrulation. Before implantation, pluripotent cell identity is governed by a gene regulatory network that includes the key transcription factors SOX2 and NANOG. However, it is unclear how the pluripotency gene regulatory network is dissolved to enable lineage restriction. Here, we show that SOX2 is required for post-implantation pluripotent identity in the mouse, and cells that lose SOX2 expression in the posterior epiblast are no longer pluripotent. Using in vitro and in vivo analyses, we demonstrate anticorrelated expression of NANOG and SOX2 preceding gastrulation, culminating in an early disappearance of pluripotent identity from posterior NANOGhigh/SOX2low epiblast. Surprisingly, Sox2 expression is repressed by NANOG and embryos with post-implantation deletion of Nanog maintain posterior SOX2 expression. Our results demonstrate that the distinctive features of post-implantation pluripotency are underpinned by altered functionality of pluripotency transcription factors, ensuring correct spatio-temporal loss of embryonic pluripotency.

Animals↗

PubMed2006

Nanog retrotransposed genes with functionally conserved open reading frames.

The Nanog gene plays a key role in the pluripotency of early embryonic cells in vitro and in vivo. In this article retrotransposed copies of Nanog, termed NanogPc and NanogPd, are identified on mouse Chromosomes 4 and 7, respectively. In contrast to the two previously characterized mouse Nanog retrogenes that contain multiple frameshifts and point mutations, NanogPc and NanogPd are 98% identical to NANOG within the open reading frame and encode proteins with activity in an embryonic stem cell self-renewal assay. Mutations common to all four retrotransposed genes but distinct from Nanog suggest divergence from a common progenitor that appears likely to be Nanog because transcripts derived from Nanog but not from the retrogenes are detected in germ-line cells. The possibility that expression of Nanog could be erroneously attributed to novel cellular sources is suggested by the high homology among Nanog, NanogPc, and NanogPd. Analysis of distinct Mus species suggests that NanogPc and NanogPd arose between divergence of M. caroli and M. spretus and indicates that Nanog retrotransposition events continue to occur at a high frequency, a property likely to extend to other germ-line transcripts.

Amino Acid Sequence↗

PubMed2006

Nanog promotes transfer of pluripotency after cell fusion.

Through cell fusion, embryonic stem (ES) cells can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ES cells. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain undifferentiated ES cell propagation. Here we report that in fusions between ES cells and neural stem (NS) cells, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ES cell characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ES cells; however, fewer colonies are obtained than from ES x NS cell fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NS x ES cell fusions elevated Nanog enables primary hybrids to develop into ES cell colonies with identical frequency to homotypic ES x ES fusion products. This means that in hybrids, increased Nanog is sufficient for the NS cell epigenome to be reset completely to a state of pluripotency. We conclude that Nanog can orchestrate ES cell machinery to instate pluripotency with an efficiency of up to 100% depending on the differentiation status of the somatic cell.

Animals↗

PubMed2004

Self-renewal of teratocarcinoma and embryonic stem cells.

Pluripotent stem cells derived from preimplantation embryos, primordial germ cells or teratocarcinomas are currently unique in undergoing prolonged symmetrical self-renewal in culture. For mouse embryonic stem (ES) cells, self-renewal is dependent on signals from the cytokine leukaemia inhibitory factor (LIF) and from either serum or bone morphogenetic proteins (BMPs). In addition to the extrinsic regulation of gene expression, intrinsic transcriptional determinants are also required for maintenance of the undifferentiated state. These include Oct4, a member of the POU family of homeodomain proteins and a second recently identified homeodomain protein, Nanog. When overexpressed, Nanog allows ES cells to self-renew in the absence of the otherwise obligatory LIF and BMP signals. Although Nanog can act independent of the LIF signal, a contribution of both pathways provides maximal self-renewal efficiency. Nanog function also requires Oct4. Here, we review recent progress in ES cell self-renewal, relate this to the biology of teratocarcinomas and offer testable hypotheses to expose the mechanics of ES cell self-renewal.

Animals↗

PubMed2004

The molecular basis of pluripotency in mouse embryonic stem cells.

Mouse embryonic stem (ES) cell self-renewal depends upon extrinsic signals from leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). These molecules activate, respectively, the nuclear localization of the latent transcription factor STAT3 and the expression of Id genes. In contrast, the homeodomain proteins Oct4 and the recently identified Nanog are intrinsic factors required for maintenance of the undifferentiated state. When overexpressed, Nanog allows ES cells to self-renew in the absence of the otherwise obligatory LIF and BMP signals. However, the highest efficiency of ES cell self-renewal occurs when Nanog is overexpressed and cells are exposed to LIF. In contrast, when Oct4 is overexpressed, ES cells differentiate in a similar manner to the differentiation that occurs upon LIF withdrawal. These observations are brought together to provide a genetic model of ES cell self-renewal centered upon interactions between Oct4, STAT3 and Nanog.

Animals↗

PubMed2003

BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.

The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3.

Animals↗

PubMed2003

Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.

Embryonic stem (ES) cells undergo extended proliferation while remaining poised for multilineage differentiation. A unique network of transcription factors may characterize self-renewal and simultaneously suppress differentiation. We applied expression cloning in mouse ES cells to isolate a self-renewal determinant. Nanog is a divergent homeodomain protein that directs propagation of undifferentiated ES cells. Nanog mRNA is present in pluripotent mouse and human cell lines, and absent from differentiated cells. In preimplantation embryos, Nanog is restricted to founder cells from which ES cells can be derived. Endogenous Nanog acts in parallel with cytokine stimulation of Stat3 to drive ES cell self-renewal. Elevated Nanog expression from transgene constructs is sufficient for clonal expansion of ES cells, bypassing Stat3 and maintaining Oct4 levels. Cytokine dependence, multilineage differentiation, and embryo colonization capacity are fully restored upon transgene excision. These findings establish a central role for Nanog in the transcription factor hierarchy that defines ES cell identity.

Animals↗

PubMed2003

Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles.

The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis. Here we applied Red/ET recombineering (using the lambda Redalpha/Redbeta recombinase pair) to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.

Alleles↗

PubMed2002

Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation.

The multilineage differentiation capacity of mouse embryonic stem (ES) cells offers a potential testing platform for gene products that mediate mammalian lineage determination and cellular specialization. Identification of such differentiation regulators is crucial to harnessing ES cells for pharmaceutical discovery and cell therapy. Here we describe the use of episomal expression technology for functional evaluation of cDNA clones during ES-cell differentiation in vitro. Several candidate cDNAs identified by subtractive cloning and expression profiling were introduced into ES cells in episomal expression constructs. Subsequent differentiation revealed that the Wnt antagonist Sfrp2 stimulates production of neural progenitors. The significance of this observation was substantiated by forced expression of Wnt-1 and treatment with lithium chloride, both of which inhibit neural differentiation. These findings reveal the importance of Wnt signaling in regulating ES-cell lineage diversification. More generally, this study establishes a path for rapid and direct validation of candidate genes in ES cells.

Animals↗

PubMed2002

Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells.

Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation.

Animals↗