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ABSTRACT: Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However, dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here we show that SMAD7, a cell-intrinsic inhibitor of TGFβ signaling, is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time-course gene expression revealed down-regulation of MAPK components, and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. FGF-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues, pluripotent cells simply revert to a program of neural conversion. Hence the "primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians, suggesting an evolutionarily conserved mechanism.
Stem Cells 10/2012; · 7.78 Impact Factor
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Cell cycle (Georgetown, Tex.) 08/2012; 11(19):3529-30. · 5.36 Impact Factor
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ABSTRACT: Transcription factor Foxd3 has been described in model systems as a key member of the pluripotency network in mice as well as being involved in the formation of many critical vertebrate cell types in vivo. Yet virtually nothing is known about roles of FOXD3 in human development and conflicting reports exist regarding its expression in human embryonic stem cells (hESCs). We find that FOXD3 is expressed at both the RNA and protein levels in undifferentiated hESCs and report a Foxd3 expression domain in paraxial mesoderm derivatives of wild-type mouse embryos. Furthermore, increasing FOXD3 activity in hESCs is sufficient for rapid and specific generation of mesenchymal cell types of the paraxial mesoderm, even under pluripotency maintenance conditions. Gene expression diagnostic of chondroblasts, skeletal myoblasts, osteoblasts, and adipoblast is observed within 48 hours of FOXD3 induction, as are morphological and genetic hallmarks of epithelial-to-mesenchymal transition. FOXD3-overexpressing cells can be maintained for several passages, while downregulation of the transgene leads to further differentiation. Loss-of-function also leads to differentiation, toward endoderm and mesoderm. Taken together, these data indicate that a balance of FOXD3 activity is required to maintain pluripotency. STEM Cells2012;30:2188-2198.
Stem Cells 08/2012; 30(10):2188-98. · 7.78 Impact Factor
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ABSTRACT: Cells in the pluripotent state have the ability to self-renew indefinitely and to differentiate to all the cells of the embryo. These cells provide an in vitro window into development, including human development, as well as holding extraordinary promise for cell-based therapies in regenerative medicine. The recent demonstration that somatic cells can be reprogrammed to the pluripotent state has raised the possibility of patient and disease-specific induced pluripotent cells. In this article, we review the molecular underpinning of pluripotency. We focus on the transcriptional and signaling networks that underlie the state of pluripotency and control differentiation. In general, the action of each of the molecular components and pathways is dose and context dependent highlighting the need for a systems approach to understanding pluripotency. WIREs Syst Biol Med 2012. doi: 10.1002/wsbm.1182 For further resources related to this article, please visit the WIREs website.
Wiley Interdisciplinary Reviews Systems Biology and Medicine 07/2012; 4(5):443-56.
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ABSTRACT: The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated R-Smads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.
Proceedings of the National Academy of Sciences 06/2012; 109(28):E1947-56. · 9.68 Impact Factor
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ABSTRACT: Multiple levels of control are in play to regulate pluripotency and differentiation in human embryonic stem cells (hESCs). At the transcriptional level, the core factors OCT4, NANOG and SOX2 form a positive autoregulatory loop that is pivotal for maintaining the undifferentiated state. At the post-transcriptional level, microRNAs (miRNAs) belonging to the miR-302 family are emerging as key players in the control of proliferation and cell fate determination during differentiation. Here, we show that the transcriptional factors OCT4 and NR2F2 (COUP-TFII) and the miRNA miR-302 are linked in a regulatory circuitry that critically regulate both pluripotency and differentiation in hESCs. In the undifferentiated state, both OCT4 and the OCT4-induced miR-302 directly repress NR2F2 at the transcriptional and post-transcriptional level, respectively. Conversely, NR2F2 directly inhibits OCT4 during differentiation, triggering a positive feedback loop for its own expression. In addition, we show that regulation of NR2F2 activity itself relies on alternative splicing and transcriptional start site choice to generate a full-length transcriptionally active isoform and shorter variants, which enhance the activity of the long isoform. During hESC differentiation, NR2F2 is first detected at the earliest steps of neural induction and thus is among the earliest human embryonic neural markers. Finally, our functional analysis points to a crucial role for NR2F2 in the activation of neural genes during early differentiation in humans. These findings introduce a new molecular player in the context of early embryonic stem cell state and cell fate determination in humans.
The EMBO Journal 01/2011; 30(2):237-48. · 9.20 Impact Factor
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ABSTRACT: In this issue of Cell Stem Cell, Warren et al. (2010) describe a new methodology, using synthetic mRNA, for efficiently generating iPSCs without compromising genomic integrity. This powerful approach can also be used for directed differentiation of iPSCs, or even for trans-differentiation to generate clinically relevant differentiated cell types.
Cell stem cell 11/2010; 7(5):549-50. · 23.56 Impact Factor
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ABSTRACT: The specification of left-right asymmetry is an evolutionarily conserved developmental process in vertebrates. The interplay between two TGFβ ligands, Derrière/GDF1 and Xnr1/Nodal, together with inhibitors such as Lefty and Coco/Cerl2, have been shown to provide the signals that lead to the establishment of laterality. However, molecular events leading to and following these signals remain mostly unknown. We find that APOBEC2, a member of the cytidine deaminase family of DNA/RNA editing enzymes, is induced by TGFβ signaling, and that its activity is necessary to specify the left-right axis in Xenopus and zebrafish embryos. Surprisingly, we find that APOBEC2 selectively inhibits Derrière, but not Xnr1, signaling. The inhibitory effect is conserved, as APOBEC2 blocks TGFβ signaling, and promotes muscle differentiation, in a mammalian myoblastic cell line. This demonstrates for the first time that a putative RNA/DNA editing enzyme regulates TGFβ signaling and plays a major role in development.
Developmental Biology 09/2010; 350(1):13-23. · 4.07 Impact Factor
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Yutaka Shimomura,
Dritan Agalliu,
Alin Vonica,
Victor Luria,
Muhammad Wajid,
Alessandra Baumer,
Serena Belli,
Lynn Petukhova,
Albert Schinzel, Ali H Brivanlou,
Ben A Barres,
Angela M Christiano
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ABSTRACT: Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.
Nature 04/2010; 464(7291):1043-7. · 36.28 Impact Factor
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ABSTRACT: Metazoan organisms can 'scale', that is, maintain similar proportions regardless of size. Ben-Zvi et al. use experiments in Xenopus to support a quantitative model that explains morphological scaling as the result of scaling of a gradient of bone morphogenetic protein (BMP) signals. We believe that the evidence for scaling in Xenopus is misinterpreted, and that their model for embryonic patterning disagrees with prior data. The experiments they present supporting their model admit alternative interpretations.
Nature 10/2009; 461(7260):E1; discussion E2. · 36.28 Impact Factor
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ABSTRACT: Unraveling the therapeutic potential of human embryonic stem cells (hESC) requires tools to modify their genome. We have engineered the PiggyBac transposable element to create an efficient system for gene delivery in hESCs. This redesigned system, named "ePiggyBac," can deliver up to 18 Kb inserts, and transgene expression is observed in almost 90% of hES cells. ePiggyBac transposons can also carry insulators, inducible expression cassettes, and short hairpin RNAs for gain- and loss-of-function approaches. In hES cells, ePiggyBac's efficiency is superior to that of viral vectors and previously described transposons, including other PiggyBac-based systems. In addition, ePiggyBac transgenes can be removed from the hESC genome without leaving any mutation. We used this system to direct hESC differentiation toward a neuronal phenotype. We then removed the transposons to obtain transgene-free neuronal precursors and neurons. The ability to create fully reversible genetic modifications represents an important step toward clinical applications of hESCs.
Cell stem cell 10/2009; 5(3):332-42. · 23.56 Impact Factor
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ABSTRACT: Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor beta (TGFbeta) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of beta-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure.
Journal of Biological Chemistry 07/2009; 284(38):26127-36. · 4.77 Impact Factor
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ABSTRACT: The role of microRNAs in embryonic cell fate specification is largely unknown. In vertebrates, the miR-430/427/302 family shows a unique expression signature and is exclusively expressed during early embryogenesis. Here, we comparatively address the embryonic function of miR-302 in human embryonic stem cells (hESCs) and its ortholog miR-427 in Xenopus laevis. Interestingly, we found that this miRNA family displays species-specific target selection among ligands of the Nodal pathway, with a striking conservation of the inhibitors, Lefties, but differential targeting of the activators, Nodals. The Nodal pathway plays a crucial role in germ layer specification. Accordingly, by gain and loss of function experiments in hESCs, we show that miR-302 promotes the mesendodermal lineage at the expense of neuroectoderm formation. Similarly, depletion of miR-427 in Xenopus embryos hinders the organizer formation and leads to severe dorsal mesodermal patterning defects. These findings suggest a crucial role for the miR-430/427/302 family in vertebrate embryogenesis by controlling germ layer specification.
Developmental cell 05/2009; 16(4):517-27. · 13.36 Impact Factor
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ABSTRACT: Within the TGF-beta superfamily, there are approximately forty ligands divided into two major branches: the TGF-beta/Activin/Nodal ligands and the BMP/GDF ligands. We studied the ligand GDF3 and found that it inhibits signaling by its co-family members, the BMPs; however, GDF3 has been described by others to have Nodal-like activity. Here, we show that GDF3 can activate Nodal signaling, but only at very high doses and only upon mRNA over-expression. In contrast, GDF3 inhibits BMP signaling upon over-expression of GDF3 mRNA, as recombinant protein, and regardless of its dose. We therefore further characterized the mechanism through which GDF3 protein acts as a specific BMP inhibitor and found that the BMP inhibitory activity of GDF3 resides redundantly in the unprocessed, predominant form and in the mature form of the protein. These results confirm and extend the activity that we described for GDF3 and illuminate the experimental basis for the different observations of others. We suggest that GDF3 is either a bi-functional TGF-beta ligand, or, more likely, that it is a BMP inhibitor that can artificially activate Nodal signaling under non-physiological conditions.
Developmental Biology 10/2008; 325(1):43-8. · 4.07 Impact Factor
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ABSTRACT: Mechanisms underlying regional specification of distinct organ precursors within the endoderm, including the liver and pancreas, are still poorly understood. This is particularly true for stages between endoderm formation and the initiation of organogenesis. In this report, we have investigated these intermediate steps downstream of the early endodermal factor Gata5, which progressively lead to the induction of pancreatic fate. We have identified TGIF2 as a novel Gata5 target and demonstrate its function in the establishment of the pancreatic region within dorsal endoderm in Xenopus. TGIF2 acts primarily by restricting BMP signaling in the endoderm to allow pancreatic formation. Consistently, we found that blocking BMP signaling by independent means also perturbs the establishment of pancreatic identity in the endoderm. Previous findings demonstrated a crucial role for BMP signaling in determining dorsal/ventral fates in ectoderm and mesoderm. Our results now extend this trend to the endoderm and identify TGIF2 as the molecular link between dorsoventral patterning of the endoderm and pancreatic specification.
Development 03/2008; 135(3):451-61. · 6.60 Impact Factor
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ABSTRACT: How does the vertebrate embryo make a nervous system? This complex question has been at the center of developmental biology for many years. The earliest step in this process - the induction of neural tissue - is intimately linked to patterning of the entire early embryo, and the molecular and embryological of basis these processes are beginning to emerge. Here, we analyze classic and cutting-edge findings on neural induction in the mouse. We find that data from genetics, tissue explants, tissue grafting, and molecular marker expression support a coherent framework for mammalian neural induction. In this model, the gastrula organizer of the mouse embryo inhibits BMP signaling to allow neural tissue to form as a default fate-in the absence of instructive signals. The first neural tissue induced is anterior and subsequent neural tissue is posteriorized to form the midbrain, hindbrain, and spinal cord. The anterior visceral endoderm protects the pre-specified anterior neural fate from similar posteriorization, allowing formation of forebrain. This model is very similar to the default model of neural induction in the frog, thus bridging the evolutionary gap between amphibians and mammals.
Developmental Biology 09/2007; 308(2):247-56. · 4.07 Impact Factor
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ABSTRACT: We describe procedures for engrafting human embryonic stem (ES) cells into blastocyst stage mouse embryos. In terms of developmental time in the mouse, the blastocyst represents a closely related in vivo embryonic environment for the human ES cells. This tool can be used to study embryological properties of human ES cells and, by using genetically modified or mutant human ES cells as graft, can be extended to study the basis of disease. Protocols are presented include: (a) production of host embryos for human ES cell incorporation, (b) preparation of pseudo-pregnant foster mothers for manipulated embryos, (c) preparation of human ES cells for morula aggregation or blastocyst injection, (d) injection or aggregation of human ES cells into appropriate stage embryos, and (e) growth in vitro as differentiating outgrowths or transfer to pseudo-pregnant foster mothers for in vivo development.Protocols for traditional, though less rigorous, methods for assessing in vivo potential of human ES cells (teratoma formation in immunocomprimised mice via kidney capsule, intramuscular and subcutaneous injection) are also included.
06/2007: pages 121 - 147; , ISBN: 9780470511619
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ABSTRACT: Formation of the left-right axis involves a symmetry-breaking signal originating in the node or its equivalents, which increases TGF-beta signaling on the left side of the embryo and ultimately leads to asymmetric patterning of the viscera. DAN domain proteins are extracellular inhibitors of TGF-beta ligands, and are involved in regulating the left-right axis in chick, mouse and zebrafish. We find that Coco, a Xenopus DAN family member, and two TGF-beta ligands, Xnr1 and derrière, are coexpressed in the posterior paraxial mesoderm at neurula stage. Side-specific protein depletion demonstrated that left-right patterning requires Coco exclusively on the right side, and Xnr1 and derrière exclusively on the left, despite their bilateral expression pattern. In the absence of Coco, the TGF-beta signal is bilateral. Interactions among the three proteins show that derrière is required for normal levels of Xnr1 expression, while Coco directly inhibits both ligands. We conclude that derrière, Xnr1, and Coco define a posttranscriptionally regulated signaling center, which is a necessary link in the signaling chain leading to an increased TGF-beta signal on the left side of the embryo.
Developmental Biology 04/2007; 303(1):281-94. · 4.07 Impact Factor
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ABSTRACT: FGF and other Ras/MAPK pathway activators counterbalance BMP action during neurogenesis, bone formation, and other aspects of vertebrate development and homeostasis. BMP receptors signal through C-terminal phosphorylation and nuclear translocation of the transcription factor Smad1, whereas MAPKs catalyze inhibitory phosphorylation in the Smad1 linker region. Here we show that linker phosphorylation restricts Smad1 activity by enabling Smad1 recognition by the HECT-domain ubiquitin ligase Smurf1. Besides causing Smad1 polyubiquitination, Smurf1 binding inhibits the interaction of Smad1 with the nuclear translocation factor Nup214. Consequently, MAPK-dependent Smurf1 binding leads Smad1 alternatively to degradation or cytoplasmic retention. Smad1 linker phosphorylation and Smurf1 act as interdependent inputs to control BMP signaling during mouse osteoblast differentiation and Xenopus neural development. Linker phosphorylation is triggered also by BMP, providing feedback control. The interplay between linker phosphorylation, Smurf-dependent ubiquitination, and nucleoporin exclusion enables regulation of BMP action by diverse signals and biological contexts.
Molecular Cell 03/2007; 25(3):441-54. · 14.18 Impact Factor
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ABSTRACT: Cancer cells have the ability to divide indefinitely and spread to different parts of the body during metastasis. Embryonic stem cells can self-renew and, through differentiation to somatic cells, provide the building blocks of the human body. Embryonic stem cells offer tremendous opportunities for regenerative medicine and serve as an excellent model system to study early human development. Many of the molecular mechanism underlying tumorigenesis in cancer and self-renewal in stem cells have been elucidated in the past decade. Here we present a systematic analysis of seven major signaling pathways implicated in both cancer and stem cells. We present on overview of the JAK/STAT, Notch, MAPK/ERK, PI3K/AKT, NF-kB, Wnt and TGF-beta pathways and analyze their activation status in the context of cancer and stem cells. We focus on their role in stem cell self-renewal and development and identify key molecules, whose aberrant expression has been associated with malignant phenotypes. We conclude by presenting a map of the signaling networks involved in cancer and embryonic stem cells.
Stem Cell Reviews and Reports 02/2007; 3(1):7-17. · 3.74 Impact Factor
