In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm Identity
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{"title"=>"In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity", "type"=>"journal", "authors"=>[{"first_name"=>"Mina", "last_name"=>"Gouti", "scopus_author_id"=>"23466699200"}, {"first_name"=>"Anestis", "last_name"=>"Tsakiridis", "scopus_author_id"=>"16687309100"}, {"first_name"=>"Filip J.", "last_name"=>"Wymeersch", "scopus_author_id"=>"24402162300"}, {"first_name"=>"Yali", "last_name"=>"Huang", "scopus_author_id"=>"55503575800"}, {"first_name"=>"Jens", "last_name"=>"Kleinjung", "scopus_author_id"=>"6602571605"}, {"first_name"=>"Valerie", "last_name"=>"Wilson", "scopus_author_id"=>"7202995750"}, {"first_name"=>"James", "last_name"=>"Briscoe", "scopus_author_id"=>"7005150612"}], "year"=>2014, "source"=>"PLoS Biology", "identifiers"=>{"issn"=>"15457885", "scopus"=>"2-s2.0-84908425180", "sgr"=>"84908425180", "pui"=>"604853549", "isbn"=>"10.1371/journal.pbio.1001937", "pmid"=>"25157815", "doi"=>"10.1371/journal.pbio.1001937"}, "id"=>"fc2f8911-5112-3380-a8bb-fc62271e6833", "abstract"=>"Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated in vitro from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate in vivo development. Similar to NMPs in vivo, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial mesoderm in vitro and in vivo. The neural cells produced by NMPs have spinal cord but not anterior neural identity and can differentiate into spinal cord motor neurons. This is consistent with the shared origin of spinal cord and somites and the distinct ontogeny of the anterior and posterior nervous system. Systematic analysis of the transcriptome during differentiation identifies the molecular correlates of each of the cell identities and the routes by which they are obtained. Moreover, we take advantage of the system to provide evidence that Brachyury represses neural differentiation and that signals from mesoderm are not necessary to induce the posterior identity of spinal cord cells. This indicates that the mesoderm inducing and posteriorising functions of Wnt signalling represent two molecularly separate activities. Together the data illustrate how reverse engineering normal developmental mechanisms allows the differentiation of specific cell types in vitro and the analysis of previous difficult to access aspects of embryo development.", "link"=>"http://www.mendeley.com/research/vitro-generation-neuromesodermal-progenitors-reveals-distinct-roles-wnt-signalling-specification-spi", "reader_count"=>218, "reader_count_by_academic_status"=>{"Unspecified"=>4, "Professor > Associate Professor"=>7, "Researcher"=>50, "Student > Doctoral Student"=>11, "Student > Ph. D. Student"=>66, "Student > Postgraduate"=>7, "Student > Master"=>34, "Other"=>7, "Student > Bachelor"=>26, "Professor"=>6}, "reader_count_by_user_role"=>{"Unspecified"=>4, "Professor > Associate Professor"=>7, "Researcher"=>50, "Student > Doctoral Student"=>11, "Student > Ph. D. Student"=>66, "Student > Postgraduate"=>7, "Student > Master"=>34, "Other"=>7, "Student > Bachelor"=>26, "Professor"=>6}, "reader_count_by_subject_area"=>{"Unspecified"=>6, "Engineering"=>8, "Biochemistry, Genetics and Molecular Biology"=>49, "Mathematics"=>1, "Agricultural and Biological Sciences"=>120, "Medicine and Dentistry"=>10, "Neuroscience"=>19, "Design"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemical Engineering"=>1, "Physics and Astronomy"=>1, "Social Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Design"=>{"Design"=>1}, "Engineering"=>{"Engineering"=>8}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>10}, "Neuroscience"=>{"Neuroscience"=>19}, "Social Sciences"=>{"Social Sciences"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>120}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>49}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>6}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"Argentina"=>1, "Sweden"=>2, "United States"=>2, "China"=>1, "Brazil"=>1, "United Kingdom"=>4, "France"=>2, "Australia"=>1, "Chile"=>1, "Switzerland"=>1}, "group_count"=>5}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1650691"], "description"=>"<p>(A) Schematic of the differentiation conditions used for transcriptome analyses. Three independent samples of RNA were collected at the indicated time points from each of the conditions and analysed by mRNA-seq. (B) Principal component analysis of the triplicate samples at each time point of differentiation shows the distinct trajectories for each of the developmental pathways. The first principal component (PC1) appears to represent the time of differentiation whereas PC2 represents the treatment regime. Note that PC3 separates N<sub>H</sub> and N<sub>P</sub> at D5. Anterior neural (N<sub>A</sub>) and hindbrain neural (N<sub>H</sub>) appear to share a common trajectory, whereas posterior neural (N<sub>P</sub>) appear to share a common trajectory with paraxial mesodermal cells (Meso). (C) Heatmap of expression levels of a subset of the genes significantly and uniquely upregulated in NMPs compared to other samples. A complete list of genes uniquely upregulated in NMPs is summarized in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s006\" target=\"_blank\">Table S1</a>. The data used to generate the heatmap can be found in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s011\" target=\"_blank\">Data S3</a>.</p>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152041, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001937.g003", "stats"=>{"downloads"=>3, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Induction_of_a_distinct_transcriptional_programme_in_neuromesodermal_progenitors_/1152041", "title"=>"Induction of a distinct transcriptional programme in neuromesodermal progenitors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-26 07:24:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1650669"], "description"=>"<p>(A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N<sub>A</sub>), Hindbrain (N<sub>H</sub>) and Spinal cord (N<sub>P</sub>) identities. (B) Relative expression levels of the indicated genes from N<sub>A</sub>, N<sub>H</sub> and N<sub>P</sub> cells at day 5 (D5) of differentiation indicate that N<sub>A</sub>, N<sub>H</sub> and N<sub>P</sub> cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N<sub>A</sub> cells express high levels of forebrain markers including <i>Otx1</i> and <i>Otx2</i>; N<sub>H</sub> cells express genes characteristic of hindbrain including <i>Mafb</i> and <i>Hoxa2</i> genes; N<sub>P</sub> cells express high levels of posterior 5′ Hox genes including <i>Hoxc8</i> and <i>Hoxc9</i>. The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N<sub>H</sub> and N<sub>P</sub> conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N<sub>P</sub> conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937-Kmita1\" target=\"_blank\">[74]</a>. In N<sub>H</sub> cells <i>Hoxb1</i> and <i>Hoxb2</i> are induced prior to <i>Hoxc4</i>. However, the more posterior Hox genes are not induced. By contrast, in N<sub>P</sub> conditions the 5′ Hox genes <i>Hoxc6</i>, <i>Hoxc8</i> and <i>Hoxc9</i> are induced at D4 and their expression is maintained at day 5. (Note log<sub>2</sub> scale). (D) Immunohistochemistry indicates that N<sub>H</sub> cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N<sub>P</sub> cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N<sub>H</sub> conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N<sub>P</sub> conditions. All data used to generate the plots of <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio-1001937-g001\" target=\"_blank\">Figure 1</a> can be found in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s009\" target=\"_blank\">Data S1</a>.</p>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152021, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001937.g001", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_neural_cells_with_specific_AP_identities_from_ESCs_/1152021", "title"=>"Generation of neural cells with specific AP identities from ESCs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-26 07:24:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1650778", "https://ndownloader.figshare.com/files/1650779", "https://ndownloader.figshare.com/files/1650780", "https://ndownloader.figshare.com/files/1650781", "https://ndownloader.figshare.com/files/1650782", "https://ndownloader.figshare.com/files/1650783", "https://ndownloader.figshare.com/files/1650784", "https://ndownloader.figshare.com/files/1650785", "https://ndownloader.figshare.com/files/1650786", "https://ndownloader.figshare.com/files/1650787", "https://ndownloader.figshare.com/files/1650789", "https://ndownloader.figshare.com/files/1650791", "https://ndownloader.figshare.com/files/1650792", "https://ndownloader.figshare.com/files/1650794", "https://ndownloader.figshare.com/files/1650795", "https://ndownloader.figshare.com/files/1650796", "https://ndownloader.figshare.com/files/1650797", "https://ndownloader.figshare.com/files/1650798"], "description"=>"<div><p>Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated <i>in vitro</i> from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate <i>in vivo</i> development. Similar to NMPs <i>in vivo</i>, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial mesoderm <i>in vitro</i> and <i>in vivo</i>. The neural cells produced by NMPs have spinal cord but not anterior neural identity and can differentiate into spinal cord motor neurons. This is consistent with the shared origin of spinal cord and somites and the distinct ontogeny of the anterior and posterior nervous system. Systematic analysis of the transcriptome during differentiation identifies the molecular correlates of each of the cell identities and the routes by which they are obtained. Moreover, we take advantage of the system to provide evidence that Brachyury represses neural differentiation and that signals from mesoderm are not necessary to induce the posterior identity of spinal cord cells. This indicates that the mesoderm inducing and posteriorising functions of Wnt signalling represent two molecularly separate activities. Together the data illustrate how reverse engineering normal developmental mechanisms allows the differentiation of specific cell types <i>in vitro</i> and the analysis of previous difficult to access aspects of embryo development.</p></div>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152112, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.1001937.s001", "https://dx.doi.org/10.1371/journal.pbio.1001937.s002", "https://dx.doi.org/10.1371/journal.pbio.1001937.s003", "https://dx.doi.org/10.1371/journal.pbio.1001937.s004", "https://dx.doi.org/10.1371/journal.pbio.1001937.s005", "https://dx.doi.org/10.1371/journal.pbio.1001937.s006", "https://dx.doi.org/10.1371/journal.pbio.1001937.s007", "https://dx.doi.org/10.1371/journal.pbio.1001937.s008", "https://dx.doi.org/10.1371/journal.pbio.1001937.s009", "https://dx.doi.org/10.1371/journal.pbio.1001937.s010", "https://dx.doi.org/10.1371/journal.pbio.1001937.s011", "https://dx.doi.org/10.1371/journal.pbio.1001937.s012", "https://dx.doi.org/10.1371/journal.pbio.1001937.s013", "https://dx.doi.org/10.1371/journal.pbio.1001937.s014", "https://dx.doi.org/10.1371/journal.pbio.1001937.s015", "https://dx.doi.org/10.1371/journal.pbio.1001937.s016", "https://dx.doi.org/10.1371/journal.pbio.1001937.s017", "https://dx.doi.org/10.1371/journal.pbio.1001937.s018"], "stats"=>{"downloads"=>9, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_Vitro_Generation_of_Neuromesodermal_Progenitors_Reveals_Distinct_Roles_for_Wnt_Signalling_in_the_Specification_of_Spinal_Cord_and_Paraxial_Mesoderm_Identity/1152112", "title"=>"<i>In Vitro</i> Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm Identity", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-08-26 07:24:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1650716"], "description"=>"<p>(A) Brachyury/Sox2 immunocytochemistry in EpiSC cultures treated with FGF/CHIR for 72 h. (B) qPCR analysis for indicated markers in mouse EpiSCs treated with FGF/CHIR. Error bars = s.d. (n = 3). n/d, not determined. Results are represented as log<sub>10</sub> ratio of expression versus untreated EpiSCs. The data used to generate the plot can be found in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s012\" target=\"_blank\">Data S4</a>. (C) Combined fluorescence/brightfield microscopy showing donor cell incorporation of grafted GFP<sup>+</sup> EpiSC differentiated for 48 h in FGF/CHIR after 48 h embryo culture. (D) Table summarizing the incorporation of grafted GFP<sup>+</sup> EpiSC differentiated for 24 h or 48 h in Fgf/Wnt within host embryos. NT, neural tube; Som, somite; PSM, presomitic mesoderm; n/a, not applicable. (E) Representative examples of donor cell incorporation (green, GFP) and differentiation (red, immunofluorescence for indicated markers). Cell nuclei were stained with DAPI (blue). White boxes indicate the position of magnified images of GFP<sup>+</sup> cells.</p>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152057, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001937.g004", "stats"=>{"downloads"=>1, "page_views"=>49, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_NMPs_from_EpiSCs_/1152057", "title"=>"Generation of NMPs from EpiSCs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-26 07:24:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1650734"], "description"=>"<p>(A) Schematic of the conditions used for mesoderm differentiation. (B) qRT-PCR analysis of the expression of <i>Tbx6</i>, <i>Cdx2</i> and <i>Hoxb1</i> relative to b-actin at D3 of differentiation in wild-type (wt) and Brachyury null cells (Bra<sup>−/−</sup>) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while <i>Cdx2</i> and <i>Hoxb1</i> continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra<sup>−/−</sup> ESCs exposed to CHIR from D2–D5 (Meso conditions). Posterior Hox genes <i>Hoxc8</i> and <i>Hoxc9</i> are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers <i>Sox1</i> and <i>Sox2</i> are expressed only in Bra<sup>−/−</sup> cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra<sup>−/−</sup> and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra<sup>−/−</sup> cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not β-Tubulin (Tuj1) whereas Bra<sup>−/−</sup> cells fail to produce Desmin/MyoD and differentiate into neurons expressing β-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N<sub>P</sub>) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log<sub>2+</sub> scale). All data used for the plots can be found in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s014\" target=\"_blank\">Data S6</a>. (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling.</p>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152074, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001937.g006", "stats"=>{"downloads"=>6, "page_views"=>114, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Brachyury_is_necessary_for_mesoderm_formation_but_not_posterior_neural_identity_/1152074", "title"=>"Brachyury is necessary for mesoderm formation but not posterior neural identity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-26 07:24:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1650687"], "description"=>"<p>(A) Schematic of differentiation protocols used to generate mesoderm and neural cells from a common NM progenitor population. (B) mRNA-seq expression values of <i>Sox2</i>, <i>Brachyury</i>, <i>Tbx6</i> and <i>Cdx2</i> following exposure to bFGF alone or bFGF/CHIR for 12 h (D2.5) and 24 h (D3). Activation of Wnt signalling with CHIR upregulated Brachyury within 12 h. Expression of <i>Tbx6</i> and <i>Cdx2</i> was also upregulated in NMPs by D3, whereas <i>Sox2</i> transcript levels were decreased. (C) Immunostaining of cells treated with FGF/Wnt revealed the coexpression of Brachyury with Sox2 (NMPs). In the absence of Wnt, NPCs express Sox2 but the expression of Brachyury is only evident in a very small proportion of cells. (D) mRNA expression values of neural (<i>Sox1</i>, <i>Sox2</i>, <i>Sox3</i>) and mesodermal progenitors markers (<i>Tbx6</i>, <i>Bra</i>, <i>Msgn1</i>) in posterior neural (N<sub>P</sub>) and mesodermal cells (Meso) at D5 show the generation of distinct populations depending on treatment after D3. Removal of Wnt at D3 results in the generation of neural cells expressing Sox1–3 whereas continued Wnt exposure induces expression of Tbx6, Brachyury and Msgn1, characteristic of paraxial mesodermal. (E) Immunostaining indicates that continued Wnt exposure generates paraxial mesodermal progenitors that express Tbx6 at D5 and Desmin and MyoD at D8. (F) Sketch of a chick embryo (HH8–9) showing the injection site (IS) of NMP or N<sub>A</sub> cells. (G) NMP cells were labelled with DiI and transplanted in the CLE region. After 24 h the cells had incorporated into both the neural tube and somites. Whole-mount and transverse sections of HH17 chick embryos show the incorporation (asterisks) in the neural tube (H) and somites (I). (J) Table summarizing the number of chick embryos that were injected at stage HH8–9 and had engrafted cells in the neural tube, the somites or both 24 h later. Injection of N<sub>A</sub> cells resulted in incorporation only in the neural tube (K, L). All data used to generate the plots of <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio-1001937-g002\" target=\"_blank\">Figure 2</a> can be found in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s010\" target=\"_blank\">Data S2</a>.</p>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152037, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001937.g002", "stats"=>{"downloads"=>0, "page_views"=>36, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transient_Wnt_and_FGF_signalling_induce_dual_fated_neuromesodermal_progenitors_/1152037", "title"=>"Transient Wnt and FGF signalling induce dual fated neuromesodermal progenitors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-26 07:24:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1650727"], "description"=>"<p>(A) Scheme describing the culture conditions employed for neural differentiation of hES cells treated for 72 h either with FGF/CHIR or subjected to dual SMAD inhibition (LDN, LDN193189; SB43, SB431542). (B) BRACHYURY/SOX2 immunocytochemistry in undifferentiated and FGF/CHIR-treated (48 h) hES cells. Corresponding graphs depict image analysis of BRACHYURY and SOX2 expression in the indicated culture conditions. Numbers: percentages of cells in each quadrant. (C) qPCR analysis for indicated markers in hES cells treated with FGF/CHIR for 72 h (D3) or 96 h (D4). Error bars = s.d. (n = 2). Results are represented as log<sub>10</sub> ratio of expression versus untreated hES cells. (D) qPCR analysis for indicated differentiation markers in hES cells differentiated in N2B27 following either an NM progenitor induction- (N<sub>P</sub>) or a dual SMAD inhibition-intermediate step (N<sub>A</sub>). Error bars = s.d. (n = 2). Anterior, anterior neural markers; PXM, paraxial mesoderm; n/d, not determined. (E) Immunocytochemistry for SOX2/HOXC8 in N<sub>A</sub> and N<sub>P</sub> culture conditions indicated in the scheme (A). (F) Quantitation of the coexpression of Hoxc8 with Sox2 in N<sub>A</sub> and N<sub>P</sub> conditions. All data used to generate the plots can be found in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s013\" target=\"_blank\">Data S5</a>.</p>", "links"=>[], "tags"=>["Vitro Generation", "differentiation", "Neuromesodermal Progenitors", "cord motor neurons", "Brachyury represses", "Distinct Roles", "vivo development", "mesodermal factor Brachyury", "cell identities", "posteriorising functions", "Paraxial Mesoderm Identity Cells", "cord cells", "Spinal cord", "Systematic analysis", "factor Sox 2", "nmp", "Wnt Signalling", "embryo development", "access aspects", "cell types", "identity", "elongating embryo"], "article_id"=>1152068, "categories"=>["Biological Sciences"], "users"=>["Mina Gouti", "Anestis Tsakiridis", "Filip J. Wymeersch", "Yali Huang", "Jens Kleinjung", "Valerie Wilson", "James Briscoe"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001937.g005", "stats"=>{"downloads"=>0, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_and_characterisation_of_hNMPs_/1152068", "title"=>"Generation and characterisation of hNMPs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-26 07:24:47"}

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