{"id":["DOI:10.1016/j.devcel.2024.10.020"],"title":["Initiation and maintenance of the pluripotent epiblast in pre-implantation human development is independent of NODAL signaling"],"publication_date":["2024-11-18"],"authors":{"ORCID:0000-0001-8371-847X":{"id":["ORCID:0000-0001-8371-847X"],"family_name":["Brumm"],"personal_name":["A. Sophie"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]},"ROR:01nrxwf90":{"organization_name":["University of Edinburgh"]}}},"ORCID:0009-0005-5450-8496":{"id":["ORCID:0009-0005-5450-8496"],"family_name":["McCarthy"],"personal_name":["Afshan"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}},"HDBItheme":["Early"]},"ORCID:0000-0002-9046-3820":{"id":["ORCID:0000-0002-9046-3820"],"family_name":["Gerri"],"personal_name":["Claudia"],"affiliations":{"ROR:05b8d3w18":{"organization_name":["Max Planck Institute of Molecular Cell Biology and Genetics"]}}},"ORCID:0000-0002-8700-3700":{"id":["ORCID:0000-0002-8700-3700"],"family_name":["Fallesen"],"personal_name":["Todd"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}}},"ORCID:0000-0003-4752-025X":{"id":["ORCID:0000-0003-4752-025X"],"family_name":["Woods"],"personal_name":["Laura"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-4358-569X":{"id":["ORCID:0000-0002-4358-569X"],"family_name":["McMahon"],"personal_name":["Riley"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-5541-1769":{"id":["ORCID:0000-0002-5541-1769"],"family_name":["Papathanasiou"],"personal_name":["Athanasios"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"ORCID:0000-0003-3510-8268":{"id":["ORCID:0000-0003-3510-8268"],"family_name":["Elder"],"personal_name":["Kay"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"AUTHTEMP:0003":{"id":["AUTHTEMP:0003"],"family_name":["Snell"],"personal_name":["Phil"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"AUTHTEMP:0004":{"id":["AUTHTEMP:0004"],"family_name":["Christie"],"personal_name":["Leila"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"AUTHTEMP:0009":{"id":["AUTHTEMP:0009"],"family_name":["Garcia"],"personal_name":["Patricia"],"affiliations":{}},"AUTHTEMP:0010":{"id":["AUTHTEMP:0010"],"family_name":["Shaikly"],"personal_name":["Valerie"],"affiliations":{}},"AUTHTEMP:0011":{"id":["AUTHTEMP:0011"],"family_name":["Taranissi"],"personal_name":["Mohamed"],"affiliations":{}},"AUTHTEMP:0006":{"id":["AUTHTEMP:0006"],"family_name":["Serhal"],"personal_name":["Paul"],"affiliations":{"ROR:05yqeks58":{"organization_name":["The Centre for Reproductive & Genetic Health"]}}},"ORCID:0000-0001-6384-2764":{"id":["ORCID:0000-0001-6384-2764"],"family_name":["Odia"],"personal_name":["Rabi A."],"affiliations":{"ROR:05yqeks58":{"organization_name":["The Centre for Reproductive & Genetic Health"]}}},"AUTHTEMP:0012":{"id":["AUTHTEMP:0012"],"family_name":["Vasilic"],"personal_name":["Mina"],"affiliations":{"ROR:05yqeks58":{"organization_name":["The Centre for Reproductive & Genetic Health"]}}},"ORCID:0000-0001-5241-1512":{"id":["ORCID:0000-0001-5241-1512"],"family_name":["Osnato"],"personal_name":["Anna"],"affiliations":{"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05f950310":{"organization_name":["KU Leuven"]}}},"ORCID:0000-0002-9601-5949":{"id":["ORCID:0000-0002-9601-5949"],"family_name":["Rugg-Gunn"],"personal_name":["Peter J."],"affiliations":{"1":{}},"HDBItheme":["Early"]},"ORCID:0000-0002-3848-2602":{"id":["ORCID:0000-0002-3848-2602"],"family_name":["Vallier"],"personal_name":["Ludovic"],"affiliations":{"ROR:0493xsw21":{"organization_name":["Berlin Institute of Health at Charité - Universitätsmedizin Berlin"]},"ROR:03ate3e03":{"organization_name":["Max Planck Institute for Molecular Genetics"]}}},"ORCID:0000-0002-8632-0480":{"id":["ORCID:0000-0002-8632-0480"],"family_name":["Hill"],"personal_name":["Caroline S."],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}}},"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]}},"abstract":["The human blastocyst contains the pluripotent epiblast from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signaling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterized NODAL signaling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signaling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signaling activity. Genome-wide transcriptional analysis showed that NODAL signaling inhibition did not decrease NANOG transcription or impact the wider pluripotency-associated gene regulatory network. These data suggest differences in the signaling requirements regulating pluripotency in the pre-implantation human epiblast compared with existing hESC culture."],"HDBItheme":["Early"],"datasets":{"BioStudies:S-BIAD1398":{"id":["BioStudies:S-BIAD1398"],"title":["NODAL signalling does not initiate or maintain the pluripotent epiblast in pre-implantation human development"],"description":["The human blastocyst contains the pluripotent epiblast, from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signalling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterised NODAL signalling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signalling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signalling activity. Genome-wide transcriptional analysis showed that NODAL signalling inhibition did not impact on NANOG transcription or the wider pluripotency-associated gene regulatory network. These data suggest differences in the signalling requirements regulating pluripotency in the pre-implantation human epiblast compared to existing hESC culture."],"authors":{"ORCID:0000-0001-8371-847X":{"id":["ORCID:0000-0001-8371-847X"],"family_name":["Brumm"],"personal_name":["A. Sophie"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]},"ROR:01nrxwf90":{"organization_name":["University of Edinburgh"]}}},"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]}},"tags":["imaging"]}},"code":{"DOI:10.25418/crick.19383428.v1":{"id":["DOI:10.25418/crick.19383428.v1"],"title":["Segmentation and image analysis pipeline for the investigation of lineage marker expression in early human development"],"description":["The pipeline makes possible the analysis of confocal imaging data acquired from preimplantation human embryos at blastocyst stage.\nThrough several stages, the pipeline first performs nuclear segmentation throughout the entire human blastocyst, including the inner cell mass. The map of nuclei is then used to track each nucleus, extract, and integrate quantitative information from the identified nuclei in 3D. The quantification of fluorescence intensity measurements from individual nuclei in several wavelengths enables the analysis of lineage marker expression, and quantification of signalling pathway activities."],"authors":{"ORCID:0000-0002-8700-3700":{"id":["ORCID:0000-0002-8700-3700"],"family_name":["Fallesen"],"personal_name":["Todd"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}}},"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]},"ORCID:0000-0001-8371-847X":{"id":["ORCID:0000-0001-8371-847X"],"family_name":["Brumm"],"personal_name":["A. Sophie"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]},"ROR:01nrxwf90":{"organization_name":["University of Edinburgh"]}}}}}},"protocols":{"DOI:10.1016/j.xpro.2025.103849":{"id":["DOI:10.1016/j.xpro.2025.103849"],"title":["Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts"],"description":["The transforming growth factor β (TGF-β) signaling superfamily includes NODAL and bone morphogenetic protein (BMP) signaling, which lead to the phosphorylation of different SMAD proteins and regulate key developmental events. Here, we present a protocol for immunofluorescence detection of phosphorylated SMAD proteins combined with other transcription factors in pre-implantation human embryos. We describe steps for segmenting the nuclei in human blastocysts and quantifying their immunofluorescence intensity. This protocol can be adapted to investigate TGF-β superfamily signaling activity in other mammalian embryos or in vitro models of their development. For complete details on the use and execution of this protocol, please refer to Brumm et al. https://doi.org/10.1016/j.devcel.2024.10.020"],"authors":{"ORCID:0000-0002-8700-3700":{"id":["ORCID:0000-0002-8700-3700"],"family_name":["Fallesen"],"personal_name":["Todd"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}}},"ORCID:0000-0001-8371-847X":{"id":["ORCID:0000-0001-8371-847X"],"family_name":["Brumm"],"personal_name":["A. Sophie"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]},"ROR:01nrxwf90":{"organization_name":["University of Edinburgh"]}}}}}}}