Peer reviewed: TrueAcknowledgements: We are grateful to Kenneth Jones, Ayaka Yanagida and Lawrence Bates for assistance with human embryo thawing; Peter Humphreys, Darran Clement (Cambridge Stem Cell Institute imaging facility) and Ann Wheeler (Institute of Genetics and Cancer) for help with microscopy, and core facilities at both Institutes; and Sophie Kraunsoe for assistance in image analysis. We thank all the staff at our participating assisted conception clinics and the patients who kindly donated their embryos to our research.Funder: Microsoft Research; doi: http://dx.doi.org/10.13039/100006112Funder: University of Cambridge; doi: http://dx.doi.org/10.13039/501100000735Funder: University of Edinburgh; doi: http://dx.doi.org/10.13039/501100000848Successful human pregnancy depends upon rapid establishment of three founder lineages: the trophectoderm, epiblast and hypoblast, which together form the blastocyst. Each plays an essential role in preparing the embryo for implantation and subsequent development. Several models have been proposed to define the lineage segregation. One suggests that all lineages specify simultaneously; another favours the differentiation of the trophectoderm before separation of the epiblast and hypoblast, either via differentiation of the hypoblast from the established epiblast, or production of both tissues from the inner cell mass precursor. To begin to resolve this discrepancy and thereby understand the sequential process for production of viable human embryos, we investigated the expression order of genes associated with emergence of hypoblast. Based upon published data and immunofluorescence analysis for candidate genes, we present a basic blueprint for human hypoblast differentiation, lending support to the proposed model of sequential segregation of the founder lineages of the human blastocyst. The first characterised marker, specific initially to the early inner cell mass, and subsequently identifying presumptive hypoblast, is PDGFRA, followed by SOX17, FOXA2 and GATA4 in sequence as the hypoblast becomes committed
