RESUMO
A crucial aspect of embryology is relating the position of individual cells to the broader geometry of the embryo. A classic example of this is the first cell-fate decision of the mouse embryo, where interior cells become inner cell mass and exterior cells become trophectoderm. Fluorescent labelling, imaging, and quantification of tissue-specific proteins have advanced our understanding of this dynamic process. However, instances arise where these markers are either not available, or not reliable, and we are left only with the cells' spatial locations. Therefore, a simple, robust method for classifying interior and exterior cells of an embryo using spatial information is required. Here, we describe a simple mathematical framework and an unsupervised machine learning approach, termed insideOutside, for classifying interior and exterior points of a three-dimensional point-cloud, a common output from imaged cells within the early mouse embryo. We benchmark our method against other published methods to demonstrate that it yields greater accuracy in classification of nuclei from the pre-implantation mouse embryos and greater accuracy when challenged with local surface concavities. We have made MATLAB and Python implementations of the method freely available. This method should prove useful for embryology, with broader applications to similar data arising in the life sciences.
Assuntos
Algoritmos , Disciplinas das Ciências Biológicas , Animais , Camundongos , Núcleo Celular , Blastocisto , Diferenciação CelularRESUMO
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
Assuntos
Callithrix , Gastrulação , Útero , Animais , Callithrix/embriologia , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Endoderma/citologia , Endoderma/embriologia , Feminino , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Until recently, naïve pluripotent stem cell lines were not captured from human embryos because protocols were based upon those devised for murine embryonic stem cells. In contrast with early lineage segregation in mouse embryos, human hypoblast specification is not solely dependent upon FGF signaling; consequently, its maturation during embryo explant culture may provide inductive signals to drive differentiation of the epiblast. To overcome this potential risk, here we describe how cells of the immature inner cell mass of human embryos can be physically separated during derivation, achieved via "immunosurgery", to eliminate the trophectoderm, followed by disaggregation of the remaining inner cell mass cells. A modification of a culture regime developed for propagation of human pluripotent stem cells reset to the naïve state is used, which comprises serum-free medium supplemented with various inhibitors of signaling pathways, polarization, and differentiation. Colonies arising from the first plating of an inner cell mass may be pooled for ease of handling, or propagated separately to allow establishment of clonal human naïve embryonic stem cell lines.
Assuntos
Células-Tronco Embrionárias , Distanciamento Físico , Animais , Blastocisto , Diferenciação Celular , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Camadas Germinativas , Humanos , CamundongosRESUMO
Classic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.
Assuntos
Blastocisto , Camadas Germinativas , Animais , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Humanos , CamundongosRESUMO
OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.