Dataset Information

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

ABSTRACT: The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

SUBMITTER: Dupont C

PROVIDER: S-EPMC9848479 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

Dupont Cathérine C Schäffers Olivier J M OJM Tan Beatrice F BF Merzouk Sarra S Bindels Eric M EM Zwijsen An A Huylebroeck Danny D Gribnau Joost J

Science advances 20230118 3

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late ga ...[more]

PMID: 36652512

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Project description:BackgroundReciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue.ResultsAs a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5.ConclusionWe propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation.

Project description:Imaging the entire cardiomyocyte network in entire small animal hearts at single cell resolution is a formidable challenge. Optical microscopy provides sufficient contrast and resolution in 2d, however fails to deliver non-destructive 3d reconstructions with isotropic resolution. It requires several invasive preparation steps, which introduce structural artefacts, namely dehydration, physical slicing and staining, or for the case of light sheet microscopy also clearing of the tissue. Our goal is to provide 3d reconstructions of the cardiomyocyte network in entire hydrated murine hearts, and to develop a methodology for quantitative analysis of heart pathologies based on X-ray phase contrast computed tomography (XPCT). We have used XPCT at two beamlines of the extremely brilliant source (EBS) at the European Synchrotron Radiation Facility (ESRF) to scan wild-type murine hearts at high resolution, as well as a series of murine hearts of different pathological models, at reduced resolution and higher throughput. All hearts were obtained from the small animal facility of the university medical center in Göttingen. The hearts were fixed in formalin, stored and measured non-destructively in phosphate buffer solution. The high resolution dataset allows to discern individual cardiomyocytes in the tissue. All datasets have been analyzed using semi-automated image segmentation of the ventricles, rotation into a common coordinate system, classification into different anatomical compartments, and finally the structure tensor approach. A 3d streamline representation of the cardiomyocyte orientation vector field is provided. The different cardiovascular disease models are analysed based on metrics derived from the 3d structure tensor. An entire hydrated murine heart has been covered at an isotropic voxel size of 1.6 μ m (distributed over several volumes). A binned and fused dataset of this heart is available at 3.2 μ m, and has been analyzed by the structure tensor approach to yield the ventricular cardiomyocyte network or mesh, i.e. the aggregation of the cardiomyocyte chains in particular in the ventricular wall. Semi-automatic determination of structural metrics is already achieved and the corresponding tools and resulting data are made publically available. XPCT using extremely brilliant undulator radiation is close to achieve single cell reconstruction in an entire small animal organ.

Dataset Information

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

Publications

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

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