, , vol.7
, pinhole 1 airy unit, and laser intensities with optical section thickness of 1.2-1.4 ?m. 16-bit resulting images were analyzed and eventually processed using Icy, Fiji and Photoshop CS5 softwares
, Fluorescence quantification, 3D stacks were analyzed using Icy software 43
, After selecting the nuclei staining channel, the protocol applies a wavelet decomposition of the 3D image volume to segregate image structures based on size and intensity. After optimizing the selection of wavelet scales and their respective thresholds, the protocol extracts one or more 3D regions of interest (ROI) for each nucleus. The selection of scales and thresholds is adapted to each embryo, depending on the signal-to-noise ratio. Finally, the protocol super-imposes the extracted ROI on the original data for visual inspection. In few instances, multiple ROI are detected inside the same nucleus, and are merged manually post-analysis. The mean of fluorescence, org/protocol/3D_mouse_embryo_quantification), for which we designed a custom semi-automated analysis protocol available here
, Blastocyst gene expression profile. mRNAs of 5 pooled blastocysts were isolated using RNeasy Plus Micro kit (Qiagen) according manufacturer instructions, reverse transcribed using Superscript VILO (Thermo Fischer Scientific) and analyzed using SYBR Green PCR Master Mix (Applied Biosystems). Mouse RT-qPCR primers were designed using Primer Blast (NCBI)
, A close look at the mammalian blastocyst: epiblast and primitive endoderm formation, Cell. Mol. Life Sci, vol.71, pp.3327-3338, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01923166
, Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst, Development, vol.135, pp.3081-3091, 2008.
, Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway, Dev. Cell, vol.10, pp.615-624, 2006.
URL : https://hal.archives-ouvertes.fr/hal-01923174
, Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst, Developmental Biology, vol.331, pp.210-221, 2009.
URL : https://hal.archives-ouvertes.fr/pasteur-01572044
, Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling, Dev. Cell, vol.21, pp.1005-1013, 2011.
URL : https://hal.archives-ouvertes.fr/hal-01917180
, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network, Development, vol.141, pp.3637-3648, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01923163
, GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst, Dev. Cell, vol.29, pp.454-467, 2014.
, Lineage Establishment and Progression within the Inner Cell Mass of the Mouse Blastocyst Requires FGFR1 and FGFR2, Dev. Cell, vol.41, pp.496-510, 2017.
, Distinct Requirements for FGFR1 and FGFR2 in Primitive Endoderm Development and Exit from Pluripotency, Dev. Cell, vol.41, pp.511-526, 2017.
, FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse, Development, vol.140, pp.267-279, 2013.
, FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst, Developmental Biology, vol.384, pp.65-71, 2013.
, Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo, Development, vol.136, pp.3215-3222, 2009.
, FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst, Development, vol.137, pp.715-724, 2010.
, Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo, Development, vol.139, pp.129-139, 2012.
, Asynchronous fate decisions by single cells collectively ensure consistent lineage composition in the mouse blastocyst, Nature Communications, vol.7, p.13463, 2016.
, Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo, Journal of Biological Chemistry, vol.276, pp.31793-31799, 2001.
, The spindle-assembly checkpoint in space and time, Nat Rev Mol Cell Biol, vol.8, pp.379-393, 2007.
, Fibroblast growth factor receptor 2 homodimerization rapidly reduces transcription of the pluripotency gene Nanog without dissociation of activating transcription factors, Journal of Biological Chemistry, vol.287, pp.30507-30517, 2012.
, The role of FGF/Erk signaling in pluripotent cells, Development, vol.137, pp.3351-3360, 2010.
, Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst, Dev. Cell, vol.18, pp.675-685, 2010.
, Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages, Nature Cell Biology, vol.16, pp.27-37, 2014.
, The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos, Open Biol, vol.3, p.130104, 2013.
, The first two cell-fate decisions of preimplantation mouse embryo development are not functionally independent, Sci. Rep, vol.5, p.15034, 2015.
, Allocation of inner cells to epiblast vs primitive endoderm in the mouse embryo is biased but not determined by the round of asymmetric divisions (8 ? 16-and 16 ? 32-cells), Developmental Biology, vol.385, pp.136-148, 2014.
, Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst, Cell Rep, vol.10, pp.1508-1520, 2015.
, , vol.7
, PEST motif sequence regulating human NANOG for proteasomal degradation, Stem Cells and Development, vol.20, pp.1511-1519, 2011.
, NANOG Is Multiply Phosphorylated and Directly Modified by ERK2 and CDK1 In Vitro, Stem Cell Reports, vol.2, pp.18-25, 2014.
, ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal, Stem Cell Research, vol.13, pp.1-11, 2014.
, Cops2 promotes pluripotency maintenance by Stabilizing Nanog Protein and Repressing Transcription, Sci. Rep, vol.6, p.26804, 2016.
, The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog, Nature Communications, vol.7, p.13594, 2016.
, G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells, Nature Cell Biology, vol.19, pp.177-188, 2017.
, DPPA5 Supports Pluripotency and Reprogramming by Regulating NANOG Turnover, Stem Cells, vol.34, pp.588-600, 2016.
, Phosphorylation stabilizes Nanog by promoting its interaction with Pin1, Proceedings of the National Academy of Sciences, vol.107, pp.13312-13317, 2010.
DOI : 10.1073/pnas.1005847107
URL : http://www.pnas.org/content/107/30/13312.full.pdf
, Differentiation of embryonic stem cells is induced by GATA factors, Genes & Development, vol.16, pp.784-789, 2002.
, Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells, BMC Developmental Biology, vol.7, p.80, 2007.
, Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells, Genes & Development, vol.29, pp.1239-1255, 2015.
, Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development, Genes & Development, vol.26, pp.1445-1458, 2012.
URL : https://hal.archives-ouvertes.fr/hal-01923171
, The RNA-binding protein Unr prevents mouse embryonic stem cells differentiation toward the primitive endoderm lineage, Stem Cells, vol.29, pp.1504-1516, 2011.
URL : https://hal.archives-ouvertes.fr/hal-01923180
, Gastric GATA-6 DNA-binding protein: proteolysis induced by cAMP, FEBS Letters, vol.408, pp.301-305, 1997.
, Characterization of cAMP-dependent proteolysis of GATA-6, Biochemical and Biophysical Research Communications, vol.332, pp.976-981, 2005.
, Concise review: role and function of the ubiquitin-proteasome system in mammalian stem and progenitor cells, Stem Cells, vol.25, pp.2408-2418, 2007.
, Treatment with the proteasome inhibitor MG132 during the end of oocyte maturation improves oocyte competence for development after fertilization in cattle, PLoS ONE, vol.7, p.48613, 2012.
, Icy: an open bioimage informatics platform for extended reproducible research, Nature Methods, vol.9, pp.690-696, 2012.