Néstor Saiz

Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo

Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and primitive endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage.

GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst.

Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.

Early cell fate decisions in the mouse embryo

During mammalian preimplantation development, the fertilised egg gives rise to a group of pluripotent embryonic cells, the epiblast, and to the extraembryonic lineages that support the development of the foetus during subsequent phases of development. This preimplantation period not only accommodates the first cell fate decisions in a mammals life but also the transition from a totipotent cell, the zygote, capable of producing any cell type in the animal, to cells with a restricted developmental potential. The cellular and molecular mechanisms governing the balance between developmental potential and lineage specification have intrigued developmental biologists for decades. The preimplantation mouse embryo offers an invaluable system to study cell differentiation as well as the emergence and maintenance of pluripotency in the embryo. Here we review the most recent findings on the mechanisms controlling these early cell fate decisions. The model that emerges from the current evidence indicates that cell differentiation in the preimplantation embryo depends on cellular interaction and intercellular communication. This strategy underlies the plasticity of the early mouse embryo and ensures the correct specification of the first mammalian cell lineages.

Atypical protein kinase C couples cell sorting with primitive endoderm maturation in the mouse blastocyst

During mouse pre-implantation development, extra-embryonic primitive endoderm (PrE) and pluripotent epiblast precursors are specified in the inner cell mass (ICM) of the early blastocyst in a ‘salt and pepper’ manner, and are subsequently sorted into two distinct layers. Positional cues provided by the blastocyst cavity are thought to be instrumental for cell sorting; however, the sequence of events and the mechanisms that control this segregation remain unknown. Here, we show that atypical protein kinase C (aPKC), a protein associated with apicobasal polarity, is specifically enriched in PrE precursors in the ICM prior to cell sorting and prior to overt signs of cell polarisation. aPKC adopts a polarised localisation in PrE cells only after they reach the blastocyst cavity and form a mature epithelium, in a process that is dependent on FGF signalling. To assess the role of aPKC in PrE formation, we interfered with its activity using either chemical inhibition or RNAi knockdown. We show that inhibition of aPKC from the mid blastocyst stage not only prevents sorting of PrE precursors into a polarised monolayer but concomitantly affects the maturation of PrE precursors. Our results suggest that the processes of PrE and epiblast segregation, and cell fate progression are interdependent, and place aPKC as a central player in the segregation of epiblast and PrE progenitors in the mouse blastocyst.

Asynchronous fate decisions by single cells collectively ensure consistent lineage composition in the mouse blastocyst

Intercellular communication is essential to coordinate the behaviour of individual cells during organismal development. The preimplantation mammalian embryo is a paradigm of tissue self-organization and regulative development; however, the cellular basis of these regulative abilities has not been established. Here we use a quantitative image analysis pipeline to undertake a high-resolution, single-cell level analysis of lineage specification in the inner cell mass (ICM) of the mouse blastocyst. We show that a consistent ratio of epiblast and primitive endoderm lineages is achieved through incremental allocation of cells from a common progenitor pool, and that the lineage composition of the ICM is conserved regardless of its size. Furthermore, timed modulation of the FGF-MAPK pathway shows that individual progenitors commit to either fate asynchronously during blastocyst development. These data indicate that such incremental lineage allocation provides the basis for a tissue size control mechanism that ensures the generation of lineages of appropriate size.

Tbx3 Controls Dppa3 Levels and Exit from Pluripotency toward Mesoderm

Summary Tbx3, a member of the T-box family, plays important roles in development, stem cells, nuclear reprogramming, and cancer. Loss of Tbx3 induces differentiation in mouse embryonic stem cells (mESCs). However, we show that mESCs exist in an alternate stable pluripotent state in the absence of Tbx3. In-depth transcriptome analysis of this mESC state reveals Dppa3 as a direct downstream target of Tbx3. Also, Tbx3 facilitates the cell fate transition from pluripotent cells to mesoderm progenitors by directly repressing Wnt pathway members required for differentiation. Wnt signaling regulates differentiation of mESCs into mesoderm progenitors and helps to maintain a naive pluripotent state. We show that Tbx3, a downstream target of Wnt signaling, fine tunes these divergent roles of Wnt signaling in mESCs. In conclusion, we identify a signaling-TF axis that controls the exit of mESCs from a self-renewing pluripotent state toward mesoderm differentiation.

Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells

During mitosis, transcription is halted and many chromatin features are lost, posing a challenge for the continuity of cell identity, particularly in fast cycling stem cells, which constantly balance self-renewal with differentiation. Here we show that, in pluripotent stem cells, certain histone marks and stem cell regulators remain associated with specific genomic regions of mitotic chromatin, a phenomenon known as mitotic bookmarking. Enhancers of stem cell-related genes are bookmarked by both H3K27ac and the master regulators OCT4, SOX2, and KLF4, while promoters of housekeeping genes retain high levels of mitotic H3K27ac in a cell-type invariant manner. Temporal degradation of OCT4 during mitotic exit compromises its ability both to maintain and induce pluripotency, suggesting that its regulatory function partly depends on its bookmarking activity. Together, our data document a widespread yet specific bookmarking by histone modifications and transcription factors promoting faithful and efficient propagation of stemness after cell division.

Completely ES Cell-Derived Mice Produced by Tetraploid Complementation Using Inner Cell Mass (ICM) Deficient Blastocysts

Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice.

BackgroundThe GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs.ResultsWe have generated a Gata6H2B-Venus knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution.ConclusionsExpression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6H2B-Venus/H2B-Venus homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.

Single cells get together: High-resolution approaches to study the dynamics of early mouse development.

Embryonic development is a complex and highly dynamic process during which individual cells interact with one another, adopt different identities and organize themselves in three-dimensional space to generate an entire organism. Recent technical developments in genomics and high-resolution quantitative imaging are making it possible to study cellular populations at single-cell resolution and begin to integrate different inputs, for example genetic, physical and chemical factors, that affect cell differentiation over spatial and temporal scales. The preimplantation mouse embryo allows the analysis of cell fate decisions in vivo with high spatiotemporal resolution. In this review we highlight how the application of live imaging and single-cell resolution analysis pipelines is providing an unprecedented level of insight on the processes that shape the earliest stages of mammalian development.