AttrActin' Attention to Early Mouse Development.

A new study by Zenker et al. uses time-lapse imaging to discover how dynamic actin movements contribute to epithelialization of living mouse embryos. Together with work from other labs, this study presents exciting new ways to think about the emergence of cell fates during mammalian development.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

The fetal lineage is susceptible to Zika virus infection within days of fertilization.

Adults contracting Zika virus (ZIKV) typically exhibit mild symptoms, yet ZIKV infection of pregnant individuals can cause miscarriage or birth defects in their offspring. Many studies have focused on maternal-to-fetal ZIKV transmission via blood and placenta. Notably, however, ZIKV is also transmitted sexually, raising the possibility that ZIKV could infect the embryo shortly after fertilization, long before the placenta is established. Here, we evaluate the consequences of ZIKV infection in mouse embryos during the first few days of embryogenesis. We show that divergent strains of ZIKV can infect the fetal lineage and can cause developmental arrest, raising concern for the developmental consequences of sexual ZIKV transmission. This article has an associated 'The people behind the papers' interview.

Efficient generation of endogenous protein reporters for mouse development.

Fluorescent proteins and epitope tags can reveal protein localization in cells and animals, yet the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. Here, we present two strategies for higher throughput production of endogenous protein reporters in mice, focusing on the blastocyst model of development. Our first strategy makes use of a split fluorescent protein, mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest, was highly efficient in embryos, potentially obviating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. Our second strategy achieves in-frame knock-in of a relatively small protein tag, which provides high efficiency and higher sensitivity protein reporting. Together, these two approaches provide complementary advantages and enable broad downstream applications.

TEAD4, YAP1 and WWTR1 prevent the premature onset of pluripotency prior to the 16-cell stage.

In mice, pluripotent cells are thought to derive from cells buried inside the embryo around the 16-cell stage. Sox2 is the only pluripotency gene known to be expressed specifically within inside cells at this stage. To understand how pluripotency is established, we therefore investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4 (Pou5f1), highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1 and Tead4 Interestingly, the repression of premature Sox2 expression was sensitive to LATS kinase activity, even though LATS proteins normally do not limit activity of TEAD4, YAP1 and WWTR1 during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1, WWTR1 and TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the four-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.

Culture conditions antagonize lineage-promoting signaling in the mouse blastocyst.

The mouse preimplantation embryo is a paradigm for discovery of the molecular principles governing formation of specific cell types during development. In this Point of View Article, we show that conditions commonly used for ex vivo culture of preimplantation development are themselves antagonistic to a pathway that is critical for blastocyst lineage commitment.

Investigations at the 'Four-Front' of Mammalian Development.

Understanding how and when cells become different during embryogenesis is a goal that is at the forefront of investigations in mammalian development. Two recent studies from the laboratories of Nicholas Plachta and Magdalena Zernicka-Goetz present evidence that cellular heterogeneities detected in four-cell mouse embryos bias the process of cell fate acquisition thereafter.

Biochemical and Cellular Analysis Reveals Ligand Binding Specificities, a Molecular Basis for Ligand Recognition, and Membrane Association-dependent Activities of Cripto-1 and Cryptic.

Transforming growth factor ß (TGF-ß) pathways are key determinants of cell fate in animals. Their basic mechanism of action is simple. However, to produce cell-specific responses, TGF-ß pathways are heavily regulated by secondary factors, such as membrane-associated EGF-CFC family proteins. Cellular activities of EGF-CFC proteins have been described, but their molecular functions, including how the mammalian homologs Cripto-1 and Cryptic recognize and regulate TGF-ß family ligands, are less clear. Here we use purified human Cripto-1 and mouse Cryptic produced in mammalian cells to show that these two EGF-CFC homologs have distinct, highly specific ligand binding activities. Cripto-1 interacts with BMP-4 in addition to its known partner Nodal, whereas Cryptic interacts only with Activin B. These interactions depend on the integrity of the protein, as truncated or deglycosylated Cripto-1 lacked BMP-4 binding activity. Significantly, Cripto-1 and Cryptic blocked binding of their cognate ligands to type I and type II TGF-ß receptors, indicating that Cripto-1 and Cryptic contact ligands at their receptor interaction surfaces and, thus, that they could inhibit their ligands. Indeed, soluble Cripto-1 and Cryptic inhibited ligand signaling in various cell-based assays, including SMAD-mediated luciferase reporter gene expression, and differentiation of a multipotent stem cell line. But in agreement with previous work, the membrane bound form of Cripto-1 potentiated signaling, revealing a critical role of membrane association for its established cellular activity. Thus, our studies provide new insights into the mechanism of ligand recognition by this enigmatic family of membrane-anchored TGF-ß family signaling regulators and link membrane association with their signal potentiating activities.

Cell signaling and transcription factors regulating cell fate during formation of the mouse blastocyst.

The first cell fate decisions during mammalian development establish tissues essential for healthy pregnancy. The mouse has served as a valuable model for discovering pathways regulating the first cell fate decisions because of the ease with which early embryos can be recovered and the availability of an arsenal of classical and emerging methods for manipulating gene expression. We summarize the major pathways that govern the first cell fate decisions in mouse development. This knowledge serves as a paradigm for exploring how emergent properties of a self-organizing system can dynamically regulate gene expression and cell fate plasticity. Moreover, it brings to light the processes that establish healthy pregnancy and ES cells. We also describe unsolved mysteries and new technologies that could help to overcome experimental challenges in the field.

XEN and the Art of Stem Cell Maintenance: Molecular Mechanisms Maintaining Cell Fate and Self-Renewal in Extraembryonic Endoderm Stem (XEN) Cell Lines.

The extraembryonic endoderm is one of the first cell types specified during mammalian development. This extraembryonic lineage is known to play multiple important roles throughout mammalian development, including guiding axial patterning and inducing formation of the first blood cells during embryogenesis. Moreover, recent studies have uncovered striking conservation between mouse and human embryos during the stages when extraembryonic endoderm cells are first specified, in terms of both gene expression and morphology. Therefore, mouse embryos serve as an excellent model for understanding the pathways that maintain extraembryonic endoderm cell fate. In addition, self-renewing multipotent stem cell lines, called XEN cells, have been derived from the extraembryonic endoderm of mouse embryos. Mouse XEN cell lines provide an additional tool for understanding the basic mechanisms that contribute to maintaining lineage potential, a resource for identifying how extraembryonic ectoderm specifies fetal cell types, and serve as a paradigm for efforts to establish human equivalents. Given the potential conservation of essential extraembryonic endoderm roles, human XEN cells would provide a considerable advance. However, XEN cell lines have not yet been successfully derived from human embryos. Given the potential utility of human XEN cell lines, this chapter focuses on reviewing the mechanisms known to govern the stem cell properties of mouse XEN, in hopes of facilitating new ways to establish human XEN cell lines.

Novel key roles for structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development.

Structural maintenance of chromosome flexible domain containing 1 (Smchd1) is a chromatin regulatory gene for which mutations are associated with facioscapulohumeral muscular dystrophy and arhinia. The contribution of oocyte- and zygote-expressed SMCHD1 to early development was examined in mice ( Mus musculus) using a small interfering RNA knockdown approach. Smchd1 knockdown compromised long-term embryo viability, with reduced embryo nuclear volumes at the morula stage, reduced blastocyst cell number, formation and hatching, and reduced viability to term. RNA sequencing analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of a small number of trophectoderm (TE)-related genes and reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 ( Skp2). Smchd1 expression was elevated in embryos deficient for Caudal-type homeobox transcription factor 2 ( Cdx2, a key regulator of TE specification), indicating that Smchd1 is normally repressed by CDX2. These results indicate that Smchd1 plays an important role in the preimplantation embryo, regulating early gene expression and contributing to long-term embryo viability. These results extend the known functions of SMCHD1 to the preimplantation period and highlight important function for maternally expressed Smchd1 messenger RNA and protein.

HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.

Maternal Cdx2 is dispensable for mouse development.

In many invertebrate and vertebrate species, cell fates are assigned through the cellular inheritance of differentially localized maternal determinants. Whether mammalian embryogenesis is also regulated by deterministic mechanisms is highly controversial. The caudal domain transcription factor CDX2 has been reported to act as a maternal determinant regulating cell fate decisions in mouse development. However, this finding is contentious because of reports that maternal Cdx2 is not essential for development. Notably, all of the previously published studies of maternal Cdx2 relied on injected RNA interference constructs, which could introduce experimental variation. Only deletion of the maternal gene can unambiguously resolve its requirement in mouse development. Here, we genetically ablated maternal Cdx2 using a Cre/lox strategy, and we definitively establish that maternal Cdx2 is not essential for mouse development.

OCT4 is expressed in extraembryonic endoderm stem (XEN) cell progenitors during somatic cell reprogramming.

During development, progenitors of embryonic stem (ES) and extraembryonic endoderm stem (XEN) cells are concomitantly specified within the inner cell mass (ICM) of the mouse blastocyst. Similarly, XEN cells are induced (iXEN cells) alongside induced pluripotent stem (iPS) cells following overexpression of Oct4, Sox2, Klf4 and Myc (OSKM) during somatic cell reprogramming. It is unclear how or why this cocktail produces both stem cell types, but OCT4 has been associated with non-pluripotent outcomes. In this report, we show that, during OSKM reprogramming, many individual Oct4-GFP-expressing cells are fated to become iXEN cells. Interestingly, SKM alone was also sufficient to induce iXEN cell formation, likely via activation of endogenous Oct4. These observations indicate that iXEN cell formation is not strictly an artifact of Oct4 overexpression. Moreover, our results suggest that a pathway to XEN may be an integral feature of establishing pluripotency during reprogramming, as in early embryo development.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential prior to implantation in the mouse.

Bone Morphogenic Protein (BMP) signaling plays an essential and highly conserved role in axial patterning in embryos of many externally developing animal species. However, in mammalian embryos, which develop inside the mother, early development includes an additional stage known as preimplantation. During preimplantation, the epiblast lineage is segregated from the extraembryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling in mouse preimplantation is imprecisely defined. We show that, in contrast to prior reports, BMP signaling (as reported by SMAD1/5/9 phosphorylation) is not detectable until implantation, when it is detected in the primitive endoderm - an extraembryonic lineage. Moreover, preimplantation development appears normal following deletion of maternal and zygotic Smad4, an essential effector of BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extraembryonic cell types drives epiblast morphogenesis post-implantation.

Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2.

The mouse blastocyst and stem cells derived from its tissue lineages provide a unique genetic system for examining the establishment and loss of pluripotency. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4, and promoting extraembryonic trophoblast fate at the blastocyst stage. However, genetic evidence has suggested that Cdx2 does not work alone in the trophoblast lineage. We have used bioinformatic and functional genomic strategies to identify the transcription factor Gata3 as a trophoblast factor. We show Gata3 to be capable of inducing trophoblast fate in embryonic stem cells and driving trophoblast differentiation in trophoblast stem cells. In addition, Cdx2 is not required for Gata3-induced expression of a subset of trophoblast genes in embryonic stem cells. We show that Gata3 is coexpressed with Cdx2 in the blastocyst, but this does not depend on Cdx2. In the embryo, expression of Gata3, like that of Cdx2, depends on Tead4, and the expression of both factors becomes restricted to trophoblast by a mechanism that does not initially rely on Oct4. These observations suggest that Gata3 and Cdx2 can act in parallel pathways downstream of Tead4 to induce the expression of common and independent targets in the trophoblast lineage, whereas Oct4 is required for continued repression of trophoblast fate in the embryonic lineage.

Distinct histone modifications in stem cell lines and tissue lineages from the early mouse embryo.

A unique property of the mammalian embryo is that stem cells can be derived from its early tissue lineages. These lineages will give rise to the fetus as well as essential extraembryonic tissues. Understanding how chromatin regulation participates in establishment of these lineages in the embryo and their derived stem cells provides insight that will critically inform our understanding of embryogenesis and stem cell biology. Here, we compare the genomewide location of active and repressive histone modifications in embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells from the mouse. Our results show that the active modification H3K4me3 has a similar role in the three stem cell types, but the repressive modification H3K27me3 varies in abundance and genomewide distribution. Thus, alternative mechanisms mediate transcriptional repression in stem cells from the embryo. In addition, using carrier chromatin immunoprecipitation we show that bivalent histone domains seen in embryonic stem cells exist in pluripotent cells of the early embryo. However, the epigenetic status of extraembryonic progenitor cells in the embryo did not entirely reflect the extraembryonic stem cell lines. These studies indicate that histone modification mechanisms may differ between early embryo lineages and emphasize the importance of examining in vivo and in vitro progenitor cells.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.