Anna Piliszek

A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse

BackgroundUnderstanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies.ResultsWe have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis.ConclusionBy combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.

FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.

The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.

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.

Anatomy of a blastocyst: Cell behaviors driving cell fate choice and morphogenesis in the early mouse embryo

The preimplantation period of mouse early embryonic development is devoted to the specification of two extraembryonic tissues and their spatial segregation from the pluripotent epiblast. During this period two cell fate decisions are made while cells gradually lose their totipotency. The first fate decision involves the segregation of the extraembryonic trophectoderm (TE) lineage from the inner cell mass (ICM); the second occurs within the ICM and involves the segregation of the extraembryonic primitive endoderm (PrE) lineage from the pluripotent epiblast (EPI) lineage, which eventually gives rise to the embryo proper. Multiple determinants, such as differential cellular properties, signaling cues and the activity of transcriptional regulators, influence lineage choice in the early embryo. Here, we provide an overview of our current understanding of the mechanisms governing these cell fate decisions ensuring proper lineage allocation and segregation, while at the same time providing the embryo with an inherent flexibility to adjust when perturbed. genesis 51:219–233.

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity.

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.

The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

Ex Utero Culture and Live Imaging of Mouse Embryos

Mouse genetic approaches when combined with live imaging tools have the potential to revolutionize our current understanding of mammalian biology. The availability and improvement of a wide variety of fluorescent proteins have provided indispensable tools to visualize cells in living organisms. It is now possible to generate genetically modified mouse strains expressing fluorescent proteins in a tissue-specific manner. These reporter-expressing strains make it possible to image dynamic cell behaviors in the context of a living embryo. Since mouse embryos develop within the uterus, live imaging experiments require culture conditions that closely mimic those in vivo. Over the past few decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation stage embryos. In this chapter, we will discuss methods for ex utero culture of preimplantation and postimplantation stage mouse embryos. In particular, we will describe protocols for collecting embryos at various stages, setting up culture conditions for imaging and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.

Suppression of ERK signalling abolishes primitive endoderm formation but does not promote pluripotency in rabbit embryo

Formation of epiblast (EPI) – the founder line of all embryonic lineages – and extra-embryonic supportive tissues is one of the key events in mammalian development. The prevailing model of early mammalian development is based almost exclusively on the mouse. Here, we provide a comprehensive, stage-by-stage analysis of EPI and extra-embryonic primitive endoderm (PrE) formation during preimplantation development of the rabbit. Although we observed that rabbit embryos have several features in common with mouse embryos, including a stage-related initiation of lineage specification, our results demonstrate the existence of some key differences in lineage specification among mammals. Contrary to the current view, our data suggest that reciprocal repression of GATA6 and NANOG is not fundamental for the initial stages of PrE versus EPI specification in mammals. Furthermore, our results provide insight into the observed discrepancies relating to the role of FGF/ERK signalling in PrE versus EPI specification between mouse and other mammals. Highlighted Article: A comprehensive analysis of rabbit preimplantation development reveals key differences between rabbit and mouse, with some aspects of lineage specification in rabbit more closely resembling that of human and primate embryos.

Differentiation of trophectoderm in rabbit embryos is initiated in the absence of Gata3 and Cdx2

In mammalian development, pivotal patterning events take place during peri-implantation period, when the embryo attaches to the uterine tissues of the mother. At this time, a layer of cells called visceral endoderm provides signals crucial for specifying the anterior-posterior (A-P) axis of the embryo. Due to the difficulty of crossing the implantation barrier in vitro, the mechanisms of generating a signaling center within the visceral endoderm itself remain elusive. Here, we present 3D-geec, a peri-implantation culture method that supports continuous mouse embryo development in vitro from preto postimplantation stages while preserving its in-vivo-like geometry. Such embryos retain the expression of lineage-specific markers, are minimally delayed in their development, preserve in-vivo-like proportions, and correctly specify A-P axis in the absence of maternal cues. Observing and manipulating embryos in this culture, we are able to explore the hidden heterogeneity within the inner cell mass of the blastocyst that predicts future body patterning. By combining 3D culture, time-lapse light sheet fluorescence microscopy, and single-cell RNA-Seq, we can now explore the development of peri-implantation embryo, and probe the lineage and character of cells that play a crucial role in the establishment of embryonic body plan.

Pre-implantation Development of Domestic Animals

During the first days following fertilization, cells of mammalian embryo gradually lose totipotency, acquiring distinct identity. The first three lineages specified in the mammalian embryo are pluripotent epiblast, which later gives rise to the embryo proper, and two extraembryonic lineages, hypoblast (also known as primitive endoderm) and trophectoderm, which form tissues supporting development of the fetus in utero. Most of our knowledge regarding the mechanisms of early lineage specification in mammals comes from studies in the mouse. However, the growing body of evidence points to both similarities and species-specific differences. Understanding molecular and cellular mechanisms of early embryonic development in nonrodent mammals expands our understanding of basic mechanisms of differentiation and is essential for the development of effective protocols for assisted reproduction in agriculture, veterinary medicine, and for biomedical research. This review summarizes the current state of knowledge on key events in epiblast, hypoblast, and trophoblast differentiation in domestic mammals.