Ivan Bedzhov

Self-Organizing Properties of Mouse Pluripotent Cells Initiate Morphogenesis upon Implantation

Summary Transformation of pluripotent epiblast cells into a cup-shaped epithelium as the mouse blastocyst implants is a poorly understood and yet key developmental step. Studies of morphogenesis in embryoid bodies led to the current belief that it is programmed cell death that shapes the epiblast. However, by following embryos developing in vivo and in vitro, we demonstrate that not cell death but a previously unknown morphogenetic event transforms the amorphous epiblast into a rosette of polarized cells. This transformation requires basal membrane-stimulated integrin signaling that coordinates polarization of epiblast cells and their apical constriction, a prerequisite for lumenogenesis. We show that basal membrane function can be substituted in vitro by extracellular matrix (ECM) proteins and that ES cells can be induced to form similar polarized rosettes that initiate lumenogenesis. Together, these findings lead to a completely revised model for peri-implantation morphogenesis in which ECM triggers the self-organization of the embryo’s stem cells.

Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo

A critical point in mammalian development is when the early embryo implants into its mothers uterus. This event has historically been difficult to study due to the fact that it occurs within the maternal tissue and therefore is hidden from view. In this review, we discuss how the mouse embryo is prepared for implantation and the molecular mechanisms involved in directing and coordinating this crucial event. Prior to implantation, the cells of the embryo are specified as precursors of future embryonic and extra-embryonic lineages. These preimplantation cell fate decisions rely on a combination of factors including cell polarity, position and cell–cell signalling and are influenced by the heterogeneity between early embryo cells. At the point of implantation, signalling events between the embryo and mother, and between the embryonic and extraembryonic compartments of the embryo itself, orchestrate a total reorganization of the embryo, coupled with a burst of cell proliferation. New developments in embryo culture and imaging techniques have recently revealed the growth and morphogenesis of the embryo at the time of implantation, leading to a new model for the blastocyst to egg cylinder transition. In this model, pluripotent cells that will give rise to the fetus self-organize into a polarized three-dimensional rosette-like structure that initiates egg cylinder formation.

In vitro culture of mouse blastocysts beyond the implantation stages

The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.

Igf1r Signaling Is Indispensable for Preimplantation Development and Is Activated via a Novel Function of E-cadherin

Insulin-like growth factor I receptor (Igf1r) signaling controls proliferation, differentiation, growth, and cell survival in many tissues; and its deregulated activity is involved in tumorigenesis. Although important during fetal growth and postnatal life, a function for the Igf pathway during preimplantation development has not been described. We show that abrogating Igf1r signaling with specific inhibitors blocks trophectoderm formation and compromises embryo survival during murine blastocyst formation. In normal embryos total Igf1r is present throughout the membrane, whereas the activated form is found exclusively at cell contact sites, colocalizing with E-cadherin. Using genetic domain switching, we show a requirement for E-cadherin to maintain proper activation of Igf1r. Embryos expressing exclusively a cadherin chimera with N-cadherin extracellular and E-cadherin intracellular domains (NcEc) fail to form a trophectoderm and cells die by apoptosis. In contrast, homozygous mutant embryos expressing a reverse-structured chimera (EcNc) show trophectoderm survival and blastocoel cavitation, indicating a crucial and non-substitutable role of the E-cadherin ectodomain for these processes. Strikingly, blastocyst formation can be rescued in homozygous NcEc embryos by restoring Igf1r signaling, which enhances cell survival. Hence, perturbation of E-cadherin extracellular integrity, independent of its cell-adhesion function, blocked Igf1r signaling and induced cell death in the trophectoderm. Our results reveal an important and yet undiscovered function of Igf1r during preimplantation development mediated by a unique physical interaction between Igf1r and E-cadherin indispensable for proper receptor activation and anti-apoptotic signaling. We provide novel insights into how ligand-dependent Igf1r activity is additionally gated to sense developmental potential in utero and into a bifunctional role of adhesion molecules in contact formation and signaling.

A Cdh1HA knock-in allele rescues the Cdh1-/- phenotype but shows essential Cdh1 function during placentation.

The specific roles of classical cadherins at key morphogenetic events during development are still not fully understood. As part of a project to study cadherin function during early mammalian development, we generated mice carrying an HA‐epitope tagged Cdh1 (E‐cadherin) cDNA knocked into the Cdh1 locus, similar to the previously described mouse mutants in which we forced Cdh2 (N‐cadherin) expression in the Cdh1 expression domain. As expected and in contrast to Cdh1Cdh2/Cdh2 and Cdh1−/−, our Cdh1HA/HA mutant embryos form proper trophectoderm, implant and undergo both gastrulation and neurulation. However, Cdh1HA/HA mice display an unexpected phenotype at embryonic day 10.5. Cdh1HA/HA embryos are smaller, paler and suffer from an insufficient nutrient supply. We detected a reduced expression of Cdh1HA specifically in the extraembryonic ectoderm and in the labyrinth layer, whereas expression in the embryo proper was normal. With this approach, we show for the first time that Cdh1 is essential for the correct formation of the placenta. Placentas without Cdh1 expression are impaired and incapable of establishing a proper connection between the embryonic and the maternal blood vessels for efficient nutrient and oxygen transport. Developmental Dynamics 239:2330–2344, 2010.

Development of the anterior-posterior axis is a self-organizing process in the absence of maternal cues in the mouse embryo

Development of the anterior-posterior axis is a self-organizing process in the absence of maternal cues in the mouse embryo

Applying the proximity ligation assay (PLA) to mouse preimplantation embryos for identifying protein-protein interactions in situ.

Analysis of protein-protein interactions in mouse preimplantation embryos is hindered by the low cell number of the embryo. Here we describe the use of the proximity ligation assay to overcome these limitations and outline how protein-protein interactions can be visualized in situ. The method is based on a normal immunofluorescence labeling protocol of preimplantation embryos. Events of binding of the two primary antibodies directed against two individual proteins close to each other are visualized. If the two primary antibodies and the corresponding oligo-linked secondary antibodies bind in close proximity a cascade of events is initiated. This includes oligo ligation, DNA amplification, and hybridization with a fluorescent probe that allows visualization of this close proximity. In contrast to normal immunofluorescence labeling, here detection of red fluorescent dots reflects protein-protein interaction.

Cell death and morphogenesis during early mouse development: Are they interconnected?

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage.

Agrobacterium - Mediated Transformation of Secondary Somatic Embryos from Rosa Hybrida L. and Recovery of Transgenic Plants

ABSTRACT A genetic transformation protocol, based on co-cultivation of Rosa hybrida L. secondary embryos with Agrobacterium was established. Transgenic phosphinothricin (ppt) - resistant plants were obtained through application of interrupted selection procedure. Following the Agrobacterium inoculation step, the somatic embryos were initially cultivated on ppt—free media containing cefatoxim for elimination of the bacteria and later transferred on media containing the selective agent. The ppt—resistant secondary somatic embryos were matured and regenerated on ppt—free media and the putative transgenic plants were subsequently selected on media contained ppt and chlorphenol red. The performed PCR—and PAT protein—assays demonstrated presence and expression of bar gene encoding the phosphinothricin acetyltransferase (PAT) in part of the selected plants. The possibilities for application of the established transformation protocol are discussed.

Correction to: ‘Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo’

[ Phil. Trans. R. Soc. B 369 , 20130538 (2014; published 27 October 2014) ([doi:10.1098/rstb.2013.0538][2])][2] The authors would like to add the following acknowledgment to their manuscript: We would like to thank Anna Hakes for her contribution to the design of figure 3 in the article. []: /