The origin of mouse extraembryonic endoderm stem cell lines

Mouse extraembryonic endoderm stem (XEN) cell lines can be derived from preimplantation embryos (pre-XEN) and postimplantation embryos (post-XEN). XEN cells share a gene expression profile and cell lineage potential with primitive endoderm (PrE) blastocysts. However, the cellular origin of XEN cells in embryos remains unclear. Here, we report that post-XEN cell lines are derived both from the extraembryonic endoderm and epiblasts of postimplantation embryos and that pre-XEN cell lines are derived both from PrE and epiblasts of blastocysts. Our strategy consisted of deriving post-XEN cells from clumps of epiblasts, parietal endoderm (PE) and visceral endoderm (VE) and deriving pre-XEN cell lines from single PrE and single epiblasts of blastocysts. Thus, XEN cell lines in the mouse embryo originate not only from PrE and PrE-derived lineages but also from epiblast and epiblast-derived lineages of blastocysts and postimplantation embryos.

Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-sequencing reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5–E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.

Vacuolar-type proton ATPase is required for maintenance of apicobasal polarity of embryonic visceral endoderm

The endocytic compartments keep their interior acidic through the inward flow of protons and anions from the cytosol. Acidification is mediated by a proton pump known as vacuolar-type ATPase (V-ATPase) and transporters conferring anion conductance to the organellar membrane. In this study, we analysed the phenotype of mouse embryos lacking the V-ATPase c-subunit. The mutant embryos differentiated embryonic epithelial tissues, primitive endoderm, epiblast, and extraembryonic ectoderm; however, the organisation of these epithelia was severely affected. The apical-basal polarity in the visceral endoderm layer was not properly established in the mutant embryos, resulting in abnormal epithelial morphology. Thus, the function of V-ATPase is imperative for the establishment and/or maintenance of epithelial cell polarity, which is required for early embryogenesis.

Characterisation of the transcriptional dynamics underpinning the function, fate, and migration of the mouse Anterior Visceral Endoderm

During early post-implantation development of the mouse embryo, the Anterior Visceral Endoderm (AVE) differs from surrounding visceral endoderm (VE) in its migratory behaviour and ability to restrict primitive streak formation to the opposite side of the egg cylinder. In order to characterise the molecular basis for the unique properties of the AVE, we combined single-cell RNA-sequencing of the VE prior to and during AVE migration, with high-resolution imaging, short-term lineage labelling, phosphoproteomics and pharmacological intervention. This revealed the transient nature of the AVE, the emergence of heterogeneities in AVE transcriptional states relative to position of cells, and its prominence in establishing gene expression asymmetries within the spatial constraints of the embryo. We identified a previously unknown requirement of Ephrin- and Semaphorin-signalling for AVE migration. These findings point to a tight coupling of transcriptional state and position in the AVE and reveal molecular heterogeneities underpinning its migratory behaviour and function.

Induction of peri-implantation stage synthetic embryos using reprogramming paradigms in ESCs

SummaryBlastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that synthetic embryo-like structures are generated solely based on transcription factor-mediated molecular reprogramming of embryonic stem cells in a simple 3D co-culture system. ESCs in these cultures self-organize into elongated, compartmentalized synthetic embryo-like structures over the course of reprogramming exhibiting anterior visceral endoderm formation and symmetry breaking. Single-cell RNA-Seq reveals transcriptional profiles resembling epiblast, visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5–E5.5. Within the epiblast, compartment marker gene expression supports primordial germ cell specification. After transplantation, synthetic embryo-like structures implant in uteri and initiate the formation of decidual tissues. This system allows for fast and reproducible generation of synthetic embryo-like structures, providing further insights into synthetic embryology.

MLL4 is required for the first embryonic collective cell migration whereas MLL3 is not required until birth

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of Mll3 and Mll4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these sister genes in mammalian development have only been incompletely reported. Here we examined the null phenotypes to establish that MLL3 is first required for lung maturation whereas MLL4 is first required for migration of the anterior visceral endoderm (AVE) that initiates gastrulation and is the first collective cell migration in development. This migration is preceded by a columnar to squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these sister genes argues against their action as general enhancer factors.Summary statementThe H3K4 methyltransferases MLL3 and MLL4 have strikingly different null phenotypes during mouse development; MLL3 is required for lung maturation whereas MLL4 is required for anterior visceral endoderm migration.

A critical role of VMP1 in lipoprotein secretion

Lipoproteins are lipid-protein complexes that are primarily generated and secreted from the intestine, liver, and visceral endoderm and delivered to peripheral tissues. Lipoproteins, which are assembled in the endoplasmic reticulum (ER) membrane, are released into the ER lumen for secretion, but its mechanism remains largely unknown. Here, we show that the release of lipoproteins from the ER membrane requires VMP1, an ER transmembrane protein essential for autophagy and certain types of secretion. Loss of vmp1, but not other autophagy-related genes, in zebrafish causes lipoprotein accumulation in the intestine and liver. Vmp1 deficiency in mice also leads to lipid accumulation in the visceral endoderm and intestine. In VMP1-depleted cells, neutral lipids accumulate within lipid bilayers of the ER membrane, thus affecting lipoprotein secretion. These results suggest that VMP1 is important for the release of lipoproteins from the ER membrane to the ER lumen in addition to its previously known functions.

Cables2 Is a Novel Smad2-Regulatory Factor Essential for Early Embryonic Development in Mice

ABSTRACTCDK5 and Abl enzyme substrate 2 (Cables2), a member of the Cables family that has a C-terminal cyclin box-like domain, is widely expressed in adult mouse tissues. However, the physiological role of Cables2 in vivo is unknown. We show here that Cables2-deficiency causes post-gastrulation embryonic lethality in mice. The mutant embryos progress to gastrulation, but then arrest, and fail to grow. Analysis of gene expression patterns reveals that formation of the anterior visceral endoderm and the primitive streak is impaired in Cables2-deficient embryos. Tetraploid complementation analyses support the critical requirement of Cables2 in both the epiblast and visceral endoderm for progression of embryogenesis. In addition, we show that Cables2 physically interacts with a key mediator of the canonical Nodal pathway, Smad2, and augments its transcriptional activity. These findings provide novel insights into the essential role of Cables2 in the early embryonic development in mice.