Gutsy moves in mice: cellular and molecular dynamics of endoderm morphogenesis

Despite the importance of the gut and its accessory organs, our understanding of early endoderm development is still incomplete. Traditionally, endoderm has been difficult to study because of its small size and relative fragility. However, recent advances in live cell imaging technologies have dramatically expanded our understanding of this tissue, adding a new appreciation for the complex molecular and morphogenetic processes that mediate gut formation. Several spatially and molecularly distinct subpopulations have been shown to exist within the endoderm before the onset of gastrulation. Here, we review findings that have uncovered complex cell movements within the endodermal layer, before and during gastrulation, leading to the conclusion that cells from primitive endoderm contribute descendants directly to gut.     

F9 teratocarcinoma aggregates express villin upon differentiation into visceral endoderm-like cells

The visceral endoderm of the mouse embryo is a polarized epithelium which has recently been shown to express villin, a major actin binding component of absorptive epitheliums. We report here that villin is induced during differentiation of aggregates of the mouse embryonal carcinoma F9, an in vitro system widely used to study extraembryonic endoderm differentiation. Identical results were obtained with a variant of F9 which carries an immortalizing vector. Villin is coexpressed with F-actin and with alpha-foetoprotein, in most of the visceral endoderm-like cells lining the aggregates. This system is potentially useful to study (i) the induction of villin expression and (ii) the establishment of polarity in the visceral endoderm epithelium.     

位点特异性重组系统及其在植物转基因研究中的应用

随着植物转基因研究的不断深入,对基因重组系统提出了新的要求。位点特异性重组系统具有高效、精确等的优点,在植物基因工程领域的应用越来越广泛。对常用的三类位点特异性重组系统的作用机制、优缺点及其应用进展进行了全面的综述,期望为植物转基因研究提供技术参考。对于目前研究较为热点的基因编辑技术CRISPR-Cas系统作了简要概述。     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

Hedgehog signals in pancreatic differentiation from embryonic stem cells: revisiting the neglected

Abstract Recent demonstrations of insulin expression by progenies of mouse and human embryonic stem (ES) cells have attracted interest in setting up these cells as alternative sources of β-cells needed in diabetes cell therapy. It is widely acknowledged that information gathered in the field of developmental biology as applied to the pancreas is of relevance for designing in vitro differentiation strategies. However, looking back at the protocols used so far, it appears that the natural route toward the pancreas, which goes via the definitive endoderm, was usually bypassed. As a consequence Hedgehog signaling, the earliest inhibitor of pancreas initiation from the endoderm, was generally not considered. A recall of the status of this pathway during ES cell differentiation appears necessary, especially in the light of findings that Activin A treatment of mouse and human ES cells coax them into definitive endoderm, a lineage showing wide Hedgehog ligands expression with the potential to hinder pancreatic programming.     

Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway

Definitive endoderm differentiation is crucial for generating respiratory and gastrointestinal organs including pancreas and liver. However, whether epigenetic regulation contributes to this process is unknown. Here, we show that the H3K27me3 demethylases KDM6A and KDM6B play an important role in endoderm differentiation from human ESCs. Knockdown of KDM6A or KDM6B impairs endoderm differentiation, which can be rescued by sequential treatment with WNT agonist and antagonist. KDM6A and KDM6B contribute to the activation of WNT3 and DKK1 at different differentiation stages when WNT3 and DKK1 are required for mesendoderm and definitive endoderm differentiation, respectively. Our study not only uncovers an important role of the H3K27me3 demethylases in definitive endoderm differentiation, but also reveals that they achieve this through modulating the WNT signaling pathway.     

Conditions affecting the differentiation of F9 teratocarcinoma cells: Potentiation of response by cyclic amp

Summary F9 cells maintained in culture were shown to have a reduced ability to differentiate. The cells produced decreased amounts of alphafetoprotein when induced with retinoic acid. We show that consistent responses can be recovered after passage of F9 cells as a tumor. In addition, optimal differentiation of F9 cells to visceral endoderm may be achieved by the addition of very low concentrations of dibutyryl cyclic AMP (dbcAMP) to the medium. This work was supported by grants HD 18782 and P30 CA 30199 from the National Institutes of Health, Bethesda, MD.