The idea that glycosphingolipids (or, briefly, glycolipids) are ubiquitous components of plasma membrane and display cell type-specific patterns perhaps stemmed from the classical studies on glycolipids of erythrocyte membranes.(1,2) Subsequently, plasma membranes of various animal cells were successfully isolated and analyzed; all were characterized by their much higher content of glycolipid than was found in intracellular membranes.(3–8) It is generally assumed that glycolipids are present at the outer leaflet of the plasma membrane bilayer, although this assumption is based only on experiments with surface-labeling by galactose oxidase-NaB[3H]4 of intact and lysed erythrocyte membranes and inside-out vesicles.(9,10) Obviously, further extensive studies of the organization of glycolipid in other eukaryotic cell membranes are necessary. Interestingly, the majority of neutral glycolipids present in plasma membranes are cryptic (see Section 4.2.1).

Glycosphingolipids in Cellular Interaction, Differentiation, and Oncogenesis

The hope that hybridoma antibodies would reveal unique cell surface antigens during embryogenesis, differentiation and oncogenesis has been replaced by the realization that such antigens are mainly carbohydrate structures of glycoproteins and glycolipids occurring in many cell types. These findings either may reflect limitations in the methods of selection of hybridoma antibodies or may point to important roles for the diverse carbohydrate structures as receptors for regulators of cell growth and differentiation.

Demonstration by Monoclonal Antibodies That Carbohydrate Structures of Glycoproteins and Glycolipids Are Onco-Developmental Antigens

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.

/pdf/development-of-definitive-endoderm-from-embryonic-stem-cells-3db20s42a1.pdf

Development of definitive endoderm from embryonic stem cells in culture

Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators.

Sox proteins belong to the HMG box superfamily of DNA-binding proteins and are found throughout the animal kingdom. They are involved in the regulation of such diverse developmental processes as germ layer formation, organ development and cell type specifi-cation. Hence, deletion or mutation of Sox proteins often results in developmental defects and congenital disease in humans. Sox proteins perform their function in a complex interplay with other transcription factors in a manner highly dependent on cell type and promoter context. They exhibit a remarkable crosstalk and functional redundancy among each other.

/pdf/from-head-to-toes-the-multiple-facets-of-sox-proteins-pxazvuohzp.pdf

From head to toes: The multiple facets of Sox proteins

Syngeneic antisera have been produced in mouse strain 129/Sv-CP males against the primitive cells of teratocarcinoma. These sera react specifically with the primitive cells and are negative on various types of differentiated teratoma cells derived from the same original tumor. They are negative on all other mouse cells tested, with the exception of male germ cells and cleavage-stage embryos. Thus, teratoma cells possess cell-surface antigens in common with normal cleavage-stage embryos.

Surface Antigens Common to Mouse Cleavage Embryos and Primitive Teratocarcinoma Cells in Culture

We have identified and characterized zebrafish eve1, a novel member of the Drosophila even-skipped (eve) gene family. eve1 RNAs are expressed initially in late blastulae with a peak during the gastrula stage, at which time expression is confined to ventral and lateral cells of the marginal zone of the zebrafish embryo. Later, eve1 transcripts are located in the most posterior part of the extending tail tip. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish, resulting in embryos with exaggerated dorsoanterior structures. In LiCl-treated embryos, eve1 transcripts are completely absent. eve1 is therefore a marker of ventral and posterior cells. In the light of its ventroposterior expression domain, the localization of eve1 transcripts was analysed in spadetail (spt) and no tail (ntl), two mutants with abnormal caudal development. In sptb140 homozygous mutants, there is an accumulation of cells in the tail region, resulting from inadequate migratory behaviour of precursors to the trunk somites. These cells, in their abnormal environment, express eve1, emphasizing the correlation between ventroposterior position and eve1 expression. In homozygous mutant embryos for the gene ntl (the homologue of mouse Brachyury, originally called Zf-T), posterior structures are missing (M. E. Halpern, C. B. Kimmel, R. K. Ho and C. Walker, 1993; Cell In press). While mutant and wild-type embryos do not differ in their eve1 transcript distribution during gastrulation, eve1 expression is absent in the caudal region of mutant ntl embryos during early somitogenesis, indicating a requirement for ntl in the maintenance of eve1 expression during tail extension. Our findings suggest that eve1 expression is correlated with a ventral and posterior cell fate, and provide first insights into its regulation.

The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos

From embryonal carcinoma cells labeled with fucose, two main classes of glycopeptide products of Pronase digestion can be distinguished by Sephadex G-50 column chromatography: one eluted near the excluded volume and a smaller one. The large fucosyl-glycopeptides are scarcely present in differentiated cells derived from embryonal carcinoma cells (i.e., fibroblastlike cells, myoblasts, and parietal yolk-sac carcinoma). During in vitro differentiation of embryonal carcinoma cells, these large glycopeptides disappear almost completely. The small glycopeptides were analyzed by paper electrophoresis, concanavalin A-Sepharose affinity chromatography, and digestion with an endoglycosidase. The major components of these glycopeptides from embryonal carcinoma cells appear to be different from complex glycopeptides known to occur in adult cells. The glycopeptide pattern of mouse preimplantation embryos resembles that of embryonal carcinoma cells. These results suggest that the carbohydrate profile changes fundamentally during early stages of mammalian development.

https://www.pnas.org/content/pnas/75/5/2315.full.pdf

Carbohydrate structure and cell differentitation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells

Hepatic nuclear factor 1 (HNF1) is a highly diverged homeoprotein that is crucial for transcription of many liver-specific genes including albumin. In particular, a minimal promoter, consisting of an HNF1-binding-site and a TATA box, is highly active only in hepatoma cell lines. The expression of the HNF1 and albumin genes has been examined in mouse embryos by in situ hybridization. At 10.5 days of gestation, the HNF1 mRNA was detected in both the hepatic primordia and visceral endoderm of the yolk sac whereas the albumin transcript was present only in the nascent liver. At later stages of development, HNF1 was detected in liver, in the epithelial cells of most of the digestive tract and in the cortex of the kidney, whereas albumin was again found only in the liver. The presence of HNF1 protein in adult kidney was demonstrated by immunodetection in gel-retardation assays and western blot analysis. These experiments show that, even though the HNF1 homeo-protein is essential for expression of many liver-specific genes, it cannot, by itself, force high expression levels of these genes, in non-hepatic tissues.

Hepatic nuclear factor 1 (HNF1) shows a wider distribution than products of its known target genes in developing mouse.

Expression of cytokeratin endo A has been analyzed during mouse blastocyst formation and embryonal carcinoma cell differentiation. To study the regulation of endo A expression, nuclease S1 mapping experiments have been performed on RNA extracted from two-cell to 7.5-day embryos. Low levels of endo A mRNA begin to be detectable in eight-cell embryos. The amount of this mRNA increases at the blastocyst stage, suggesting that endo A expression is regulated at the mRNA level during blastocyst formation. At this stage, in situ hybridization studies show that endo A mRNA is present in the trophectoderm but not in the inner cell mass. In 7.5-day embryos, endo A mRNAs are also detectable in the endoderm layer and in the amnion.

Hubert Condamine

Papers

Surface Antigens Common to Mouse Cleavage Embryos and Primitive Teratocarcinoma Cells in Culture

The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos

Carbohydrate structure and cell differentitation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells

Hepatic nuclear factor 1 (HNF1) shows a wider distribution than products of its known target genes in developing mouse.

Expression of the cytokeratin endo A gene during early mouse embryogenesis.