The engrailed locus of Drosophila: structural analysis of an embryonic transcript

cDNA clones originating from the engrailed gene of Drosophila have been isolated from recombinant phage libraries that were made using poly(A)+ RNA extracted from early embryos. The DNA sequence of one of these clones includes a homeo box, a 180 bp sequence present in several other Drosophila genes important in formation of body pattern during development. The homeo boxes found in the other Drosophila genes, as well as in cognate sequences from a wide range of segmented animals, including higher vertebrates, are highly conserved. By contrast, the homeo box within the engrailed gene diverges substantially and, unlike the other homeo boxes, is interrupted by an intervening sequence. The engrailed homeo box is located near the 3' end of a 1700 bp open reading frame. If translated, this sequence would produce a protein of unusual composition. We also show that a neighboring gene has a large region with strong homology to engrailed, and that it also contains a homeo box.     

Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications

Abstract.  Embryonal carcinoma and embryonic stem cells have served as models to understand basic aspects of neuronal differentiation and are promising candidates for regenerative medicine. Besides being well characterized regarding the capability of embryonal carcinoma and embryonic stem cells to be precursors of different tissues, the molecular mechanisms controlling neuronal differentiation are hardly understood. Neuropeptide and neurotransmitter receptors are expressed at early stages of differentiation prior to synaptogenesis, triggering transient changes in calcium concentration and inducing neurone-specific gene expression. In vitro neuronal differentiation of embryonal carcinoma and embryonic stem cells closely resembles early neuronal development in vivo . Murine P19 EC cells are a well-characterized model for in vitro differentiation, which upon treatment with retinoic acid differentiate into neurones. Expression and activity of various receptor proteins is regulated during their differentiation. Stimulation of kinin-B2, endothelin-B, muscarinic acetylcholine, and N-methyl-D-aspartate receptors results in transient increases of intracellular free calcium concentration [Ca²⁺]_i in P19 cells undergoing neuronal differentiation, whereas embryonal cells do not respond or show a smaller change in [Ca²⁺]_i than differentiating cells. Receptor inhibition, as studied with the example of the kinin-B2 receptor, aborts neuronal maturation of P19 cells, demonstrating the crucial importance of B2 receptors during the differentiation process. Future success in obtaining desired neuronal phenotypes from pluripotent cells in vitro may offer new therapeutic perspectives for curing genetic and acquired dysfunctions of the developing and adult nervous system.     

Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage-specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage-specific carbohydrates are the lactoseries structure (Gal beta 1----4GlcNAc) and the globoseries structure (Gal alpha 1----4Gal). Notably, the glycoprotein-bound large carbohydrate of poly-N-acetyllactosamine-type ([Gal beta 1----4GlcNAc beta 1----3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage-specific embryonic antigen-1 (SSEA-1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N-acetylglucosaminide alpha 1----3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA-1. Accumulating evidence suggests that poly-N-acetyllactosamine-type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.     

The small heat shock protein hsp25 is accumulated in P19 embryonal carcinoma cells and embryonic stem cells of line BLC6 during differentiation

Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6.     

Sequence analysis of the murine Hox-2.2, -2.3, and -2.4 homeo boxes: evolutionary and structural comparisons

We have determined the nucleotide sequences and deduced the amino acid sequences of three tandemly arranged murine boxes of the Hox-2 homeo box gene complex on mouse chromosome 11 (Hox-2.2, -2.3, and -2.4). The type and position of differences with other sequenced homeo boxes were analyzed. Hox-2.2 is nearly identical with its cognate human homeo box Hu-2. Hox-2.3 shares 59 of 61 amino acids with the Antennapedia homeo domain of Drosophila and the MM-3 homeo domain of Xenopus and shows 60 of 61 amino acid identity with human HuC1. Hox-2.3, MM-3, and HuC1 also share a stretch of six glutamic acid residues followed by a stop codon 15-20 amino acids 3' of the homeo domain. Hox-2.4 is relatively divergent from most of the other homeo boxes sequenced to date; however, it matches the Hox-3.1 murine homeo domain at 60 of 61 positions. Sequence comparisons with other murine homeo domains, together with previous studies of their genomic organization and chromosomal location, provide support for the hypothesis of a large-scale duplication resulting in the two major murine homeo box gene complexes Hox-1 and Hox-2.     

Altered activation by calcitonin and inhibition by somatostatin of human embryonal carcinoma cell adenylate cyclase with retinoic acid-induced differentiation

Human teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cyclase with differentiation in response to 10 microM retinoic acid. Two cell lines CL12 and CL13, cloned from Tera 2 cells by Dr. C. F. Graham, have been used in these studies. Adenylate cyclase of CL12 and CL13 cells is stimulated in the presence of 10 microM GTP by epinephrine and calcitonin, with calcitonin being the most potent stimulator of cyclic AMP production. Exposure of these cells to retinoic acid leads to an arrest in growth and within 6 days to a differentiated cell population with a stable nonreversible phenotype. No changes in basal, GTP- and fluoride-stimulated adenylate cyclase activities are observed with retinoic acid treatment, but the cyclase of differentiated cells exhibits a greater stimulation by calcitonin (7.5-fold) and the appearance of a somatostatin inhibitory effect. Somatostatin specifically inhibits, by 25%, the hormonal stimulation of adenylate cyclase of cells treated for 5 days with retinoic acid. The increase in calcitonin stimulation of adenylate cyclase activity of the differentiated cells is related to an increase (congruent to 3-fold) in the number of hormonal receptors and not to a significant change in receptor binding affinity (Kd 4.6 X 10(8) M-1). These alterations in calcitonin and somatostatin responsiveness suggest a possible regulatory role for these hormones during embryonic development. Furthermore, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of human embryonal carcinoma cell differentiation.     

Expression of metalloproteinases and inhibitors in the differentiation of P19CL6 cells into cardiac myocytes

P19CL6 are a clonal derivative of P19 embryonal carcinoma cells, a euploid, multipotent mouse cell line, that differentiate efficiently into cardiac myocytes, with spontaneous beating evident within 10 days, following DMSO treatment. Using real-time quantitative RT-PCR we have profiled the expression of the complete matrix metalloproteinase and tissue inhibitor of metalloproteinase gene families during P19CL6 differentiation to cardiac myocytes. The genes subdivide into eight groups based upon their expression profile. Their expression was both qualitatively and quantitatively highly homologous to that seen during mouse heart development.     

Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early stages of development

The patterns of protein synthesis in teratocarcinoma stem cells (embryonal carcinoma cells) and in mouse embryos at various stages of preimplantation development were studied using SDS-polyacrylamide slab gel electrophoresis with autoradiography. Significant differences were observed in comparisons of embryonal carcinoma cells with isolated inner cell masses (ICMs) or with embryonic cells at earlier stages of development. However, no such differences in the overall pattern of protein synthesis were found when the embryonal carcinoma cells were compared with the embryonic ectoderm (that portion of the ICM which remains after endoderm differentiation). Both synthesize at least one prominent 55,000-dalton protein that is not detected in embryonic cells at earlier stages of development. This protein can thus be used as a biochemical marker of ectoderm formation during embryonic development. The pattern of protein synthesis common to embryonal carcinoma cells and embryonic ectoderm is not shared by other cultured cell types.     

Assessment of pluripotency and multilineage differentiation potential of NTERA-2 cells as a model for studying human embryonic stem cells

Embryonal carcinoma cells are pluripotent stem cells derived from teratocarcinomas and are considered to be the malignant counterparts of human embryonic stem cells. As there are few reliable experimental systems available to study the molecular mechanisms governing normal embryogenesis, well-characterized human embryonal carcinoma stem cell lines may provide a robust and simple model to study certain aspects of pluripotency and cellular differentiation. Here, we have analysed NTERA-2 cL.D1 cells at molecular and cellular levels during expansion and differentiation, via formation of cell aggregates similar to embryoid bodies in embryonic stem cells. Thus, human embryonal carcinoma cells may provide a valuable insight into cell fate determination, into the embryonic ectoderm, mesoderm and endoderm and their downstream derivatives.     

The glycosidic antigen recognised by a novel monoclonal antibody, 75.12, is developmentally regulated on mouse embryonal carcinoma cells

Monoclonal antibody 75.12 raised against the human ovarian teratocarcinoma cell line PA1 detects a 'Y' or iso-leb glycosidic structure. Using the 75.12 antibody we have established that the Y antigen is expressed on some but not all mouse embryonal carcinoma (EC) lines. The Y or 75.12 antigen-positive EC cell lines F9 and PCC4 cease to express the antigen after differentiation induced with retinoic acid and this decreased expression parallels the morphological differentiation of the EC cells. These results support not only the idea that carbohydrate structures present on embryonic cells undergo marked alteration during differentiation, but also that established mouse EC cells may differ in their differentiation states.