In erythropoietic differentiation, mature red blood cells are generated from specific progenitor cells through the action of specific growth regulatory molecules. To know the mechanism of differentiation, it is important to examine the control of gene expression in these progenitor cells in combination with growth regulatory molecules. We have cloned two genes expressing at a maximal level in the CFU-E (colony forming unit-erythroid), one of the erythroid progenitor cells from novel murine erythroleukemia (MEL) cell line (TSA8) which can be induced to CFU-E in vitro. The expression of these genes is well correlated with the appearance of CFU-E during induction of TSA8 cells, and is higher in the CFU-E-cells enriched from mouse fetal livers than in the more differentiated erythroid cells. Combining these with our previous results, it is suggested that in the erythropoiesis the progenitor cells have distinct patterns of gene expression. This expression is replaced through each progenitor cell rather than by the continuous increase in the expression of a set of genes specific to the mature erythroid cell following the commitment process.
In this report we examine the accumulation of glycoconjugates in the extracellular medium and insoluble matrices surrounding developing cells of the cellular slime mold Dictyostelium discoideum. Conditions were employed which permitted advanced development (slug stage and beyond) in suspension culture. Under these conditions, up to one-third of the total culture protein appeared as non-sedimentable, extracellular material over the course of 48 h of incubation. Most of the secreted molecules expressed carbohydrate antigens (glycoantigens) as detected by Western blotting, using a panel of six monoclonal antibodies. Since the glycoantigens are secreted, immunoelectron microscopy was used to localize the glycoantigens in the extracellular matrices surrounding normally developing cells, including the slime sheath, stalk tube, inner spore coat, outer spore coat, and intercellular fluid between spores. Each glycoantigen had a characteristic distribution, and each extracellular matrix space contained a unique combination of glycoantigens. Thus, although each of these matrices (except inter-spore fluid) contains cellulose as a primary component, they could be distinguished on the basis of their glycoantigen and, by inference, glycoprotein compositions. Furthermore, there were differences between anterior and posterior regions of both slime sheats and stalk tubes. These observations show that secretion as detected in suspension culture occurs under normal conditions as a part of the process of depositing extracellular matrices around the cells. The distributions show that the cell aggregate positionally regulates the expression and deposition of secretory glycoproteins; the resultant patterns of expression of unique protein-linked carbohydrate structures imply a functional role in matrix organization and possibly cell activity which can now be explored.
The chorio-allantoic grafts analysed were prepared from avian proventricular endoderm combined with its own or pancreatic mesenchyme and from re-associated pancreatic layers. Intestine developed ectopically in some grafts: in these, endocrine cells typical of intestine differentiated irrespective of the source of the endoderm or mesenchyme. In addition, endocrine cells inappropriate for the surrounding histology were detected in small numbers in grafts of all categories. Clearly it is not the mesenchyme that is responsible but perhaps some aspect of the procedure, which may relate to stressful stimuli thought to provoke intestinal metaplasia. The differentiation of inappropriate cells aids in understanding the occurrence of ectopic endocrine tumours.
Proteins in oocytes, meiotically mature eggs and zygotes of Xenopus laevis were examined to determine whether proteins in the peripheral region differ from those in the central region of these cells. We show that different regions contain different amounts of newly synthesized proteins and that during meiotic maturation and fertilization the periphery of the animal hemisphere becomes the site where most newly synthesized proteins are found. Examination of two-dimensional gels indicates that most of these proteins are found in all parts of the egg, but certain proteins demonstrate patterns of distribution which are indicative of (1) polarity, (2) developmental stage, and (3) the position within the hemisphere (central or peripheral). These results suggest that the periphery of oocytes, eggs, and zygotes is a site of greater metabolic activity compared with the central region.
This paper reviews the constituents of the cytoskeleton in the cells of the preimplantation mouse embryo and how they change as the development proceeds. The cytoskeleton can be divided into two distinct groups, that in the cytosplasm and that associated with the membrane. The first and better-known group contains microfilaments, microtubules and intermediate filaments, the second such components of the cell and nuclear membrane as spectrin-like protein and nuclear lamin. The filamentous components of the cytoplasmic cytoskeleton adhere to the nuclear and cell membrane at attachment points where specific proteins such as vinculin may mediate the interaction. Each cell of the early embryo has all of these components, but their morphological organization and molecular constitution alter as the embryo develops. These modifications are especially pronounced when the cleavage-stage embryo compacts and when the blastocyst forms and differentiates. These events represent the most critical stages of morphogenesis and cytodifferentiation in the preimplantation embryo. The cytoskeleton may thus have an important role in the control of the early mammalian development.
A monoclonal antibody, FiN1, obtained by immunization of a mouse with homogenates of embryonic quail nodose ganglia, was found to react with a surface antigenic determinant, both in quail and chick, present on practically all neurons of the spinal cord and of the peripheral nervous system and on a subpopulation of fibroblasts. An ontogenetic study performed on tissue sections, cell suspensions and cultures showed that FiN1 defines a differentiation marker which appears relatively late in development, during the second half of embryonic life, and persists after hatching. The onset and evolution of its expression during development varies in a tissue-specific manner.
Progesterone stimulates the activity of a potent Ca2+- and cyclic nucleotide-independent histone kinase in Xenopus oocytes. Microinjection of ATP-gamma-S during the course of meiotic maturation or after the oocytes had arrested at second meiotic metaphase resulted in a further increase of this histone kinase activity in progesterone-stimulated oocytes. ATP-gamma-S microinjection also increased and stabilized the activity of the maturation-promoting factor. The possible mechanism of ATP-gamma-S action is discussed.
The results of this study indicate that the induction of the central nervous system in Xenopus laevis depends on the close juxtaposition of inducing chordamesoderm and reacting ectoderm, which is necessary for the short distance migration of neural inducing factors. The examination of the neuroectoderm-chordamesoderm interface at intervals of 1 h up to 5 h showed that the onset of neural induction is correlated to the degree of contact formation between ectodermal and mesodermal cells. In the ectoderm cells the number of coated pits, a feature of receptor-mediated endocytosis, is increased. Furthermore there exist telophase bridges between some ectoderm cells, which are possibly correlated to secondary cell interactions.
The appearance of sialoconjugates in developing rat kidney glomeruli was studied using lectins and neuraminidase-lectin staining sequences. In the early S-shaped bodies, binding of Maclura pomifera (MPA; specific for galactosaminyl residues of glycoconjugates) could be detected in the presumptive podocyte layer at the apex of these cells, but notably no binding of lectins specific for sialic acid could be seen. During further morphologic maturation of the S-shaped bodies, binding of Limax flavus (LFA; specific for sialic acids) and Triticum vulgaris (WGA; specific for sialic acids and N-acetyl glucosaminyl moieties) appeared at the apex of podocytes and extended subsequently along the lateral membranes to the base of these cells. In morphologically mature glomeruli, LFA stained not only the base of podocytes but also glomerular basement membranes. WGA and MPA bound to the capillary endothelia as well as to the structures bound by LFA. The intensity of WGA binding increased considerably after 5 days of postnatal life, seemingly in parallel with the decrease and ultimate disappearance of MPA binding. In addition to showing individual appearance pattern for various lectin binding sites, these studies give evidence of previously unrecognized postnatal completion of the components of glomerular filtration barrier.
Specific qualitative and quantitative changes in protein synthesis occur from the fertilization to the onset of diapause in the silkworm. We have used two-dimensional gel electrophoresis to analyse the patterns of proteins synthesized in prediapausing eggs of Bombyx. This analysis has been carried out with in vivo labelled polypeptides and with proteins synthesized in vitro by RNA isolated at different stages. The oocyte contains an abundant supply of diverse mRNA which are translatable in vitro. A set of proteins with molecular weight range of 68000 to 74000 and isoelectric points of 5.85–5.95 (hereafter referred to as No. 30) is specific of the germ-anlage stage. Transcripts encoding the No. 30 proteins are not detectable in oocytes, and inhibition of transcription by actinomycin D indicates that No. 30 mRNA are synthesized de novo. Treating eggs at the germ-anlage stage with 4 N HCl at 46°C prevents diapause and is accompanied by overproduction of No. 30 protein. The induction of No. 30 synthesis is also the main event of the heat shock response. The implications of these findings in relation to early embryonic development and prevention of diapause are discussed.
Alveolar type II-like colonies were obtained after a low density plating (5 × 103/60 mm tissue culture dish) of primary type II cells. These colonies were formed only when type II cells were either cocultured with alveolar macrophages or with conditioned media generated by alveolar macrophages. Cells in the colonies appeared homogeneous and kept their lamellar bodies over a period of 8 weeks and more, as observed by electron microscopy. These cells reacted immunocytochemically with antibodies directed against the 32–38 kDa protein fractions of rat surfactant.
We describe muscarinic receptors and intracellular Ca2+ mobilization after cholinergic stimulation in cell suspensions prepared from chick embryos between day 2 (stage 1213 and day 13 (stage 40) of development. Cell suspensions are prepared from whole chick embryos and from embryonic hearts, heads or brains, limb buds, and trunks. Muscarinic receptors are measured using [3H]quinuclidinylbenzilate as specific ligand. Intracellular Ca2+ mobilization is determined by changes of chlorotetracycline fluorescence. (1) Considerable amounts of muscarinic receptors are found in all parts of the embryo and at all stages tested. (2) The intracellular Ca2+ response after stimulation by muscarinic agonist shows a peak at day 3–4 (stage 23). (3) The pharmacological profile of the Ca2+ response remains constant during embryonic development and differs from the profiles of most adult systems. (4) The ‘embryonic muscarinic system’ is uniformly expressed in cells from neural and non-neural tissues. It appears and disappears independently of innervation.
The data we present here permit us to affirm that a 48 kDa phosphoprotein is the target of extracellular Ca2+ during fusion. It is detected only in fusion-competent L6 myoblasts and not in the fusion-defective spontaneous stable variants we isolated. The phosphorylation of this protein species can be totally inhibited by culturing myoblasts in a medium containing low Ca2+ concentrations (0.250 mM). However, under such conditions myoblasts do not fuse, but withdraw from the cell cycle and accumulate the muscle isoform of creatine kinase (M-CK). The results we have obtained support the following conclusions: (1) in fusion-competent cells, overall Ca2+-dependent phosphorylation of cell surface proteins appears to be necessary, but is not sufficient by itself for myoblast fusion; (2) the phosphorylation of a 48 kDa protein species is required for cell fusion; and (3) the phosphorylation of this 48 kDa protein is independent of other main events of cellular differentiation.
Qualitative and quantitative changes in the liver tissue composition have been studied during prenatal development of the Wistar rat by electron microscopy and stereologic methods. The absolute volume of the fetal liver is multiplied by 84 between days 13 and 20 of gestation. In the meantime, the average hepatocyte volume is multiplied by 1.5 between days 12 and 20. The volumetric fraction of hepatocytes increases from 35% of the volumetric fraction of the liver on day 12 to 66% on day 20 of gestation. The non-hepatocyte cells decrease from 49% on day 12 to 25% on day 20. By days 12 and 13, the rough endoplasmic reticulum and the Golgi apparatus are well differentiated, indicating that young fetal hepatocytes are able to synthesize and export plasma proteins. The volumetric fraction of free ribosomes decreases from 38% of the hepatocytic cytoplasm on days 12 and 13 to 6% on day 20. The mitochondrial compartment occupies about 10% of the hepatocyte cytoplasm. The mitochondria, small and round on days 12, 13 and 14, become oblong from day 18 of gestation. The shape of hepatocytes changes during the prenatal development, from potato-like on days 12, 13, 14 to cubic on day 20, with an intermediate, more spheric, stage on day 18.
This investigation was conducted to assess the effects of mouse serum on chick skeletal muscle cell differentiation. In light of earlier findings of altered membrane phospholipid metabolism following mouse serum treatment of Friend erythroleukemic and chick chondrogenic cells, it was of interest to determine whether similar changes would modulate the fusion of mononucleated myoblasts, which is necessary for the formation of multinucleated skeletal muscle fibers. When mouse serum is added to low density cultures of enriched chick myoblasts shortly following cell attachment to the substratum, fusion is inhibited and neutral lipid accumulation ensues. There is an early inhibitory effect on DNA synthesis but not on protein synthesis. There is no increase in the uptake of 2-deoxyglucose following insulin stimulation of the cells, which suggests that while the cells are accumulating large amounts of lipid, they are not being converted into typical adipocytes. Finally, even in cultures of mouse serum-treated cells that undergo significant fusion, one observes thinner myotubes that do not spontaneously contract as do those of control cultures, as well as a disorganization of fluorescently stained actin and myosin myofilaments. These findings demonstrate that mouse serum acts in a dose-dependent manner, is not cytotoxic to the cells, but is capable of modulating normal developmental events of myoblasts as reported for other cell and tissue types.
Retina cognin (R-cognin) is an intrinsic membrane protein of vertebrate retinal cells which supports tissue-specific cell adhesion and mediates cell type-specific associations during development. As a first step in understanding how R-cognin mediates specific adhesion of retinal cell membranes, we asked if cognin bound to another cognin molecule or to a different macromolecule, a possible cognin-binding protein. To do this, we constructed an affinity column with retinal cell membrane proteins (enriched for cognin) bound to the matrix. Proteins in a detergent extract of retinal cell membranes were exposed to this matrix and those which bound specifically eluted and identified by immunoelectrophoresis. Most prominent among these was a protein with an apparent mass of 64 kDa. The binding of this material to the column was blocked by cognin antibody. To eliminate possible artifacts of molecular interactions in vitro, we sought independent confirmation that 64 kDa protein actually bound R-cognin. Using a modified retina membrane vesicle system, we asked what proteins could be photoaffinity cross-linked to cognin during vesicle aggregation. Cross-linking produced a 114 kDa complex on gels which could be resolved into a 50 kDa (cognin) and a 64 kDa band under reducing conditions. Identification of a 64 kDa protein by independent techniques suggests that cognin promotes association of embryonic chick neural retina cells by binding to this macromolecule or these molecules. Identification of a second component in the mechanism should allow elucidation of cognin's role in mediating cell-cell interactions in developing neural retina.
