Showing papers on "Cellular differentiation published in 1993"
TL;DR: There are many compelling examples of control of cell differentiation and gene expression through adhesive interactions with extracellular matrix, including activation of T-lymphocytes through the T-cell antigen receptor is markedly enhanced by integrin-mediated adhesion to fibronectin or laminin.
Abstract: Adhesive interactions between cells and the insoluble meshwork of extracellular matrix proteins play a vital role in embryonic morphogenesis (33, 36, 94, 109, 135, 145), and in the regulation of gene expression in cells of the adult organism (1, 6, 105, 124). While the overall phenomenology ofextracellular matrix (ECM) 1 effects on cell differentiation is well known, the biochemical and molecular bases for these effects have remained elusive. It is clear that many of the interactions between cells and the ECM are mediated by the integrin family of cell surface receptors (2, 3, 13, 72). However, the precise mechanism(s) whereby signals from ECM proteins are transduced via integfins to the intraceUular machinery that controls cell growth, behavior, and differentiation, remains poorly defined. There are many compelling examples of control of cell differentiation and gene expression through adhesive interactions with extracellular matrix. In fibroblasts, cell attachment has been reported to rapidly increase expression of c-los and pro al(I) collagen messages (26, 27). Adhesion to fibronectin fragments, or cross-linking of the integfin oe5/~l fibronectin receptor with antibody, induced the expression of metalloprotease genes in fibroblastic cells; interestingly, intact fibronectin did not provoke this response nor did fibronectin fragments in solution (137). In a somewhat similar vein, stimulation of the C~v//~3 integrin in melanoma cells induced the expression of type IV collagenase and increased the invasive ability of these cells (115). The capacity of breast epithelial cells to express milk proteins in response to hormonal stimuli is quite dependent on the presence of an appropriate ECM (124). Studies in this system have led to the preliminary identification of matrix-dependent elements in the promoter region of the ~ casein gene (111). In the immune system, activation of T-lymphocytes through the T-cell antigen receptor is markedly enhanced by integrin-mediated adhesion to fibronectin or laminin (85, 97, 119). This process is part of a complex dialogue involving adhesive receptors occurring between mature T-cells and antigen presenting cells, as well as during lymphocyte differentiation (40, 132, 133). There is extensive signaling \"cross talk\" between
1,710 citations
TL;DR: The results suggest that in addition to regulating cell growth and differentiation, the ECM also functions as a survival factor for many cell types.
Abstract: Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. In many cases, PCD is induced by the withdrawal of specific hormones or growth factors that function as survival factors. In this study, we have investigated the potential role of the extracellular matrix (ECM) as a cell survival factor. Our results indicate that in the absence of any ECM interactions, human endothelial cells rapidly undergo PCD, as determined by cell morphology, nuclei fragmentation, DNA degradation, protein cross-linking, and the expression of the PCD-specific gene TRPM-2. PCD was blocked by plating cells on an immobilized integrin beta 1 antibody but not by antibodies to either the class I histocompatibility antigen (HLA) or vascular cell adhesion molecule-1 (VCAM-1), suggesting that integrin-mediated signals were required for maintaining cell viability. Treatment of the cells in suspension with the tyrosine phosphatase inhibitor sodium orthovanadate also blocked PCD. When other cell types were examined, some, but not all, underwent rapid cell death when deprived of adhesion to the ECM. These results suggest that in addition to regulating cell growth and differentiation, the ECM also functions as a survival factor for many cell types.
1,550 citations
TL;DR: This simple model fits the understanding of the interactions of growth factors with hematopoietic progenitors and risks oversimplification of a very complex process.
1,404 citations
TL;DR: A great variety of responses has been reported to result from CD44 ligation, which indicates that downstream events following ligand binding by CD44 may vary depending on the cell type expressing CD44 and on the environment of that cell.
Abstract: Publisher Summary This chapter focuses on CD44 and its interaction with extracellular matrix. CD44 is a broadly distributed family of cell surface glycoprotein that has been studied independently by many investigators in a variety of systems and under a variety of names. Two reviews clarify the diverse historical nomenclature and present the evidence that has brought this assortment of molecules and their proposed functions together under the designation CD44. Study of the structure of the single gene that encodes CD44 reveals the way the great variety of molecular forms of CD44 may be generated. CD44 is involved in lymphocyte development, during which it participates both in the earliest stages of T and B cell differentiation and in later stages of T and B cell activation in response to immunological stimuli. In these and other contexts, CD44 seems to function by mediating cell–cell or cell–substrate interactions through recognition of elements of the extracellular matrix, intercellular milieu, and pericellular layer. CD44 is expressed on cells in the early stages of hematopoiesis and has been shown to participate in at least some aspects of the hematopoietic process. In mature lymphocytes, CD44 is upregulated in response to antigenic stimuli and may participate in the effector stage of immunological responses.
1,069 citations
TL;DR: The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that finding in the embryo.
Abstract: We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.
968 citations
TL;DR: This report describes a gene, MCL1, that is isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway and shows that it has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(14;18) chromosome translocation.
Abstract: During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCL1, that we isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression of MCL1 increases early in the induction, or "programming," of differentiation in ML-1 (at 1-3 hr), before the appearance of differentiation markers and mature morphology (at 1-3 days). They further show that MCL1 has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(14;18) chromosome translocation. MCL1 and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCL1 and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCL1 broadens our perspective on an emerging MCL1/BCL2 gene family and will allow further comparison with oncogene families.
966 citations
TL;DR: A mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex is suggested.
Abstract: Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex.
932 citations
TL;DR: The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells, and shows that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis.
749 citations
TL;DR: It is concluded that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor that plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.
Abstract: We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately 50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.
737 citations
TL;DR: Cloned and characterized preadipocyte factor 1 (pref-1), a novel member of the epidermal growth factor (EGF)-like family of proteins, indicates that pref-1 functions as a negative regulator of adipocyte differentiation, possibly in a manner analogous to EGF-like proteins that govern cell fate decisions in invertebrates.
668 citations
TL;DR: R Rearranged V genes from single human B cells, isolated from histological sections of two such structures by micromanipulation, were amplified and sequenced and may indicate a late phase of the germinal centre reaction.
Abstract: Germinal centres are areas of intense B lymphocyte proliferation inside primary B cell follicles in spleen and lymph nodes Rearranged V genes from single human B cells, isolated from histological sections of two such structures by micromanipulation, were amplified and sequenced Cells from the follicular mantle were clonally diverse and largely expressed germline V genes Germinal centres were dominated by a few large B cell clones dispersed throughout these structures and exhibiting intraclonal diversity by ongoing somatic hypermutation Pronounced counterselection of replacement mutations seen in one of the germinal centres may indicate a late phase of the germinal centre reaction A polyclonal population of activated B cells expressing unmutated antibodies in the dark zone of the other germinal centre may represent the initial founder cells
TL;DR: The activated Ki-ras gene plays a key role in colorectal tumorigenesis through altered cell differentiation and cell growth.
Abstract: Point mutations that activate the Ki-ras proto-oncogene are presented in about 50 percent of human colorectal tumors. To study the functional significance of these mutations, the activated Ki-ras genes in two human colon carcinoma cell lines, DLD-1 and HCT 116, were disrupted by homologous recombination. Compared with parental cells, cells disrupted at the activated Ki-ras gene were morphologically altered, lost the capacity for anchorage-independent growth, grew more slowly both in vitro and in nude mice, and showed reduced expression of c-myc. Thus, the activated Ki-ras gene plays a key role in colorectal tumorigenesis through altered cell differentiation and cell growth.
TL;DR: Expressing the PML-RAR alpha protein in U937 myeloid precursor cells showed that they lost the capacity to differentiate under the action of different stimuli, acquired enhanced sensitivity to retinoic acid, and exhibited a higher growth rate consequent to diminished apoptotic cell death.
TL;DR: B cell studies, in conjunction with evidence for a similar developmental switch for T cells and erythrocytes, suggest that evolution has created a "layered" immune system in which successive progenitors (HSC) reach predominance during development and give rise to differentiated cells (B, T, etc) responsible for progressively more complex immune functions.
Abstract: Until recently, the hematopoietic stem cells (HSC) that appear early in ontogeny were thought to constitute a homogeneous, self-replenishing population whose developmental potential remains constant throughout the life of the animal. Studies reviewed here, however, demonstrated clear differences in the developmental potential of fetal and adult progenitor populations (including FACS-sorted HSC). These studies, which chart the ability of various progenitor sources to reconstitute functionally distinct B cell populations, define three B cell lineages: B-1a cells (CD5 B cells), derived from progenitors that are present in fetal omentum and fetal liver but are largely absent from adult bone marrow; B-1b cells ("sister" population), derived from progenitors that are present in fetal omentum, fetal liver, and also in adult bone marrow; and conventional B cells, whose progenitors are missing from fetal omentum but are found in fetal liver and adult bone marrow. B-1a and B-1b cells share many properties, including self-replenishment and feedback regulation of development. These B cell studies, in conjunction with evidence for a similar developmental switch for T cells and erythrocytes, suggest that evolution has created a "layered" immune system in which successive progenitors (HSC) reach predominance during development and give rise to differentiated cells (B, T, etc) responsible for progressively more complex immune functions.
TL;DR: Differences in gene expression during B lymphopoiesis at two distinct ontogenic timings are reported, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal Liver.
Abstract: The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre-Pro-B, early Pro-B, late Pro-B/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate the levels of several B lineage associated genes in each of these fractions by RT-PCR, demonstrating different patterns of expression. We find that expression of terminal deoxynucleotidyl transferase (TdT), lambda 5, and VpreB is predominantly restricted to the Pro-B stages. Rag-1 and Rag-2 expression is also tightly regulated, and is found largely in the Pro-B through small Pre-B stages. Mb-1 is present from Pro-B throughout the pathway at high levels. Finally, Bcl-2 is expressed at high levels only at the pre-Pro-B and mature B stages, whereas it is low during all the intermediate stages. We also correlate this expression data with an analysis of the onset of Ig gene rearrangement as assessed by amplifying D-JH, VH-DJH, and VK-JK. Finally, we report differences in gene expression during B lymphopoiesis at two distinct ontogenic timings, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal liver. In contrast, lambda 5, VpreB, Rag-1, and Rag-2 are expressed at comparable levels.
TL;DR: Three systems in which ECM has been shown to play a crucial role in functional differentiation are described, but mainly the work from the own laboratory is emphasized to provide a more in‐depth analysis of one system.
Abstract: Extracellular matrix (ECM) is an intricate network composed of an array of macromolecules, the importance of which is becoming increasingly apparent. The ECM is an integral part of the machinery that regulates cell function; its role in cell differentiation and tissue-specific gene expression, although essential, is not yet understood. It can act as a positive as well as a negative regulator of functional differentiation depending on the cell type and the genes studied. It also acts in a hierarchical fashion, exacting higher and higher degrees of stringency to achieve full functional differentiation. Regulation by ECM is closely interrelated with the action of other regulators of cellular function, such as growth factors and hormones. But ECM may exert its regulation of gene expression by mechanisms distinct from those known for soluble transcription factors. In this short review, we describe three systems in which ECM has been shown to play a crucial role in functional differentiation, but we emphasize mainly the work from our own laboratory to provide a more in-depth analysis of one system. The three systems are: mouse mammary epithelial cells, rat hepatocytes, and human keratinocytes. The crucial role of ECM in normal cell differentiation implies that its alteration may have serious consequences in malignancies and other diseases. The current functional cell culture models could provide powerful tools not only for understanding regulation of normal cell function but also for the studies of tumorigenesis and possibly cancer therapy.
TL;DR: Results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.
Abstract: The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.
TL;DR: Molecular analysis identifies a nuclear protein expressed in progenitor cells prior to differentiation in eya mutants, suggesting that eya activity influences the distribution of cells between differentiation and death.
TL;DR: This work provides the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts, and proper development was not impaired by the cointegration of alarge portion of the yeast genome with the YAC.
Abstract: Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.
TL;DR: The presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro.
TL;DR: The analyses of TCR γ and δ genes in the mutant mice suggest that intracellular mechanisms acting at the level of DNA rearrangement play key roles in the differential δ and γ gene rearrangements and in the generation of the highly restricted junctional sequences during fetal thymic development.
TL;DR: It is proposed that the earliest oligodendrocyte precursors in the spinal cord originate in a very restricted region of the ventricular zone during a brief window of time around embryonic day 14 (E14).
Abstract: During rat embryogenesis, PDGF alpha receptor (PDGF-alpha R) mRNA is expressed in the ventral half of the spinal cord in two longitudinal columns, one each side of the central canal. Initially, these columns are only two cells wide but the cells subsequently appear to proliferate and disseminate throughout the spinal cord. Our previous studies of PDGF-alpha R expression in the developing CNS suggested that PDGF-alpha R may be a useful marker of the oligodendrocyte lineage in situ. The data presented here complement those studies and lead us to propose that the earliest oligodendrocyte precursors in the spinal cord originate in a very restricted region of the ventricular zone during a brief window of time around embryonic day 14 (E14). In the embryonic brain, migrating PDGF-alpha R+ cells appear to originate in a localized germinal zone in the ventral diencephalon (beneath the foramen of Monro). Our data demonstrate that gene expression and cell fate can be regulated with exquisite spatial resolution along the dorsoventral axis of the mammalian neural tube.
TL;DR: It is reported that bFGF, in a dose-dependent manner, can induce the survival and proliferation of embryonic hippocampal progenitor neurons in vitro and the ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system.
Abstract: Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic fibroblast growth factor (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-20 ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serum-free medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained > 5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.
TL;DR: The results in this model system indicate that a single protein factor can cause differentiation of a stem cell line to multiple phenotypes, that phenotypes induced can be regulated by factor concentration, and that other factors can also influence BMP-2 induced differentiation.
Abstract: C3H10T1/2 cells are an established mesenchymal stem cell line which can differentiate into muscle, fat and cartilage cells when treated with azacytidine. Bone morphogenetic protein-2 (BMP-2) caused a dose dependent differentiation of these cells into fat, cartilage and bone cells—low concentrations favoring adipocytes and high concentrations chondrocytes and osteoblasts. The differentiated phenotypes were stable in the absence of BMP-2. Furthermore, the addition of other growth factors during the differentiation process altered the frequency of the differentiated colony formation. Transfection of the C3H10T1/2 cells with a BMP-2 cDNA also induced a phenotypic change from the parental fibroblast to adipocytes and osteoblasts. Our results in this model system indicate that a single protein factor can cause differentiation of a stem cell line to multiple phenotypes, that phenotypes induced can be regulated by factor concentration, and that other factors can also influence BMP-2 induced differentiation.
TL;DR: PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.
Abstract: Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells. Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen. The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit. Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family. The PEDF gene contains a typical signal-peptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., alpha 1-antitrypsin). It lacks homology, however, at the putative serpin reactive center. Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.
TL;DR: TT2 cells can serve as a valuable vehicle for the production of mutant mice by segregating inside the blastomeres, localized in an inner cell mass of blastocysts, and colonized efficiently in each tissue of the pups.
TL;DR: Recent experimental evidences suggesting that LPS might share a pathway of NF-kappa B activation with other inducers of the factor, which may involve reactive oxygen intermediates (ROI) as messenger molecules.
TL;DR: Detailed analysis of the lit mouse anterior pituitary reveals spatially distinct proliferative zones of growth hormone-producing stem cells and mature somatotrophs, each regulated by a different trophic factor.
Abstract: The molecular basis for the little (lit) mouse phenotype, characterized by a hypoplastic anterior pituitary gland, is the mutation of a single nucleotide that alters Asp 60 to Gly in the growth hormone releasing factor receptor. Detailed analysis of the lit mouse anterior pituitary reveals spatially distinct proliferative zones of growth hormone-producing stem cells and mature somatotrophs, each regulated by a different trophic factor. This sequential growth factor requirement for a specific cell type may exemplify a common strategy for regulating cellular proliferation in other mammalian organs.
TL;DR: Data from this study are consistent with the hypothesis that Xwnt-8 plays a role in the mesodermal differentiation of ventral marginal zone cells during normal development and may ventralize the response of lateral mesodmal cells to dorsalizing signals from the organizer, thus contributing to the graded nature of the final body pattern.
Abstract: This study analyzes the hierarchy of signals that spatially restrict expression of Xenopus Xwnt-8 to mesodermal cells outside of the Spemann organizer field and examines the potential role that endogenous Xwnt-8 may play in dorsoventral patterning of the embryonic mesoderm. The effects of ectopic introduction of a Nieuwkoop center-like activity or of ectopic expression of goosecoid, on the distribution of endogenous Xwnt-8 transcripts were analyzed. The results of these studies are consistent with the hypothesis that maternally derived signals from the Nieuwkoop center function to positively regulate expression of the homeo box gene goosecoid in Spemann organizer cells, leading to a subsequent repression of Xwnt-8 expression in these cells. This exclusion of Xwnt-8 from cells of the organizer field may be important for normal dorsal development, in that ectopic expression of Xwnt-8 in organizer cells after the midblastula stage, by injection of plasmid DNA, ventralizes the fate of these cells. This is distinct from the previously observed dorsalizing effect of Xwnt-8 when expressed prior to the midblastula stage by injection of RNA. The effects of plasmid-derived Xwnt-8 on isolated blastula animal cap ectoderm were also analyzed. Expression of Xwnt-8 in animal pole ectoderm after the midblastula stage ventralizes the response of dorsal animal pole cells to activin and allows naive ectodermal cells to differentiate as ventral mesoderm in the absence of added growth factors. Collectively, these data are consistent with the hypothesis that Xwnt-8 plays a role in the mesodermal differentiation of ventral marginal zone cells during normal development. Furthermore, endogenous Xwnt-8 may ventralize the response of lateral mesodermal cells to dorsalizing signals from the organizer, thus contributing to the graded nature of the final body pattern.
TL;DR: It is proposed that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons.
Abstract: We have identified a human cell line with a phenotype resembling committed CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expressed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule-associated protein (MAP) expressed in human neuroepithelium. NT2/D1 cells also expressed the cell adhesion molecules NCAM and N-cadherin which are thought to be important in cell-cell interactions within the neuroepithelium. These NT2/D1 cells also expressed small amounts of NF-L, alpha-internexin, NF-M, and MAP2c, indicating that they are committed to a neuronal fate. Previous studies have shown that, following RA treatment, a proportion of NT2/D1 cells terminally differentiate into neurons and that this occurs via an asymmetric stem cell mode of differentiation. In light of the identification of the neuroepithelial phenotype of NT2/D1 cells we decided to examine more closely the relationship of in vitro neurogenesis in NT2/D1 cells, during RA treatment to that of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and increased phosphorylation of NF-M. By 10-14 days, these cells began to resemble neurons morphologically, i.e., with rounded cell bodies and processes. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons began to express MAP2b and tau and extinguished their expression of nestin. Recently, we developed a method for obtaining pure cultures of neurons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cells. Given the data obtained in this study and the characterization of the neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons.