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Showing papers on "Cellular differentiation published in 1996"


Journal ArticleDOI
12 Jul 1996-Science
TL;DR: A monoclonal antibody raised to the mouse homolog of CD34 (mCD34) was used to purify mouse HSCs to near homogeneity to enable analysis of the self-renewal and multilineage differentiation of individual HSCS.
Abstract: Hematopoietic stem cells (HSCs) supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. A monoclonal antibody raised to the mouse homolog of CD34 (mCD34) was used to purify mouse HSCs to near homogeneity. Unlike in humans, primitive adult mouse bone marrow HSCs were detected in the mCD34 low to negative fraction. Injection of a single mCD34(lo/-), c-Kit+, Sca-1(+), lineage markers negative (Lin-) cell resulted in long-term reconstitution of the lymphohematopoietic system in 21 percent of recipients. Thus, the purified HSC population should enable analysis of the self-renewal and multilineage differentiation of individual HSCs.

2,134 citations


Journal ArticleDOI
02 Aug 1996-Science
TL;DR: In vitro application of Hedgehog or P THrP protein to normal or PTHrP (−/−) limb explants demonstrated that PTHRP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.
Abstract: Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone—related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (−/−) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (−/−) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.

1,930 citations


Journal ArticleDOI
14 Jun 1996-Science
TL;DR: Heritable mutations in BCNS patients and a somatic mutation in a sporadic BCC were identified in a human homolog of the Drosophila patched (ptc) gene, which appears to be crucial for proper embryonic development and for tumor suppression.
Abstract: The basal cell nevus syndrome (BCNS) is characterized by developmental abnormalities and by the postnatal occurrence of cancers, especially basal cell carcinomas (BCCs), the most common human cancer. Heritable mutations in BCNS patients and a somatic mutation in a sporadic BCC were identified in a human homolog of the Drosophila patched (ptc) gene. The ptc gene encodes a transmembrane protein that in Drosophila acts in opposition to the Hedgehog signaling protein, controlling cell fates, patterning, and growth in numerous tissues. The human PTC gene appears to be crucial for proper embryonic development and for tumor suppression.

1,898 citations


Journal ArticleDOI
TL;DR: This study describes the first demonstration, through clonal and population analyses in vitro, of a mammalian CNS stem cell that proliferates in response to an identified growth factor (EGF) and produces the three principal cell types of the CNS.

1,462 citations


Journal ArticleDOI
18 Apr 1996-Nature
TL;DR: Stat6-l- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE, indicating the lack of a non-redundant function in normal development.
Abstract: Signal transducers and activators of transcription (Stats) are activated by tyrosine phosphorylation in response to cytokines, and are thought to mediate many of their functional responses. Stat6 is activated in response to interleukin (IL)-4 and may contribute to various functions including mitogenesis, T-helper cell differentiation and immunoglobulin isotype switching. To evaluate the role of Stat6, we generated Stat6-null mice (Stat6 -/-) by gene disruption in embryonic stem cells. The mice were viable, indicating the lack of a non-redundant function in normal development. Although naive lymphoid cell development was normal, Stat6 -/- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE. In contrast, IL-4-mediated proliferation was only partly affected.

1,314 citations


Journal ArticleDOI
TL;DR: It was found that ARPE-19 cells exhibit morphological polarization when plated on laminin-coated Transwell-COL filters in medium with a low serum content and has structural and functional properties characteristic of RPE cells in vivo, suggesting that this cell line will be valuable for in vitro studies of retinal pigment epithelium physiology.

1,269 citations


Journal ArticleDOI
TL;DR: A simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells that are suitable for the formation of intracardiac grafts, and should be applicable to all ES-derived cell lineages.
Abstract: This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.

1,230 citations


Journal ArticleDOI
TL;DR: A simple model in which transient exposure to extrinsic factors acting through known pathways initiates fate decisions by multipotential CNS stem cells is supported.
Abstract: Identifying the signals that regulate stem cell differentiation is fundamental to understanding cellular diversity in the brain. In this paper we identify factors that act in an instructive fashion to direct the differentiation of multipotential stem cells derived from the embryonic central nervous system (CNS). CNS stem cell clones differentiate to multiple fates: neurons, astrocytes, and oligodendrocytes. The differentiation of cells in a clone is influenced by extracellular signals: Platelet-derived growth factor (PDGF-AA, -AB, and -BB) supports neuronal differentiation. In contrast, ciliary neurotrophic factor and thyroid hormone T3 act instructively on stem cells to generate clones of astrocytes and oligodendrocytes, respectively. Adult stem cells had remarkably similar responses to these growth factors. These results support a simple model in which transient exposure to extrinsic factors acting through known pathways initiates fate decisions by multipotential CNS stem cells.

1,224 citations


Journal ArticleDOI
TL;DR: While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.
Abstract: PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3-5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic-treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

1,135 citations


Journal ArticleDOI
20 Dec 1996-Science
TL;DR: Results indicate that covalent modification of PPARγ by serum and growth factors is a major regulator of the balance between cell growth and differentiation in the adipose cell lineage.
Abstract: Adipocyte differentiation is an important component of obesity and other metabolic diseases. This process is strongly inhibited by many mitogens and oncogenes. Several growth factors that inhibit fat cell differentiation caused mitogen-activated protein (MAP) kinase-mediated phosphorylation of the dominant adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and reduction of its transcriptional activity. Expression of PPARγ with a nonphosphorylatable mutation at this site (serine-112) yielded cells with increased sensitivity to ligand-induced adipogenesis and resistance to inhibition of differentiation by mitogens. These results indicate that covalent modification of PPARγ by serum and growth factors is a major regulator of the balance between cell growth and differentiation in the adipose cell lineage.

1,064 citations


Journal ArticleDOI
TL;DR: Data indicate that ADD1 plays an important role in fat cell gene expression and differentiation, and suggest that it may function by augmenting a step in PPARgamma-mediated transcription.
Abstract: Adipocyte determination and differentiation-dependent factor 1 (ADD1) is a member of the basic helix-loop-helix leucine zipper (bHLH-LZ) family of transcription factors that binds to two distinct DNA sequences and has been associated with both adipocyte development and cholesterol homeostasis (where it has been termed SREBP1). To investigate the biological role of ADD1, we expressed wild-type and dominant negative forms of this protein with retroviral vectors in preadipocytes and nonadipogenic cells. A dominant-negative form of ADD1 with a point mutation in the DNA-binding domain sharply represses the differentiation of 3T3-L1 cells as observed morphologically or by the expression of adipocyte-specific mRNAs. When NIH-3T3 cells ectopically expressing ADD1 are cultured under hormonal conditions not favoring differentiation, they do not overtly differentiate but still activate expression of mRNAs for fatty acid synthase (FAS) and lipoprotein lipase (LPL), two key genes that regulate fatty acid metabolism. Under culture conditions permissive for differentiation including a PPAR activator, 15%-20% of the cells expressing ADD1 undergo adipogenesis while 2%-3% of cells containing a control vector differentiate. Simultaneous expression of ADD1 with PPARgamma increases the transcriptional activity of this adipogenic nuclear hormone receptor, suggesting involvement of ADD1 in this pathway. These data indicate that ADD1 plays an important role in fat cell gene expression and differentiation, and suggest that it may function by augmenting a step in PPARgamma-mediated transcription.

Journal ArticleDOI
15 Nov 1996-Cell
TL;DR: The view of eye development is broadened to include the whole ommatidium and it is suggested that reiterative activation of DER is critical for triggering the differentiation of all cell types.

Journal ArticleDOI
TL;DR: These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.

Journal ArticleDOI
TL;DR: Purification of the SWI‐SNF2 homologs demonstrates that it is heterogeneous with respect to subunit composition, and certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.
Abstract: We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.

Journal ArticleDOI
TL;DR: A null mutation of the only known mammalian DNA methyltransferase gene is generated in mouse embryonic stem cells and it is found that the development of the homozygous embryos is arrested prior to the 8-somite stage, suggesting the existence of a second DNA methyl transferase in mammalian cells.
Abstract: It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

Journal ArticleDOI
TL;DR: Evidence is obtained for maturation in vivo in response to the bacterial product lipopolysaccharide (LPS), which is interpreted to mean that LPS can cause DC in the marginal zone to mature and to migrate into and then out of the T cell areas.
Abstract: Dendritic cells (DC) are described as "nature's adjuvant," since they have the capacity to sensitize T cells in vivo upon first encounter with the antigen. The potent accessory properties of DC appear to develop sequentially. In particular, the ability to process antigens and to sensitize native T cells develops in sequence, a process termed "maturation" that is well described in vitro. Here, we obtain evidence for maturation in vivo in response to the bacterial product lipopolysaccharide (LPS). Before LPS treatment, many DC are found at the margin between the red and white pulp. These cells lack the M342 and DEC-205 markers, but process soluble proteins effectively. 6 h after LPS, DC with the M342 and DEC-205 markers are found in increased numbers in the T cell areas. These cells have a reduced capacity to process proteins, but show increases in the B7 costimulator and T cell stimulatory capacity. 48 h after LPS, the number of DC in the spleen is reduced markedly. We interpret these findings to mean that LPS can cause DC in the marginal zone to mature and to migrate into and then out of the T cell areas.

Journal ArticleDOI
TL;DR: In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages.
Abstract: Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Journal ArticleDOI
26 Jan 1996-Cell
TL;DR: Cell differentiation in the neural tube of embryos in which ISL1 expression has been eliminated by gene targeting shows that ISl1 is required for the generation of motor neurons and suggests that motor neuron generation is requiredfor the subsequent differentiation of certain interneurons.

Journal ArticleDOI
01 Dec 1996-Immunity
TL;DR: The lymphoid defects detected in Ikaros null mice reveal critical molecular differences between fetal and postnatal hematopoietic progenitors that dictate their ability to give rise to T cells and establish IkarOS as a tumor suppressor gene acting during thymocyte differentiation.

Journal ArticleDOI
TL;DR: The data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids.
Abstract: The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.

Journal ArticleDOI
TL;DR: Despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.
Abstract: During terminal differentiation of skeletal myoblasts, cells fuse to form postmitotic multinucleated myotubes that cannot reinitiate DNA synthesis. Here we investigated the temporal relationships among these events during in vitro differentiation of C2C12 myoblasts. Cells expressing myogenin, a marker for the entry of myoblasts into the differentiation pathway, were detected first during myogenesis, followed by the appearance of mononucleated cells expressing both myogenin and the cell cycle inhibitor p21. Although expression of both proteins was sustained in mitogen-restimulated myocytes, 5-bromodeoxyuridine incorporation experiments in serum-starved cultures revealed that myogenin-positive cells remained capable of replicating DNA. In contrast, subsequent expression of p21 in differentiating myoblasts correlated with the establishment of the postmitotic state. Later during myogenesis, postmitotic (p21-positive) mononucleated myoblasts activated the expression of the muscle structural protein myosin heavy chain, and then fused to form multinucleated myotubes. Thus, despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.

Journal ArticleDOI
05 Dec 1996-Nature
TL;DR: The results show that GATA-3−/− ES cells can contribute to the development of the mature ery-throid, myelomonocytic and B-cell lineages, but fail to give rise to thymocytes or mature peripheral T cells, suggesting that Gata-3 is an essential and specific regulator of early thymocyte development.
Abstract: THE zinc-finger transcription factor GATA-3 is expressed in haematopoietic cells and in the developing kidney and nervous system1–7. Within the haematopoietic lineages, expression of GATA-3 is restricted to thymocytes and T cells. Functionally important GATA-3 binding sites have been identified in multiple T-cell-specific genes1,6–8. Mice containing homozygous null mutations of the GATA-3 gene die on embryonic day 12, precluding a detailed assessment of the role of GATA-3 in haematopoietic development9. Here we have used murine embryonic stem (ES) cells containing homozygous mutations in the GATA-3 gene (GATA-3−/− ) in conjunction with the RAG-2−/− (ref. 10) and C57BL/6 complementation systems to study the role of GATA-3 in mammalian haematopoiesis. Our results show that GATA-3−/− ES cells can contribute to the development of the mature ery-throid, myelomonocytic and B-cell lineages, but fail to give rise to thymocytes or mature peripheral T cells. The differentiation of GATA-3−/−T cells is blocked at or before the earliest double-negative (CD4−/CD8−) stage of thymocyte development, such that the GATA-3−/− ES cells are unable to contribute measurably to the double-negative thymocyte population. These findings suggest that GATA-3 is an essential and specific regulator of early thymocyte development.

Journal ArticleDOI
01 Jan 1996-Immunity
TL;DR: Mice deficient in interleukin-5 (IL-5-/- mice) were generated by gene targeting in embryonal stem cells, indicating that increased eosinophils do not play a significant role in the host defence in this parasite model.

Journal ArticleDOI
TL;DR: IL‐6 generates both growth‐enhancing signals and growth arrest‐ and differentiation‐inducing signals at the same time, suggesting Stat3 may be a key molecule which determines the cellular decision from cell growth to differentiation in M1 cells.
Abstract: Interleukin-6 (IL-6) induces either differentiation or growth of a variety of cells. Little is known about the molecular basis of this cellular decision. The family of signal transducer and activator of transcription (Stat) proteins are involved in signaling through a variety of cytokine and growth factor receptors, although their biological roles have not been established. To address whether Stat proteins play roles in IL-6-induced growth or differentiation, we introduced two types of mutant Stat3 acting in a dominant-negative manner into M1 leukemic cells which respond to IL-6 with growth arrest and terminal differentiation. We show that dominant-negative forms of Stat3 inhibited both IL-6-induced growth arrest at G(0)/G1 and macrophage differentiation in the M1 transformants. Blocking of Stat activation resulted in inhibition of IL-6-induced repression of c-myb and c-myc. Furthermore, IL-6 enhanced the growth of M1 cells primarily through shortening the length of the G1 period when Stat3 was suppressed. Thus IL-6 generates both growth-enhancing signals and growth arrest- and differentiation-inducing signals at the same time. Stat3 may be a key molecule which determines the cellular decision from cell growth to differentiation in M1 cells.

Journal ArticleDOI
TL;DR: It is shown that mice carrying the orJ allele have a premature stop codon in the homeobox of the Chx1O gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells, which indicates that Chx10 is an essential component in the network of genes required for the development of the mammalian eye.
Abstract: Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

Journal ArticleDOI
TL;DR: It is proposed that Cdx2 is part of a regulatory network that orchestrates a developmental program of proliferation, morphogenesis, and gene expression in the intestinal epithelium by supporting transcription of differentiated gene products.
Abstract: Precise regulation of cellular proliferation, differentiation, and senescence results in the continuous renewal of the intestinal epithelium with maintenance of a highly ordered tissue architecture. Here we show that an intestine-specific homeobox gene, Cdx2, is a transcription factor that regulates both proliferation and differentiation in intestinal epithelial cells. Conditional expression of Cdx2 in IEC-6 cells, an undifferentiated intestinal cell line, led to arrest of proliferation for several days followed by a period of growth resulting in multicellular structures containing a well-formed columnar layer of cells. The columnar cells had multiple morphological characteristics of intestinal epithelial cells. Enterocyte-like cells were polarized with tight junctions, lateral membrane interdigitations, and well-organized microvilli with associated glycocalyx located at the apical pole. Remarkably, there were also cells with a goblet cell-like ultrastructure, suggesting that two of the four intestinal epithelial cell lineages may arise from IEC-6 cells. Molecular evidence for differentiation was shown by demonstrating that cells expressing high levels of Cdx2 expressed sucrase-isomaltase, an enterocyte-specific gene which is a well-defined target for the Cdx2 protein. Taken together, our data suggest that Cdx2 may play a role in directing early processes in intestinal cell morphogenesis and in the maintenance of the differentiated phenotype by supporting transcription of differentiated gene products. We propose that Cdx2 is part of a regulatory network that orchestrates a developmental program of proliferation, morphogenesis, and gene expression in the intestinal epithelium.

Journal ArticleDOI
TL;DR: Alongside the functional roles of stromal cells, considerable progress is being made in unraveling the nature of the signaling pathways involved in T cell development, and identification of the pre-T cell receptor and associated signaling molecules marks an important advance.
Abstract: Interactions between stromal cells and thymocytes play a crucial role in T cell development. The thymic stroma is complex and consists of epithelial cells derived from the pharyngeal region during development, together with macrophages and dendritic cells of bone marrow origin. In addition, fibroblasts and matrix molecules permeate the whole framework. It is now apparent that these individual stromal components play specialized roles at different stages of T cell differentiation. Thus, at the early CD4-8- stage of development, T cell precursors require fibroblast as well as epithelial cell interactions. Later, at the CD4+8+ stage, as well as providing low avidity TCR/MHC-peptide interactions, thymic epithelial cells have been shown to possess unique properties essential for positive selection. Dendritic cells, on the other hand, are probably efficient mediators of negative selection, but they may not be solely responsible for this activity. Alongside the functional roles of stromal cells, considerable progress is being made in unraveling the nature of the signaling pathways involved in T cell development. Identification of the pre-T cell receptor (pre-TCR) and associated signaling molecules marks an important advance in understanding the mechanisms that control gene rearrangement and allelic exclusion. In addition, a better understanding of the signaling pathways that lead to positive selection on the one hand and negative selection on the other is beginning to emerge. Many issues remain unresolved, and some are discussed in this review. What, for example, is the nature of the chemotactic factor(s) that attract stem cells to the thymus? What is the molecular basis of the essential interactions between early thymocytes and fibroblasts, and early thymocytes and epithelial cells? What is special about cortical epithelial cells in supporting positive selection? These and other issues are ripe for analysis and can now be approached using a combination of modern molecular and cellular techniques.

Journal ArticleDOI
S K Masur1, H S Dewal, T T Dinh, I Erenburg, S Petridou 
TL;DR: It is concluded that myofibroblast differentiation can be induced in fibroblasts by plating at low density, in which at least two factors interact: loss of cell contact and the presence of TGF-beta.
Abstract: Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha-actin phenotype was lost within 3 days. Furthermore, initially 60% of the low density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enriched cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast-enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density-dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.

Journal ArticleDOI
19 Jul 1996-Science
TL;DR: Data indicate that induction of Cdk inhibitors may serve to protect differentiating myocytes from programmed cell death as well as play a role in establishing the postmitotic state.
Abstract: Proliferating murine C2C12 myoblasts can undergo either terminal differentiation or programmed cell death under conditions of mitogen deprivation Unlike myoblasts, differentiated myotubes were resistant to apoptosis During myogenesis the appearance of the apoptosis-resistant phenotype was correlated with the induction of the cyclin-dependent kinase (Cdk) inhibitor p21CIP1 but not with the appearance of myogenin, a marker expressed earlier in differentiation Forced expression of the Cdk inhibitors p21CIP1 or p16INK4A blocked apoptosis during myocyte differentiation These data indicate that induction of Cdk inhibitors may serve to protect differentiating myocytes from programmed cell death as well as play a role in establishing the postmitotic state

Journal ArticleDOI
01 Nov 1996-Bone
TL;DR: Recent findings concerning bone marrow adipocyte morphology and physiology, the transcriptional and cytokine mechanisms regulating their differentiation, and the interrelationships existing betweenBone marrow adipocytes, hematopoiesis, and osteogenesis lend support to a "plastic" model of bone marrow stromal cell differentiation.