A functional analysis of imprinting in parthenogenetic embryonic stem cells

A detailed analysis of the developmental potential of parthenogenetic embryonic stem cells (PGES) was made in vivo and in vitro, and a comparison was made with the development of cells from parthenogenetic embryos (PG). In vivo, in chimeras with normal host cells (N), PGES cells showed a restricted tissue distribution consistent with that of PG cells, suggesting faithful imprinting in PGES cells with respect to genes involved in lineage allocation and differentiation. Restricted developmental potential was also observed in teratomas formed by ectopic transfer under the kidney capsule. In contrast, the classic phenotype of growth retardation normally observed in PG<==>N chimeras was not seen, suggesting aberrant regulation in PGES cells of genes involved in growth regulation. We also analysed the expression of known imprinted genes after ES cell differentiation. Igf2, H19 and Igf2r were all appropriately expressed in the PGES derived cells following induction of differentiation in vitro with all-trans retinoic acid or DMSO, when compared with control (D3) and androgenetic ES cells (AGES). Interestingly, H19 was found to be expressed at high levels following differentiation of the AGES cells. Due to the unexpected normal growth regulation of PGES<==>N chimeras we also examined Igf2 expression in PGES derived cells differentiated in vivo and found that this gene was still repressed. Our studies show that PGES cells provide a valuable in vitro model system to study the effects of imprinting on cell differentiation and they also provide invaluable material for extensive molecular studies on imprinted genes. In addition, the aberrant growth phenotype observed in chimeras has implications for mechanisms that regulate the somatic establishment and maintenance of some imprints. This is of particular interest as aberrant imprinting has recently been invoked in the etiology of some human diseases.     

Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated in vitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS.     

Induction of cyclooxygenase (COX)-2 but not COX-1 gene expression in apoptotic cell death

In this study we assessed the regulation of cyclooxygenase (COX)-2 in models of apoptotic cell death in vivo and in vitro. By 6 h after hippocampal colchicine injection in rat, COX-2 (but not COX-1) mRNA expression was elevated. The induction of COX-2 mRNA expression preceded temporally and overlapped anatomically the cellular morphological features of apoptosis in the granule cell layer of the dentate gyrus. Similarly, in an established in vitro model of apoptosis in P19 cells, COX-2 induction preceded apoptosis in response to serum deprivation by 12 h. These studies suggest that COX-2 may be involved in the early mechanisms leading to apoptosis.     

LMP-1, a LIM-domain protein, mediates BMP-6 effects on bone formation

Glucocorticoids can promote osteoblast differentiation from fetal calvarial cells and bone marrow stromal cells. We recently reported that glucocorticoid specifically induced bone morphogenetic protein-6 (BMP-6), a glycoprotein signaling molecule that is a multifunctional regulator of vertebrate development. In the present study, we used fetal rat secondary calvarial cultures to determine genes induced during early osteoblast differentiation as initiated by glucocorticoid treatment. Glucocorticoid, and subsequently BMP-6, was found to induce a novel rat intracellular protein, LIM mineralization protein-1 (LMP-1), that in turn resulted in synthesis of one or more soluble factors that could induce de novo bone formation. Blocking expression of LMP-1 using antisense oligonucleotide prevented osteoblast differentiation in vitro. Overexpression of LMP-1 using a mammalian expression vector was sufficient to initiate de novo bone nodule formation in vitro and in sc implants in vivo. These data demonstrate that LMP-1 is an essential positive regulator of the osteoblast differentiation program as well as an important intermediate step in the BMP-6 signaling pathway.     

Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium

Hematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34(+) cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin-CD34(+)Sca-1(+)c-Kit+ phenotype and was proficient at maintaining fetal liver-derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.     

Genes expressed after retinoic acid-mediated differentiation of embryoid bodies are likely to be expressed during embryo development

In order to test if retinoic acid-mediated differentiation of embryoid bodies can be used as an in vitro preselection method for ES cell lines generated by gene trap, we correlated gene expression after in vitro differentiation and in 11.5-day embryos. Fifty-two genes captured by gene trap and expressed in undifferentiated embryonic stem cells were analyzed. Most genes expressed after differentiation in vitro were also expressed during embryo development. In order to correlate the expression patterns in vitro and in vivo, the in vitro expression in the center and in the periphery of the embryoid body outgrowths was observed. This allowed us to distinguish, according to in vitro expression, not expressed genes from those expressed widely in 11.5-day embryos. Consequently, with this parameter we increased the probability to obtain the restricted expression patterns in vivo. This study demonstrates the potential of the differentiation procedure in combination with the gene trap to select in vitro for genes expressed during embryo development.     

Suppression of fibroblast growth factors 1 and 2 by antisense oligonucleotides in embryonic chick retinal cells in vitro inhibits neuronal differentiation and survival

As retinal histogenesis proceeds there is a pronounced increase in the expression of fibroblast growth factor (FGF), reaching its maximum in the mature retina and largely in terminal differentiated retinal neurons. Recent in vivo evidence suggests that exogenous FGF functions as a differentiation and survival factor for a wide variety of cell types including CNS neurons and that endogenous FGF may perform similar functions. We have examined the consequences of selectively and independently inhibiting FGF1 or FGF2 expression using antisense oligonucleotides in embryonic chick retinal cells, differentiating in vitro. Whether FGF1 or FGF2 expression was inhibited the results were the same: a marked reduction in neuronal photoreceptor cells differentiation, an increase in programmed cell death, but no effects on cell proliferation. Even although these two related factors promote the same final effect on retinal cells, namely, neuronal differentiation and survival, their normal combined activities or levels appear to be important in achieving this effect. Stimulation with either exogenous FGF1 or FGF2 served to increase endogenous levels of both FGF1 and FGF2 and reversed the effects of antisense blockade of either FGF1 or FGF2. Our data suggest that although other sources of FGF exist within the eye, the function of endogenous FGF in differentiating retinal neurons may be to stimulate their differentiation and promote their survival.     

Dominant negative ATF1 blocks cyclic AMP-induced neurite outgrowth in PC12D cells

Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)-induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB-induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D-ATF1 RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D-ATF1RL cells were proved to be defective at cAMP-induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D-ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP-induced differentiation of PC12 cells.     

Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors

Tissue-specific bHLH proteins play important roles in the specification and differentiation of neural cell lineages in invertebrate and vertebrate organisms. Two groups of bHLH proteins, atonal and achaete-scute, have proneural activities in Drosophila, and the mouse achaete-scute homolog MASH1 is required for the differentiation of several neural lineages. In a screen for proteins interacting with MASH1, we have isolated a novel bHLH protein related to atonal, named MATH4A, which is broadly expressed in neural precursor cells in the mouse embryonic CNS and PNS. Interaction assays in yeast and in vitro demonstrate that MATH4A interacts efficiently with both MASH1 and the ubiquitous bHLH protein E12. MATH4A-E12 heterodimers, but not MATH4A-MASH1, bind to a consensus E-box sequence. Math4A expression is restricted to undifferentiated neural precursors and is complementary to that of Mash1 in most regions of the nervous system. In particular, Math4A is transcribed at high levels in the cerebral cortex, dorsal thalamus, and epibranchial placodes, which present little or no Mash1 expression. However, expression of the two genes shows limited overlap in certain CNS regions (retina, preoptic area of the hypothalamus, midbrain, hindbrain). Its structure and expression pattern suggest that MATH4A may regulate an early step of neural development, either as a partner of ubiquitous bHLH proteins or associated with other neural-specific bHLH proteins.     

High-level expression of the ER-MP58 antigen on mouse bone marrow hematopoietic progenitor cells marks commitment to the myeloid lineage

Studies on the early events in the differentiation of the nonspecific immune system require the identification and isolation of myeloid-committed progenitor cells. Using the monoclonal antibodies (mAb) ER-MP12 and ER-MP20, generated against immortalized macrophage precursors, we have shown previously that the earliest macrophage colony-stimulating factor (M-CSF)-responsive cells in the bone marrow have the ER-MP12hi 20- phenotype. In addition, we found that the ER-MP12hi 20- subset (comprising about 2 % of total nucleated marrow) contains progenitor cells of all hematopoietic lineages. Aiming at the identification and purification of the myeloid progenitor cells within the ER-MP12hi 20-subset, we used ER-MP58, a marker expressed at high level by all M-CSF-responsive bone marrow progenitors. With this marker the ER-MP12hi 20- cell population could be divided into three subfractions: one with absent or low level ER-MP58 expression, one with intermediate, and one with high ER-MP58 expression. These subfractions were isolated by fluorescence-activated cell sorting and tested in vitro and in vivo for their differentiation capacities. In addition, the expression of ER-MP58 on stem cell subsets was examined in the cobblestone area-forming cell (CAFC) assay. Our data indicate that in the ER-MP12hi 20- subpopulation myeloid-committed progenitors are characterized by high-level expression of the ER-MP58 antigen, whereas cells with other or broader differentiation capacities have an ER-MP58 negative/low or intermediate phenotype. These myeloid-committed progenitors have no significant repopulating ability in vivo, in contrast to the ER-MP58 intermediate cells. Primitive CAFC-28/35, corresponding to cells providing long-term hematopoietic engraftment in vivo, also did not express the ER-MP58 Ag at a high level. Thus, cells committed to the myeloid lineage can be separated from progenitor cells with other differentiation capacities by means of multiparameter cell sorting using ER-MP58 in combination with ER-MP12 and ER-MP20.     

Expression of human aldehyde dehydrogenase-3 associated with hepatocellular carcinoma: promoter regions and nuclear protein factors related to the expression

Studies in our laboratory have shown that as early as day 8.5 of development, mouse yolk sac cells can generate T cells when placed in a thymic microenvironment. At this stage, yolk sac cells can also differentiate into myeloid cells in vitro. B cell differentiation in vitro was achieved with day 9 yolk sac by providing a bone marrow stromal feeder layer. We have now established endothelial cell lines and clones from yolk sacs of day 8-12 mouse embryos. These vary in their ability to support stem cell maintenance and differentiation. Our principal work has been carried out with day 12 cloned endothelial cell lines. One clone supported the > 100 fold expansion of yolk sac hematopoietic stem cells that subsequently could generate B cells, T cells and myeloid cells both in vitro and in vivo. Preliminary experiments with endothelial cells from younger embryos are also described.     

Inactivating FSH receptor mutations and gonadal dysfunction

CNS neurogenesis involves a critical transition where neuronal progenitors exit the cell cycle and initiate terminal differentiation. Recent experiments have suggested that depolarization inhibits DNA synthesis in cortical progenitors. Depolarization of proliferating neuronal progenitors may thus activate mechanisms that prevent proliferation and allow the initiation of terminal differentiation. We present evidence that depolarizing concentrations of KCl (25-50 mM) reduce proliferation of developing postnatal cerebellar granule cells in culture. These studies show that KCl antagonizes the mitogenic response of granule cells to insulin-like growth factor-I (IGF-I) and that this reduction in proliferating cells is not the result of a selective cell death. We also examined the differentiation of granule cell cultures using Brn-5 expression as an early differentiation marker. In vivo Brn-5 expression occurs soon after developing granule cells exit the cell cycle and begin their final differentiation. In control cultures and cultures treated with high concentrations of KCl Brn-5 expression increased over 24-48 h of culture. Our results suggest depolarizing concentrations of KCl antagonize proliferation of cerebellar granule neuron progenitors however allow their continued differentiation.     

Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog

An early step in the development of vertebrae, ribs, muscle, and dermis is the differentiation of the somitic mesoderm into dermomyotome dorsally and sclerotome ventrally. To analyze this process, we have developed an in vitro assay for somitic mesoderm differentiation. We show that sclerotomal markers can be induced by a diffusible factor secreted by notochord and floor plate and that heterologous cells expressing Sonic hedgehog (shh/vhh-1) mimic this effect. In contrast, expression of dermomyotomal markers can be caused by a contact-dependent signal from surface ectoderm and a diffusible signal from dorsal neural tube. Our results extend previous studies by suggesting that dorsoventral patterning of somites involves the coordinate action of multiple dorsalizing and ventralizing signals and that a diffusible form of Shh/Vhh-1 mediates sclerotome induction.