Stigma development in Nicotiana tabacum. Cell death in transgenic plants as a marker to follow cell fate at high resolution

Pistil development was studied in transgenic tobacco plants in which the stigma is ablated by expression of a stigma-specific cytotoxic gene. These plants offer a tool to investigate the process of differentiation of the secretory zone, in that cell death caused by barnase activity provides a marker to follow cell fate at high resolution. After fusion of the carpel walls in the region most distal from the ovary, the epidermal cells begin to divide in both wild-type and stigmaless plants. Divisions of the L1 layer of the pistil are immediately followed by the morphogenetic events that lead to three different cell types: rounded-angular cells showing an equal number of anti- and periclinal divisions, cells that are more oblong forming the transition zone, and the square cells of the transmitting tissue dividing mostly anticlinally with respect to the original carpel wall. In the stigmaless plants, cell death caused by the expression ofSTIG 1-barnase begins at stage –1 and proceeds gradually, but is always associated with round epidermal cells and with angular-rounded cells underneath them. Studies at the ultrastructural level show that cell death caused by barnase activity occurs first in solitary cells and gradually extends to groups of cells.In situ hybridizations using the STIG 1 RNA probe in wild-type pistils confirm these results. Most likely, the cells in whichSTIG 1 is expressed are those that have just differentiated into the secretory cell type. Our results indicate that the transition zone or neck is autonomously differentiated from the secretory zone and the transmitting tissue. Furthermore, our results indicate that in both wild-type and stigmaless pistils secretion of lipids most likely occurs through the plasmodesmata. This observation suggests that bulk transport can occur via plasmodesmata.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 × 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5–13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [³⁵S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis

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.     

A transient role for ciliary neurotrophic factor in chick photoreceptor development

Previous studies suggest that ciliary neurotrophic factor (CNTF) may represent one of the extrinsic signals controlling the development of vertebrate retinal photoreceptors. In dissociated cultures from embryonic chick retina, exogenously applied CNTF has been shown to act on postmitotic rod precursor cells, resulting in an two- to fourfold increase in the number of cells acquiring an opsin-positive phenotype. We now demonstrate that the responsiveness of photoreceptor precursors to CNTF is confined to a brief phase between their final mitosis and their terminal differentiation owing to the temporally restricted expression of the CNTF receptor (CNTFRα). As shown immunocytochemically, CNTFRα expression in the presumptive photoreceptor layer of the chick retina starts at embryonic day 8 (E8) and is rapidly down-regulated a few days later prior to the differentiation of opsin-positive photoreceptors, both in vivo and in dissociated cultures from E8. We further show that the CNTF-dependent in vitro differentiation of rods is followed by a phase of photoreceptor-specific apoptotic cell death. The loss of differentiated rods during this apoptotic phase can be prevented by micromolar concentrations of retinol. Our results provide evidence that photoreceptor development depends on the sequential action of different extrinsic signals. The time course of CNTFRα expression and the in vitro effects suggest that CNTF or a related molecule is required during early stages of rod differentiation, while differentiated rods depend on additional protective factors for survival. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 672–683, 1998     

Differential accumulation of the mRNA of the auxin-repressed gene CsGRP1 and the auxin-induced peg formation during gravimorphogenesis of cucumber seedlings

When cucumber seeds are germinated horizontally, an outgrowth (peg) develops on the lower side of the transition zone between the hypocotyl and the root for pulling the cotyledons and plumule out of the seed coat. We previously suggested that gravistimulation suppresses peg formation on the upper side of the transition zone when placed in a horizontal position. In the gravistimulated transition zone, auxin and the mRNAs of auxin-inducible genes are more abundant in the lower side than in the upper side. Here, using fluorescent differential display, we identified Cucumis sativus glycine-rich protein1(CsGRP1) as a gene whose mRNA accumulated more abundantly on the upper side than on the lower side of the transition zone in response to gravistimulation. Auxin starvation increased CsGRP1 mRNA in segments of the transition zone, and inhibition of polar auxin transport with 2,3,5-triiodobenzoic acid (TIBA) prevented the asymmetric accumulation of CsGRP1 mRNA. These results suggest that gravistimulation increases not only the expression of auxin-inducible genes on the lower side of the transition zone, but also the expression of auxin-repressed genes, such as CsGRP1, on the upper side of cucumber seedlings. In the hypocotyls of 3-day-old seedlings, neither gravistimulation nor changes in auxin level influenced the accumulation of CsGRP1 mRNA. These results suggest that the transition zone responds to gravistimulation in a specific manner by an asymmetric expression of CsGRP1 gene during regulation of peg formation.     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells. Dev. Genet. 22:132–140, 1998. © 1998 Wiley-Liss, Inc.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

MiR-185 inhibits 3T3-L1 cell differentiation by targeting SREBP-1

Adipogenesis involves a highly orchestrated series of complex events in which microRNAs (miRNAs) may play an essential role. In this study, we found that the miR-185 expression increased gradually during 3T3-L1 cells differentiation. To explore the role of miR-185 in adipogenesis, miRNA agomirs and antagomirs were used to perform miR-185 overexpression and knockdown, respectively. Overexpression of miR-185 dramatically reduced the mRNA expression of the adipogenic markers, PPARγ, FABP4, FAS, and LPL, and the protein level of PPARγ and FAS. MiR-185 overexpression also led to a notable reduction in lipid accumulation. In contrast, miR-185 inhibition promoted differentiation of 3T3-L1 cells. By target gene prediction and luciferase reporter assay, we demonstrated that sterol regulatory element binding protein 1 (SREBP-1) may be the target of miR-185. These results indicate that miR-185 negatively regulates the differentiation of 3T3-L1 cells by targeting SREBP-1, further highlighting the importance of miRNAs in adipogenesis.     

cDNA cloning and function analysis of two novel erythroid differentiation related genes

Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen λ-gtll human cDNA expression library of fetal liver in order to obtain the relevant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' functions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a prote     

Expression of Focal Adhesion Proteins in the Developing Rat Kidney

Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and hydrogen peroxide–inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that the protein levels of FAK, p-FAK³⁹⁷, paxillin, p-paxillin¹¹⁸, and Hic-5 were high in embryonic kidneys, while ILK expression persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK³⁹⁷, paxillin, and p-paxillin¹¹⁸ were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney development might play a role in morphogenesis and cell differentiation.     

The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian morphogenesis during Drosophila metamorphosis

 Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the ecdysone receptor (EcR) and its partner ultraspiracle (USP). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of USP and the EcR-A isoform in all but one of eight mesoderm-derived somatic cell types. The one exception is the larval terminal filament (TF) cells, in which only USP is detectable during cell differentiation. In cells destined to form the basal stalks and anterior oviduct, USP colocalizes with what appears to be the EcR-B2 isoform. Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. Our results demonstrate that proper expression of the ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects. Received: 12 February 1998 / Accepted: 5 May 1998     

Targeted expression of SbMATE in the root distal transition zone is responsible for sorghum aluminum resistance

Aluminum (Al) toxicity is one of the major limiting factors for crop production on acid soils that comprise significant portions of the world's lands. Aluminum resistance in the cereal crop Sorghum bicolor is mainly achieved by Al‐activated root apical citrate exudation, which is mediated by the plasma membrane localized citrate efflux transporter encoded by SbMATE. Here we precisely localize tissue‐ and cell‐specific Al toxicity responses as well as SbMATE gene and protein expression in root tips of an Al‐resistant near‐isogenic line (NIL). We found that Al induced the greatest cell damage and generation of reactive oxygen species specifically in the root distal transition zone (DTZ), a region 1–3 mm behind the root tip where transition from cell division to cell elongation occurs. These findings indicate that the root DTZ is the primary region of root Al stress. Furthermore, Al‐induced SbMATE gene and protein expression were specifically localized to the epidermal and outer cortical cell layers of the DTZ in the Al‐resistant NIL, and the process was precisely coincident with the time course of Al induction of SbMATE expression and the onset of the recovery of roots from Al‐induced damage. These findings show that SbMATE gene and protein expression are induced when and where the root cells experience the greatest Al stress. Hence, Al‐resistant sorghum plants have evolved an effective strategy to precisely localize root citrate exudation to the specific site of greatest Al‐induced root damage, which minimizes plant carbon loss while maximizing protection of the root cells most susceptible to Al damage.     

Dividing neuron precursors express neuron-specific tubulin

Neuronal differentiation involves specific molecular and morphological changes in precursors and results in mature, postmitotic neurons. The expression of neuron-specific β tubulin, as detected by the monoclonal antibody TuJ1, begins during the period of neurogenesis. Indeed, TuJ1 expression precedes that of the 160 kD neurofilament protein in both the central and peripheral nervous systems. In the embryonic rat spinal cord, bipolar cells and some mitotic cells in the ventricular zone were TuJ1 immunoreactive (IR). Sensory ganglia also contained cells with TuJ1-IR mitotic spindles in situ. In embryonic rat sensory and sympathetic ganglion cell cultures pulsed with the thymidine analog bromodeoxyuridine (BrdU), TuJ1 label was detected in the spindle of mitotic cells and in the midbody of cells joined at cytokinesis, indicating that neuron-specific tubulin expression was initiated during or before the final mitosis of neuronal progenitors. Dorsal root ganglion cultures included TuJ1-IR cells with several shapes that may reflect morphological transitions, from flattened stellate neural crest-like cells to differentiated bipolar neurons. Indeed, the presence of flattened TuJ1-IR cells was correlated with neurogenesis. Some sympathetic neuron precursors possessed long TuJ1-IR neurites, as well as TuJ1-IR spindle microtubules and BrdU-labeled chromosomes, indicating that these precursors can possess long processes during metaphase. These results support the hypothesis that neuron-specific tubulin expression represents an early molecular event in neuronal differentiation exhibited by a wide range of neuronal precursors. The cessation of proliferation can occur at different points during neuronal differentiation, as TuJ1-IR was detected in cells undergoing mitosis. Future studies directed toward understanding the molecules that initiate neuron-specific tubulin expression may lead to the factors that control the initial phases of neuronal differentiation. © 1995 John Wiley & Sons, Inc.     

The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development

 Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan’s closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species. Received: 3 November 1998 / Accepted: 9 December 1998     

Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation

Mesenchymal stem cells (MSCs) from a variety of sources are being used in pre-clinical and clinical studies. The choice of optimal source for treatment of diseases requires quantitative evaluation of self-renewal, proliferation and differentiation potencies of MSCs. For this purpose, quantitative real-time polymerase chain reaction (qRT-PCR) technique is used to determine the expression of genes. qRT-PCR requires the normalization of the gene expression levels by the use of reference genes in order to obtain accurate and reliable results. There is a limited number of studies focused on the selection of reference genes that are appropriate and reliable for MSCs. Thus, no single reference gene has yet been found for use in the in vitro proliferation and differentiation of MSCs. The aim of this study is to investigate the stability of the expression of widely used reference genes during the in vitro proliferation and differentiation of human adipose-derived mesenchymal stem cells (hASCs). For this purpose, 13 reference genes commonly used in MSC studies were selected. As a result, the expression stabilities of EF1α, RPLP0 and RPL13A genes were found to be high and were predicted to be suitable for use as reference genes for normalization in hASC studies. The GAPDH was identified as the gene with the lowest expression stability and evaluated to be an unsuitable reference gene for hASC differentiation studies. This piece of information could be crucial for the selection of appropriate reference genes and accurate measurement of gene expression in hASC studies.