Developmental Stage-dependent Manner Research Articles

Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions.

Cardiogenesis is a multistep process regulated by a hierarchy of factors defining each developmental stage of the heart. One of these factors, leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, is expressed in embryonic and neonatal cardiomyocytes and induces cardiomyocyte hypertrophy. Many aspects of embryogenesis are faithfully recapitulated during in vitro differentiation of embryonic stem cells in embryoid bodies. We exploited this model to study effects of growth factors on commitment and differentiation of cardiomyocytes and on maintenance of their phenotype. We identified LIF as a factor affecting commitment and differentiation of cardiomyocytes in an opposite manner. Diffusible LIF inhibited mesoderm formation and hampered commitment of cardiomyocytes. Lack of both the diffusible and matrix-bound isoforms of LIF in lif-/- embryoid bodies did not interfere with commitment, but it severely suppressed early differentiation of cardiomyocytes. Onset of differentiation was rescued by very low concentrations of diffusible LIF; however, consecutive differentiation was attenuated in a concentration-dependent manner by diffusible LIF both in wild-type and lif-/- cardiomyocytes. Differentiation of cardiomyocytes was severely hampered but not completely blocked in lifr-/- embryoid bodies, suggesting additional, LIF-receptor ligand independent pathways for commitment and differentiation of cardiomyocytes. At the fully differentiated state, both paracrine and autocrine LIF promoted proliferation and increased longevity of cardiomyocytes. These findings suggest that both paracrine and autocrine and both diffusible and matrix-bound isoforms of LIF contribute to the modulation of cardiogenesis in a subtle, opposite, and developmental stage-dependent manner and control proliferation and maintenance of the differentiated state of cardiomyocytes.

Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina.

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.

Molecular cloning, expression, and characterization of a novel mouse liver SULT1B1 sulfotransferase.

A mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3,4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3,3',5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.

Differential expression of D-type G1 cyclins during mouse development and liver regeneration in vivo.

D-type G1 cyclins are the primary cell cycle regulators of G1/S transition in eukaryotic cells, and are differentially expressed in a variety of cell lines in vitro. Little is known, however, about the expression patterns of D-type G1 cyclins in normal mouse in vivo. Thus, in the present study, tissue-specific expressions of cyclin D1 and D3 genes were examined in several tissues derived from adult male mice, and stage-specific expression of cyclin genes was studied in brain, liver, and kidney of developing mice from embryonic day 13 to postnatal day 11. Cell cycle-dependent expression of cyclins was also examined in regenerating livers following partial hepatectomy. Our results indicate that (1) cyclins D1 and D3 are expressed in a tissue-specific manner, with cyclin D1 being highly expressed in kidney and D3 in thymus; (2) cyclin D3 mRNA is abundantly expressed in young proliferating tissues and is gradually reduced during development, whereas cyclin D1 mRNA fluctuates during development; and (3) compensatory regeneration of liver induces cyclin D1 gene expression 12 hr after partial hepatectomy, and cyclin D3 gene expression from 36 to 42 hr (at the time of G1/S transition). In conclusion, this study indicates that cyclin D1 and D3 genes are differentially expressed in vivo in a tissue-specific, developmental stage-dependent, and cell cycle-dependent manner.

Dexamethasone stimulates luciferase gene expression through the rat bone sialoprotein gene promoter in transgenic mice.

Bone sialoprotein (BSP) is expressed by differentiated osteoblasts during the initial formation and mineralization of bone matrix. Studies using transgenic mice harboring 2.7 kb of the rat BSP promoter linked to a luciferase reporter gene have shown luciferase activity in bone and other mineralized tissues while most soft tissues tested expressed a much lower level of the reporter gene. To study regulation of the transgene, mice were administered dexamethasone (dex) by intramuscular injection. After 4 h and 24 h, various tissues were dissected from the treated mice as well as from untreated transgenic littermates. Luciferase assays showed that dex stimulated expression of the transgene significantly. In bone tissues, dex increased the average luciferase activity 1.6- to 11-fold compared with control tissues from untreated transgenic mice. The luciferase activity in lung, liver and kidney remained at a low level and showed no increase with dex treatment. In some animals, however, the luciferase activity in brain and skin was also increased after dex administration. These experiments indicate that a transgene comprising 2.7 kb of the rat BSP promoter linked to a luciferase reporter is regulated in a tissue and developmental stage-dependent manner and that glucocorticoid-induced stimulation of BSP gene expression may be mediated within this region of the promoter.

Molecular cloning of a developmentally regulated brain protein, chicken drebrin A and its expression by alternative splicing of the drebrin gene

Drebrins are developmentally regulated proteins found in the chicken brain and are classified into three forms, E1, E2 and A. Previously we isolated two cDNAs corresponding to the embryonic drebrin mRNAs from a chick embryo cDNA library. They differed in that an internal 129-nucleotide sequence, designated ins1, was inserted in the cDNA encoding drebrin E2 and was deleted in the other cDNA encoding drebrin E1. To search for the cDNA clone encoding drebrin A, a cDNA library of 1-day-old chick brains was screened using embryonic drebrin cDNA fragments as probes. Consequently, a novel cDNA was isolated, the sequence of which was entirely identical with that of drebrin E2 except for the insertion of a 138-nucleotide sequence, designated ins2, in the 5′ direction immediately upstream from ins1. Since the translation product of the entire coding region was similar to that of drebrin A, this cDNA should correspond to the mRNA for drebrin A. Sequencing analysis of three drebrin cDNAs clearly indicated that the heterogeneity of chicken drebrins was caused by the insertion or deletion of the two sequences, ins1 and ins2. The amino-terminal half region including ins2 and two short sequences in the carboxyl-terminal region of the predicted drebrin A were highly evolutionarily conserved. Cloning and sequencing of the drebrin gene revealed that ins1 and ins2 were independently encoded by separate exons and three drebrin isoforms were thought to arise by alternative splicing from a single drebrin gene. The difference in the time course of expression and tissue distribution of each drebrin suggests that the machinery of alternative splicing site selection of the drebrin gene is regulated in a developmental stage-dependent and tissue-specific manner.

Kappa-Opioid agonist modulation of [3H]thymidine incorporation into DNA: evidence for the involvement of pertussis toxin-sensitive G protein-coupled phosphoinositide turnover.

A body of evidence has indicated that mu-opioid agonists can inhibit DNA synthesis in developing brain. We now report that kappa-selective opioid agonists (U69593 and U50488) modulate [3H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner, kappa agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the kappa-selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3.5-fold increase was evident. Cell labeling by [3H]thymidine was also inhibited by the kappa-opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbinaltorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of kappa agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that kappa agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.