Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Expression of Frizzled10 in mouse central nervous system

Frizzled transmembrane proteins (Fzd) are receptors of Wnts, and they play key roles during central nervous system (CNS) development in vertebrates. Here we report the expression pattern of Frizzled10 in mouse CNS from embryonic stages to adulthood. Frizzled10 is expressed strongly at embryonic days E8.5 and E9.5 in the neural tube and tail bud. At E10.5, Frizzled10 is expressed in the forebrain vesicle, the fourth ventricle and the dorsal spinal cord. From E12.5 to E16.5, Frizzled10 expression is mainly observed in the cortical hem/fimbria, the neuroepithelium of the third ventricular zone, midbrain, developing cerebellum, and dorsal spinal cord. At P0, with the exception of expression in the fimbria, Frizzled10 mRNA expression is limited to specific nuclei including the ventral posterior thalamic nucleus (VP) and the dorsal lateral geniculate nucleus (DLG) in the developing thalamus as well as in the proliferative ventricular zone of the developing cerebellum. From P20 to adult, Frizzled10 mRNA is detected only in the internal capsule (ic). Our data show that expression of Frizzled10 is very strong during embryonic development of the CNS and suggest that Frizzled10 may play an essential role in spatial and temporal regulation during neural development.     

The roles of ERAS during cell lineage specification of mouse early embryonic development

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.     

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.     

巢蛋白mRNA在小鼠中枢神经系统发育过程中的表达

巢蛋白属于中等纤维基因家族,在增殖较快的神经前体细胞中表达。该基因被克隆后,作为神经前体的标记基因得到广泛应用。本文中,我们根据小鼠巢蛋白ｃＤＮＡ序列,设计了一对引物,在确定了反轩录ＰＣＲ反应的最佳反应条件后,详细地考察了小鼠巢蛋白ｍＲＮＡ在中枢神经系统发育过程中的表达规律。     

Developmental expression of P2X5 receptors in the mouse prenatal central and peripheral nervous systems

The functions of P2X purinoceptors (P2X1-7) in the nervous system of adults have been widely studied. However, little is known about their roles during embryonic development. Our previous work has reported an extensive expression of P2X5 receptors in the adult mouse central nervous system. In the present study, we have examined the expression pattern of P2X5 receptor mRNA and protein during prenatal development of the mouse nervous system (from embryonic day E8 to E17). P2X5 receptors appeared in the neural tube as early as E8 and were gradually confined to new-born neurons in the cortical plate and ventral horn of the spinal cord. Heavy signals for P2X5 receptors were also found in dorsal root ganglia (DRG), retina, olfactory epithelium, and nerve fibers in skeletal muscles. In conclusion, P2X5 receptors were strongly represented in the developing mouse nervous system. The transient high expression pattern of P2X5 receptors in epithelium-like structures suggests a role during early neurogenesis.     

Developmental expression of sorting nexin 3 in the mouse central nervous system

We previously reported that sorting nexin 3 (SNX3), a protein belonging to the sorting nexin family, regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The snx3 gene is disrupted in patients with microcephaly, microphthalmia, ectrodactyly, and prognathism (MMEP) and mental retardation, demonstrating that SNX3 plays an important role in the genesis of these organs during development. The present study was designed to determine the expression pattern of snx3 mRNA, particularly in the mouse central nervous system (CNS), from the embryonic stage to adulthood. Whole mount in situ hybridization of embryonic day (E) 9.5 and 10.5 mouse embryos revealed strong positive signals for snx3 mRNA in the forebrain, pharyngeal arches, eyes, and limb buds. In situ hybridization analyses of embryonic and neonatal brain sections revealed that snx3 mRNA is mainly expressed in the cerebral cortex, hippocampus, piriform cortex, cerebellum, and spinal cord. In adulthood, the expression of snx3 mRNA is observed in the cerebral cortex, hippocampus, piriform cortex, and cerebellar neurons. Thus, snx3 mRNA is expressed during neural development and in adult neural tissues, suggesting that SNX3 may play an important role in the development and function of the CNS.     

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells

BackgroundThe heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature.MethodsToxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds.ResultsObtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels.ConclusionThis study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.     

Embryonic Expression of Human Keratin 18 and K18-β-Galactosidase Fusion Genes in Transgenic Mice

During embryogenesis, EndoB, the mouse form of human keratin 18 (K18), is expressed in a complex spatial and temporal pattern in various embryonic epithelia. We have compared the expression of transgenic human K18 to the endogenous mouse homolog and to the coexpressed, complementary keratin 8 homolog, EndoA, during postimplantation mouse embryogenesis and fetal development in order to determine the developmental expression pattern of the human gene in a mouse environment. The tissue distribution of K18 protein was identical to that of endogenous EndoB in both 7.5- and 13.5-day-old embryos, except for certain heart, eye, and extraembryonic mesodermal tissues in which K18 was not detected. These results indicate that the 10-kb K18 gene specifies appropriate developmental expression in the mouse and support previously reported differences in K18 expression in human and mouse fetal heart. We have also compared the expression patterns of K18 to a series of constructions that utilize the Escherichia coli gene for β-galactosidase (lacZ) as a reporter gene. Some of these constructions were regulated correctly in embryos during development of the germ layers. However, none was expressed consistently in extraembryonic or in adult tissues. Analysis with methylation-sensitive restriction enzymes revealed that hypermethylation of the CpG-rich prokaryotic reporter gene was not the cause of its silence in adult transgenic liver. However, the repressed state of K18-LacZ transgenes in adult liver was correlated with a different chromatin state that lacked diagnostic DNase hypersensitive sites found in K18 transgenic liver. Expression of the lacZ reporter gene did not accurately reflect the developmental pattern of K18 even in constructions that used all available K18 sequences. We conclude that in these contexts, the lacZ gene was not a developmentally neutral reporter gene.     

Conserved expression of the PRELI domain containing 2 gene (<Emphasis Type="Italic">Prelid2</Emphasis>) during mid-later-gestation mouse embryogenesis

Prelid2, which belongs to the PRELI domain containing family, is identified as a conserved evolution gene. The expression and regulation during embryonic development of the prelid2 gene is unknown. In this study, we investigated the prelid2 gene expression and regulation using mouse embryos model, by in situ hybridization analysis, RT-PCR and bisulfite sequencing. In situ hybridization analysis showed that prelid2 gene expression were found in midbrain, spinal cord, optic eminence, otic vesicle and tail at E9.5 and E10.5 embryos, in forebrain, hindbrain, heart, lung, liver and kidney at E13.5 and E15.5 embryos. Real-time quantitative RT-PCR results verified the expression pattern in the four major mouse organs, brain, heart, lung, and liver during organs differentiation and formation. Bisulfite sequencing illustrated the consistent result of expression and its unmethylation status in the genomic promoter region at E12.5, E18.5, and new born. Thus, the prelid2 gene is a widely-spread, persistently expressed and unmethylated gene in mouse embryonic development. Our results suggest that the PRELI domain containing 2 gene is involved in mouse embryonic development.