Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development

DNA methylation-dependent gene silencing, mediated by DNA methyltransferases (DNMTs), is essential for normal mammalian development and its dysregulation has been implicated in neurodevelopmental disorders. Despite this, little is known about DNMTs in the developing or mature nervous system. Here, we show that DNMT1, 3a and 3b are expressed at discrete developmental stages in the olfactory neuron lineage, coincident with key shifts in developmental gene expression. DNMT1 is induced in cycling progenitors and is retained in post-mitotic olfactory receptor neurons (ORNs). DNMT3b is restricted to mitotic olfactory progenitors, whereas DNMT3a is expressed only in post-mitotic immature neurons prior to ORN terminal maturation, coincident with histone deacetylase 2 (HDAC2), a key downstream effector of methylation-dependent chromatin condensation. Similar stage-specific expression of DNMT3b and 3a was also found in other developing sensory and CNS neurons. This suggests that progressive lineage restriction regulated by methylation-dependent silencing could be a highly conserved mechanism shared by multiple lineages in the developing nervous system.     

Stage-specific effects of cAMP signaling during distal lung epithelial development

cAMP signaling is postulated to play a role in distal lung epithelial differentiation based on several observations. First, it enhances fibroblast growth factor-induced transdifferentiation of early tracheal epithelium into respiratory epithelium. Second, there are cAMP-responsive elements in the heterologous promoters of Sftpb and Sftpa genes. Third, cAMP augments the effect of dexamethasone in maintaining differentiation of human fetal type II pneumocyte culture. However, this concept has not been thoroughly tested in vivo. In the current study, we modulated cAMP signaling in developing distal lung epithelium in vivo using an inducible transgenic system that expressed a mutant form of Galpha(s) (Galpha(s)Q227L). We failed to demonstrate the ability of cAMP to promote distal epithelial maturation during embryonic stages. The results argue against its physiological role in this process. In addition, induction of cAMP signaling at the late pseudoglandular stage but not during the canalicular or saccular stage surprisingly delayed distal differentiation by suppressing the expression of Sftpc, Sftpa, and Aquaporin5 as well as the formation of lamellar bodies. This stage-specific inhibitory effect was observed in the absence of cellular toxicity or changes in branching. Transgenic lungs did not show significant changes in the known pathways that are important for distal differentiation. Therefore, we propose the existence of yet-to-be identified cAMP-sensitive novel regulators of early distal lung epithelial differentiation. Although the delay of differentiation seemed to be reversible at later stages, it still led to pronounced permanent postnatal airspace enlargement due to impaired paracrine function of distal epithelium in regulating alveolar myofibroblast development.     

Stage-specific changes in protein phosphorylation during preimplantation development in the mouse

To gain insight into the role of protein phosphorylation during early mammalian development, seven mouse preimplantation stages were metabolically labeled with radioactive orthophosphate and the radiolabeled proteins identified using gel electrophoresis and autoradiography. The results obtained indicate that there are marked differences in protein phosphorylation patterns between the zygote and two-cell stage and between the morula and blastocyst stage. In addition, there is a compaction-specific change in the phosphorylation profile of three components of Mr 37,000. This compaction-specific change takes place during compaction in the eight-cell embryo; thus, it is the first biochemical change specifically correlated to this important event of early development.     

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.     

Assessment of cytoplasmic effects on the development of mouse embryonic nuclei transferred to enucleated zygotes

In this study, cytoplasmic effects on the development of nuclear transplant embryos were examined. In addition, the production of offspring from nuclear transplant embryos was attempted. Nuclei from cleavage-stage embryos were transplanted to enucleated zygotes at different cell cycle stages and with different cytoplasmic volumes. A greater developmental rate to the blastocyst stage was observed in reconstituted late stage zygotes that received nuclei from late 2-cell stage embryos than in early stage zygotes (46.3% vs. 16.9%). A further increase in developmental rate to the blastocyst stage (85.5%) and in cell number was obtained in reconstituted late stage zygotes with reduced cytoplasmic volume. However, developmental potential of nuclei from 4- and 8-cell stage embryos was very limited, although they were transferred to enucleated late stage zygotes with reduced cytoplasm. After the transfer of blastocysts derived from nuclear transplant embryos to recipient females, live young were obtained from reconstituted embryos that received nuclei from late 2-cell stage embryos (28.6%). These results confirm that the development of nuclear transplant embryos can be affected by recipient cell cycle stage and cytoplasmic volume. Furthermore, the nuclei from late 2-cell stage embryos in which activation of the embryonic genome had occurred can be reprogrammed to a certain extent when transplanted into enucleated zygotes, especially late stage zygotes with reduced cytoplasmic content.     

CTGF expression during mouse embryonic development

Connective tissue growth factor (CTGF) is a potent fibroblast mitogen and angiogenic factor which plays an important role in wound healing, cancerogenesis and fibrotic and vascular disease. Here we explored the regulation and the cellular site of the mRNA synthesis for this growth factor in the developing mouse embryo by in situ hybridisation. Strong and persistent CTGF gene expression was limited to three types of tissue: the vascular endothelium, particularly the high-pressure part of the cardiovascular system, condensed connective tissue around bone and cartilage, and maturing layer VII neurons in the cerebral cortex. With few exceptions (late tooth bud, neuroepithelium) epithelial tissue was negative. Very transient but strong expression was observed early during formation of cartilage, in late stages during perichondral ossification, on cerebral neuroepithelium, and in several discrete stages of tooth formation, on mesenchymal precursors of odontoblasts condensing on inner dental epithelium, and later on apposing regions of ameloblast and odontoblast epithelium. Altogether, the current study suggests that CTGF performs a dual role: a continuous function in the cardiovascular system, bone and cartilage-associated mesenchyme and maturing layer VII neurons, but also a more transient function associated with the formation of cartilage, bone, tooth and cerebral nerve cells.     

Stage-specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands

Abstract The ichneumonid endoparasitoid Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) injects a polydnavirus when it oviposits into a host. We compared the development of Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae parasitized in the penultimate (fourth) stadium with those parasitized in the last (fifth) stadium by C.sonorensis and show that hosts stung in the fifth stadium exhibited arrested or delayed development compared to the controls. Parasitoids developed normally to the point of emergence in larvae stung in the fifth stadium but most did not successfully emerge from the host. The prothoracic glands in all successfully parasitized fifth stadium hosts and most unsuccessfully parasitized fifth stadium hosts showed some degree of virally-induced degeneration. Larvae stung in the fourth stadium developed more slowly than controls and either did not moult or developed to a fifth and sometimes a supernumerary sixth stadium before parasitoid emergence. Unsuccessfully parasitized hosts were delayed in their development but eventually moulted to the fifth and, in some cases, a supernumerary sixth stadium before pupating. Hosts stung in the fourth stadium showed no signs of prothoracic gland degeneration whether successfully parasitized or not. In addition, calyx fluid injections into early fourth stadium hosts did not cause prothoracic gland degeneration even after these hosts moulted to the fifth stadium, suggesting that degeneration induced by polydnavirus is specific to the last stadium of the host.     

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

For several secretory proteins, it has beenhypothesized that disulfide-bonded loop structures are required forsorting to secretory granules. To explore this hypothesis, we employeddithiothreitol (DTT) treatment in live pancreatic islets, as well as inPC-12 andGH₄C₁cells. In islets, disulfide reduction in the distal secretory pathwaydid not increase constitutive or constitutive-like secretion ofproinsulin (or insulin). In PC-12 cells, DTT treatment caused adramatic increase in unstimulated secretion of newly synthesizedchromogranin B (CgB), presumably as a consequence of reducing thesingle conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, inGH₄C₁cells that also synthesize CgB endogenously, DTT treatment reducednewly synthesized prolactin and blocked its export, whereas newlysynthesized CgB was routed normally to secretory granules. Moreover, ontransient expression inGH₄C₁cells, CgA and a CgA mutant lacking the conserved disulfide bond showedcomparable multimeric aggregation properties and targeting to secretorygranules, as measured by stimulated secretion assays. Thus theconformational perturbation of regulated secretory proteins caused bydisulfide disruption leads to consequences in protein trafficking thatare both protein and cell type dependent.

     

Drapc1 expression during mouse embryonic development

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.     

早期小鼠胚胎发育的基因表达

受精是新个体发育的时 -空点 ,从一个形态单纯的单细胞受精卵发育成能独立生活的个体动物 ,形态上出现一系列的变化 ,而更重要的是基因表达。基因组是机体内惟一确定的 ,为所有各类细胞共同拥有 ,但基因组内各个基因表达的选择性和程度随时间、位置和环境条件的不同而发生改变 (严云勤等 ,2 0 0 2 ;胡静等 ,2 0 0 1;范衡宇等 ,2 0 0 1)。个体的发育和分化、内环境的稳定性、对外界刺激的应答、细胞循环的调节、衰老和程序化细胞死亡等正常的发育过程以及疾病的病理学过程 ,包括癌症的病理学过程 ,无论是由一个基因的突变引起的或是由于多基…     

Expression of smoothened in mouse embryonic maxillofacial development

Abstract

Hedgehog (Hh) signaling plays many key roles in the development of Drosophila and vertebrate embryos including regulation of craniofacial development. The seven-transmembrane protein, smoothened (Smo) transduces the Hh signal across the plasma membrane as an essential receptor of PTCHED1/2. There are few studies that evaluate the detailed expression of Smo in mouse embryonic craniofacial development. We investigated the expression patterns of Smo during murine embryonic craniofacial development using in situ hybridization (ISH), studies of whole-mounts and sections, immunohistochemistry, quantitative real time PCR, and Western blot analysis. We found that Smo mRNA was expressed in the face of mouse embryos at 11 and 12.5 days post coitum (dpc). After 13.5 dpc, the expression decreased to a low level and was faintly detected after birth. Smo protein could be detected also in embryos at 11, 12.5, and 14.5 dpc. After 15.5 dpc, the expression was very faint and paralleled the gene expression studies. No expression was detected in whisker follicle during facial development and faint signal was detected in Meckel's cartilage. These findings concerning Smo expression should guide further investigation of sonic Hh signaling pathway gene function during maxillofacial development.     

Inhibition of mouse embryonic development by calmodulin antagonists

Two-cell mouse embryos were incubated in the presence of calmodulin inhibitors to determine their effect on embryonic development to the blastocyst stage. Calmodulin, a Ca²⁺-dependent regulatory protein, has been localized in the cytoplasm and has been implicated in regulation of many cellular events, such as mitosis. Several concentrations of either commercial or synthesized calmodulin inhibitors were tested. Several phenothiazine sulfoxide derivatives were more effective than the three naphthalene sulonic acid derivatives tested; 2-chloro-10-aminopropyl phenothiazine and 10-aminopropyl phenothiazine were the most potent phenothiazines to inhibit embryonic development at the two-cell stage. The interesting aspect of this study is that phenothiazine sulfoxide derivatives are not potent inhibitors of calmodulin, however, they were successful in inhibiting embryonic development. Potent inhibitors of calmodulin apparently did not penetrate the embryonic membranes because they had no effect.