Expression profiles of extracellular superoxide dismutase during mouse organogenesis

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.     

Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo

Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the delta-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins.     

Spatiotemporal expression of the selenoprotein P gene in postimplantational mouse embryos

Selenoprotein P (Sepp) is an extracellular glycoprotein which functions principally as a selenium (Se) transporter and antioxidant. In order to assess the spatiotemporal expression of the Sepp gene during mouse embryogenesis, quantitative RT-PCR and in situ hybridization analyses were conducted in embryos and extraembryonic tissues, including placenta. Sepp mRNA expression was detected in all embryos and extraembryonic tissues on embryonic days (E) 7.5 to 18.5. Sepp mRNA levels were high in extraembryonic tissues, as compared to embryos, on E 7.5-13.5. However, the levels were higher in embryos than in extraembryonic tissues on E 14.5-15.5, but were similar in both tissues during the subsequent periods prior to birth. According to the results of in situ hybridization, Sepp mRNA was expressed principally in the ectoplacental cone and neural ectoderm, including the neural tubes and neural folds. In whole embryos, Sepp mRNA was expressed abundantly in nervous tissues on E 9.5-12.5. Sepp mRNA was also expressed in forelimb and hindlimb buds on E 10.5-12.5. In the sectioned embryos, on E 13.5-18.5, Sepp mRNA was expressed persistently in the developing limbs, gastrointestinal tract, nervous tissue, lung, kidney and liver. On E 16.5-18.5, Sepp mRNA expression in the submandibular gland, whisker follicles, pancreas, urinary bladder and skin was apparent. In particular, Sepp mRNA was detected abundantly in blood cells during all the observed developmental periods. These results show that Sepp may function as a transporter of selenium, as well as an antioxidant, during embryogenesis.     

Developmental expression of plasma glutathione peroxidase during mouse organogenesis

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.     

Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse.

Fibroblast growth factors (FGFs) can influence the growth and differentiation of cultured cells derived from neuroectoderm, ectoderm or mesenchyme. The FGFs interact with a family of at least four closely related receptor tyrosine kinases that are products of individual genes. To investigate the role of FGFs in the growth and differentiation of embryonic tissues and to determine whether the individual FGF receptor genes might have specific functions, we compared the localization of mRNA for two FGF receptor genes, FGFR1 (the flg gene product) and FGFR2 (the bek gene product), during limb formation and organogenesis in mouse embryos (E9.5-E16.5). Although the two genes were coexpressed in some tissues, the differential expression of FGFR1 and FGFR2 in most embryonic tissues was striking. FGFR1 was expressed diffusely in mesenchyme of limb buds, somites and organ rudiments. In contrast, FGFR2 was expressed predominantly in the epithelial cells of embryonic skin and of developing organs. The differential expression of FGFR1 and FGFR2 in mesenchyme and epithelium respectively, suggests the receptor genes are independently regulated and that they mediate different functions of FGFs during development.     

Developmentally regulated expression of mouse HtrA3 and its role as an inhibitor of TGF-beta signaling

The expression of mouse HtrA1 is developmentally regulated and restricted in embryo tissues which depend largely on TGF-beta signaling for their differentiation. We examined whether mouse HtrA3, another HtrA family member very close to HtrA1, shows similar expression patterns. HtrA3 and -1 were expressed mostly in the same embryonic organs but exhibited complementary patterns in various tissues; the lens epithelial cells in day 12.5 embryo expressed HtrA3 whereas the ciliary body and pigment retina expressed HtrA1. In the vertebrae of day 14.5 embryo, HtrA3 was expressed in the tail region, but HtrA1 was predominantly expressed in the thoracic and lumbar regions. Similar to HtrA1, HtrA3 bound to various TGF-beta proteins and inhibited the signaling of BMP-4, -2 and TGF-beta 1. HtrA3 did not inhibit signaling originated from a constitutively active BMP receptor, indicating that the inhibition occurred upstream of the cell surface receptor. HtrA3 also showed proteolytic activities indistinguishable from those of HtrA1 toward beta-casein and some extracellular matrix (ECM) proteoglycans. The protease activity was absolutely required for the TGF-beta signal inhibition activity. All these data suggest that HtrA3 and -1 have the overlapping biological activities but can function in complementary fashion in certain types of tissues.     

印记基因Dlk1在小鼠胚胎发育中的表达分析

目的:研究印记基因Dlk1在小鼠胚胎发育过程中的动态表达模式,以揭示Dlk1与胚胎发育的关系。方法:通过半定量PCR和定量PCR分析Dlk1在小鼠胚胎发育E8.5～E19.5的基因表达模式,并选取Dlk1表达量最高的时期进行胚胎切片原位杂交和组织定量PCR分析。结果:在小鼠胚胎发育E8.5～E15.5时,Dlk1的表达逐渐升高,在E15.5时表达量达到最高;E15.5～E19.5时,Dlk1表达有所下降,但仍然维持较高水平。E15.5切片原位杂交显示,垂体、肺脏、软骨、舌和背侧肌肉组织中Dlk1表达较高,组织定量PCR实验进一步证实了原文杂交的结果。结论:Dlk1在小鼠胚胎发育中后期持续表达,并呈现一定的组织特异性,对胚胎发育可能起重要的调节作用。     

Changes in expression of serine proteases HtrA1 and HtrA2 during estrogen-induced oxidative stress and nephrocarcinogenesis in male Syrian hamster

Serine proteases HtrA1 and HtrA2 are involved in cellular stress response and development of several diseases, including cancer. Our aim was to examine the involvement of the HtrA proteins in acute oxidative stress response induced in hamster kidney by estrogen treatment, and in nephrocarcinogenesis caused by prolonged estrogenization of male Syrian hamster. We used semi-quantitative RT-PCR to estimate the HtrA1 and HtrA2 mRNA levels in kidney tissues, and Western blotting to monitor the amount of the HtrA proteins. Within the first five hours following estrogen administration both HtrA1 mRNA and the protein levels were increased significantly. No changes in the expression of HtrA2 were observed. This indicates that HtrA1 may be involved in the response against oxidative stress induced by estrogen treatment in hamster kidney. During prolonged estrogenization, a significant reduction of the HtrA1 mRNA and protein levels was observed after 6 months of estradiol treatment, while the expression of HtrA2 was significantly elevated starting from the third month. This suggests an involvement of the HtrA proteins in estrogen-induced nephrocarcinogenesis in hamster. Using fluorescence in situ hybridization we localized the HtrA1 gene at the qb3-4 region of Syrian hamster chromosome 2, the region known to undergo a nonrandom deletion upon prolonged estrogenization. It is possible that the reduced level of HtrA1 expression is due to this chromosomal aberration. A full-length cDNA sequence of the hamster HtrA1 gene was obtained. It codes for a 50 kDa protein which has 98 and 96% identity with mouse and human counterparts, respectively.     

The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development

Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.     

Expression of the EMILIN-1 gene during mouse development.

Expression of EMILIN-1, the first member of a newly discovered family of extracellular matrix genes, has been investigated during mouse development. EMILIN-1 mRNA is detectable in morula and blastocyst by RT-PCR. First expression of the gene is found by in situ hybridization in ectoplacental cone in embryos of 6.5 days and in extraembryonic visceral endoderm at 7.5 days. The allantois is also labeled. Staining of ectoplacental cone-derived secondary trophoblast giant cells and spongiotrophoblast is strong up to 11.5 days and then declines. In the embryo, high levels of mRNA are initially expressed in blood vessels, perineural mesenchyme and somites at 8.5 days. Later on, intense labeling is identified in the mesenchymal component of organs anlage (i.e. lung and liver) and different mesenchymal condensations (i.e. limb bud and branchial arches). At late gestation staining is widely distributed in interstitial connective tissue and smooth muscle cell-rich tissues. The data suggest that EMILIN-1 may have a function in placenta formation and initial organogenesis and a later role in interstitial connective tissue.     

Regulation of Pcsk6 expression during the preantral to antral follicle transition in mice: opposing roles of FSH and oocytes

Several secreted products of the TGFbeta superfamily have important roles during follicular development and are produced by both oocytes and somatic cells (granulosa and theca) in the follicle. The proprotein convertases are a family of seven known proteins that process TGFbeta ligands and other secreted products to their mature active form. The present study examined the regulation of steady-state levels of Pcsk6 mRNA, which encodes a convertase protein known to process members of the TGFbeta superfamily, during mouse follicular development. Pcsk6 mRNA and protein were expressed in preantral but not cumulus or mural granulosa cells. Pcsk6 mRNA levels in preantral granulosa cells were not regulated by growing oocytes of preantral follicles, but were elevated by FSH. Furthermore, Pcsk6 mRNA in preantral granulosa cells was potently suppressed by factor(s) secreted by fully grown oocytes from antral follicles, in part through SMAD2/3-mediated pathways. Oocytes acquired the ability to suppress the steady-state levels of Pcsk6 mRNA in granulosa cells during the preantral to antral follicle transition. Suppression of Pcsk6 mRNA by oocytes could reflect a change in the mechanism(s) regulating the activity of members of the TGFbeta superfamily.     

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.     

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.