Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.     

A long region upstream of the IME1 gene regulates meiosis in yeast

Summary Meiosis and sporulation in yeast are subject to two types of regulation. The first depends on environmental conditions. The second depends on a genetic pathway which involves the control of the positive regulatory gene IME1 by RME1, which is in turn controlled by the MAT locus. The presence of IME1 on a multicopy plasmid enables cells to undergo meiosis regardless of their genotype at MAT or RME1. We show here that a multicopy plasmid carrying IME1 also enables meiosis, regardless of the environment. Therefore, both kinds of regulation appear to act through IME1. Furthermore, the behavior of multicopy plasmids carrying various segments from the IME1 region suggests that the region upstream of IME1 contains both positive and negative regulatory sites. Control of IME1 by the environment and by the MAT pathway both act through negative regulatory sites.     

Cyclin E-Cdk2 temporally regulates centrosome assembly and establishment of polarity in Caenorhabditis elegans embryos

Establishment of polarity in C. elegans embryos is dependent on the centrosome. The sperm contributes a pair of centrioles to the egg and these centrioles remain incapable of polarizing the cortex while the egg completes meiosis. Coincident with the establishment of polarity, the centrioles recruit centrosomal proteins, several of which are required for polarity, suggesting that the temporal regulation of centrosome assembly may control the initiation of polarization. We found that cyclin E-Cdk2 is required for the establishment of polarity. Cyclin E-Cdk2 controls the recruitment of centrosomal proteins specifically at the time of polarity establishment. Cyclin E is required for several examples of asymmetric cell division and fate determination in C. elegans and Drosophila. Here, we suggest a possible mechanism for cyclin E-Cdk2-dependent differentiation: the establishment of cortical polarity by the centrosome.     

Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.     

Targeted disruption of the Fgf2 gene does not affect vascular growth in the mouse ischemic hindlimb.

Ischemic revascularization involves extensive structural adaptation of the vasculature, including both angiogenesis and arteriogenesis. Previous studies suggest that fibroblast growth factor (FGF)-2 participates in both angiogenesis and arteriogenesis. Despite this, the specific role of endogenous FGF-2 in vascular adaptation during ischemic revascularization is unknown. Therefore, we used femoral artery ligation in Fgf2(+/+) and Fgf2(-/-) mice to test the hypothesis that endogenous FGF-2 is an important regulator of angiogenesis and arteriogenesis in the setting of hindlimb ischemia. Femoral ligation increased capillary and arteriole density in the ischemic calf in both Fgf2(+/+) and Fgf2(-/-) mice. The level of angiographically visible arteries in the thigh was increased in the ischemic hindlimb in all mice, and no significant differences were observed between Fgf2(+/+) and Fgf2(-/-) mice. Additionally, limb perfusion progressively improved to peak values at day 35 postsurgery in both genotypes. Given the equivalent responses observed in Fgf2(+/+) and Fgf2(-/-) mice, we demonstrate that endogenous FGF-2 is not required for revascularization in the setting of peripheral ischemia. Vascular adaptation, including both angiogenesis and arteriogenesis, was not affected by the absence of FGF-2 in this model.     

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.