Search

Copyright of Zywnosc is the property of Polish Society of Food Technologists - Scientific Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Advances in Biochemistry / Postepy Biochemii is the property of Polish Biochemical Society / Acta Biochimica Polonica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Mammalian development is a process, whereby cells from a totipotent zygote gradually lose their potency, i.e. their ability to differentiate, in the environment of the developing embryo. An ideal model for testing the real potential of cells is the experimental production of chimaeras. The first experimentally produced mammalian (murine) chimaeras were created by Tarkowski [1961] and since then many new methods of chimaera production have been developed, including injecting cells into the blastocyst's cavity or into cleaving embryos. This review describes how different cell types, depending on the developmental stage or culture conditions, manifest their potential to contribute to chimaeras. Cell developmental potential has been analysed in pluripotent blastomeres, which can contribute to all embryonic and extra-embryonic lineages, albeit differently depending on their developmental stage. This is the case in blastocyst lineages, with various developmental potentials depending on the surrounding cells, and in more differentiated cells from different stages of pregnancy, which in some cases may colonise chimaeric animal tissue. Cell potential has also been analysed in embryonic stem and embryonal carcinoma cells, which are pluripotent and efficiently contribute to chimaeras; in multipotent fetal and adult stem cells, which can also participate in chimaera formation; and in somatic mouse embryonic fibroblasts (MEFs), which can also be reprogrammed in the environment of the cleaving embryo. Interspecies chimaera studies have also demonstrated the pluripotency of foreign cells. Experiments with chimaeras have shown that not only pluripotent embryonic cells are capable of contributing to chimaeras, so are adult cells, which plasticity is now well-documented. [ABSTRACT FROM AUTHOR]

In an overwhelming majority of experiments, both mammalian embryonic and somatic cloning have relied on introducing exogenous nuclei into enucleated metaphase II (MII) oocytes. Since attempts at cloning using interphase zygotes as recipient cells have failed, these cells were - until quite recently - commonly regarded as poor recipients for nuclear transfer. However, we have recently shown that interphase zygotes can be successfully used as recipients of embryonic nuclei. In a previous study, we used our original method of selective enucleation (SE), in which the pronuclear envelope with attached chromatin is removed while the liquid pronuclear contents and nucleoli in the zygote's cytoplasm are left intact, to obtain fertile mice upon transfer of 8-cell (1/8) nuclei into SE zygotes. Here we report that 16-cell (1/16) nuclei can also support full-term development. Additionally, full pre-implantation development, albeit to a limited degree, was obtained after transfer of embryonic stem (ES) cell and foetal fibroblast (FF) nuclei (2.4% and 2.5%, respectively). Sporadically, SE zygotes reconstructed with FF nuclei were able to implant, but they never developed beyond midpregnancy. Our results clearly indicate that SE zygotes can be successfully used as competitive recipients of embryonic nuclei from, at least, the 16-cell (morula) stage. However, their use as recipients of ES cell and somatic cell nuclei seems to be questionable. [ABSTRACT FROM AUTHOR]

The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker coexpression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineagebiased cells. [ABSTRACT FROM AUTHOR]

The embryonic development of placental mammals takes place inside the mother’s womb, which requires the formation of appropriate supportive structures by both the mother’s organism and the developing embryo. The first stages of mammalian embryonic development, preceding implantation, are the period of differentiation of the first cell lineages – epiblast (which will give rise to the embryo proper), and extra-embryonic lineages: trophectoderm (responsible for implantation and formation of the placenta) and primitive endoderm (giving rise to the yolk sac). Their differentiation is necessary for further development, and is a common feature of the development of all placental mammals, but the timing and molecular mechanisms responsible for these processes differ between mammalian species.

Mouse genetic approaches when combined with live imaging tools are revolutionizing our current understanding of mammalian developmental biology. The availability and improvement of a wide variety of genetically encoded fluorescent proteins have provided indispensable tools to visualize cells and subcellular features in living organisms. It is now possible to generate genetically modified mouse lines expressing several spectrally distinct fluorescent proteins in a tissue-specific or -inducible manner. Such reporter-expressing lines make it possible to image dynamic cellular behaviors in the context of living embryos undergoing normal or aberrant development. As with all viviparous mammals, mouse embryos develop within the uterus, and so live imaging experiments require culture conditions that closely mimic the in vivo environment. Over the past decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation-stage mouse embryos. In this chapter, we discuss routine methods for ex utero culture of preimplantation- and postimplantation-stage mouse embryos. In particular, we describe protocols for collecting mouse embryos of various stages, setting up culture conditions for their ex utero culture and imaging, and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.

Mouse genetic approaches when combined with live imaging tools have the potential to revolutionize our current understanding of mammalian biology. The availability and improvement of a wide variety of fluorescent proteins have provided indispensable tools to visualize cells in living organisms. It is now possible to generate genetically modified mouse strains expressing fluorescent proteins in a tissue-specific manner. These reporter-expressing strains make it possible to image dynamic cell behaviors in the context of a living embryo. Since mouse embryos develop within the uterus, live imaging experiments require culture conditions that closely mimic those in vivo. Over the past few decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation stage embryos. In this chapter, we will discuss methods for ex utero culture of preimplantation and postimplantation stage mouse embryos. In particular, we will describe protocols for collecting embryos at various stages, setting up culture conditions for imaging and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.