An electrophoretic investigation was carried out on protein variation in different tissues of Apodemus sylvaticus. In addition to some ontogenetic studies, physicochemical methods were employed to characterise esterase enzymes and other proteins. The distribution of variant forms from thirteen localities in Iceland, Ireland, Scotland, Norway and Sweden were studied. Finally it was possible in some cases to acquire breeding data and clarify modes of inheritance. Protein: Serum pre-albumins, albumin and ceruloplasmin are monomorphic, whilst post-albumin and transferring may be polymorphic. R-cell proteins A and B are polymorphic. Other tissue proteins were found to be uniform, except for a cathodally migrating protein fraction in striated muscle. Esterases: Most esterase fractions were found to be nonspecific carboxylesterases. Each tissue has its own esterase pattern, but also shares fractions with other tissues. The physicochemical tests, breeding, ontogenetic and population data make it feasable to propose a mode of inheritance for many esterase fractions. Thus, the proposed loci Es-1, Es-2, Es-3, Es-4, Es-7 and Es-8 have each two codominant alleles. Es-6 has one dominant silent allele, and one recessive producing allele. These loci produce esterase in more than one tissue at the same time. There is a difference between all populations tested, the Iceland populations differing most from the rest. It is suggested that chance founder effects is the chief factor for the uneven/uneven distribution of protein markers. The phvsicochemical characters of proteins in Apodemus sylvatcus are similar to those established for other rodents.

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The aim of the project was to optimize cell cycle co-ordination in murine embryos reconstructed by nuclear transfer. The project also involved studies of oocyte activation, cell cycle length and synchronization, evaluation of MPF, and DNA replication in early embryos. Embryos were successfully arrested at mitosis by culturing them up to 16 hours with 2.5 mM 6-dimethylaminopurine (DMAP). A comparative study of different concentrations of 6-DMAP and 10 μM nocodazole for holding the embryos at mitosis for 12 hours showed a greater effectiveness of nocodazole. In most of the experiments 25 mM strontium chloride was used as the method for the artificial activation of cytoplasts and reconstituted embryos. Later on, a new method for parthenogenetic activation of metaphase II oocytes or recipient cytoplasts was established. The effect of meiosis/mitosis/maturation promotion factor (MPF) on the development of embryos reconstructed at different times in relation to activation was investigated and its level was studied by looking at nuclear envelope breakdown (NEBD) and nuclear envelope breakdown and premature chromatin condensation. After determining the timing of G1 duration and S-phase initiation of 3<SUP>rd</SUP> and 4<SUP>th</SUP> cell cycle, nuclear transfer experiments were performed to investigate the effect of nuclear donor cell cycle stage and its interaction with recipient cell cycle stage. The studies confirm the importance of cell cycle co-ordination in the development of reconstructed embryos. By appropriate co-ordination of cell cycle stages it has been possible for the first time to obtain similar development with 4-cell blastomeres with donor cell nuclei at any stage of the cell cycle. Moreover, the use of strontium chloride + 6-DMAP activated cytoplasts with 4-cell donor nuclei from different cell cycle can produce embryos that will develop with a higher frequency to morula/blastocyst than strontium chloride alone.

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A conserved feature of early vertebrate embryos is the formation of simple epithelial layer of cells which surrounds the embryo and protects it from the external environment. This epithelium is called the trophectoderm in mammals, the superficial layer in Xenopus and the enveloping layer in zebrafish. This project investigates what promotes differentiation of this cell type. In Xenopus embryos aPKC and Notch signalling were found to be unable to promote differentiation of the superficial layer. In contrast, BMP signalling can promote expression of a number of transcriptional regulators, including members of the Grhl and Msx families and differentiation of the superficial layer. This pathway is initiated in the underlying deep cells, but not all target genes are activated so differentiation does not occur in these cells. The role of BMP signalling in mouse development was investigated by using mouse embryonic stem cells (mESCs) as the model. BMP4 is sufficient to induce mESCs to form a polarised epithelial cell type and that these epithelial cells appear trophoblast in fate. BMP signalling activates Grhl and Msx genes in mESCs, as it does in Xenopus embryos. This suggests that similar target genes are activated by BMP signalling in the first epithelium of Xenopus and mouse. Based on this data it is tempting to propose that BMP signalling acts in a conserved manor to promote differentiation of the first epithelium in diverse vertebrates.

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