The developing eye of the cockroach, Periplaneta americana, and the locust, Schistocerca gregaria, was chosen for an experimental investigation of factors involved in the development of nervous tissue and the formation of ordered nerve connections. Grafting experiments between eye colour mutants of P. americana showed that the retina develops postembryonically from a region of head epidermis which differs from other larval head epidermis in being competent to respond to a recruiting signal produced by adjacent eye tissue. Recruited cells divide and segregate into clusters which differentiate into ommatidia. The specific fate of individual cells is probably assigned according to their position within the cluster. Quantitative analysis of cell proliferation and death in the compound eye of S. gregaria under normal and experimental conditions showed that the retina develops normally in the absence of the optic lobe. In the absence of innervation from the retina, lamina ganglion cells are generated normally but do not differentiate and subsequently degenerate. Experiments on S. gregaria in which spatial or temporal relationships between retina and lamina were altered, showed that axons tend to grow along the paths of immediately adjacent axons, with no indication of specificity for their normal target sites. The following model was proposed for the development of the retina-lamina projection. Formation of the retina by recruitment of epidermis ensures that sensory axons develop in an orderly spatiotemporal sequence. Newly-formed axons grow along preceeding ones and, in turn, provide a conducting tract for suceeding axons, so that the orderly pattern of axon outgrowth is maintained in the pattern of axon arrival in the lamina. As both retina and lamina grow anteriorly, new axons arrive among new lamina cells. Lamina cells innervated by retinal axons differentiate into discrete synaptic units; excess, uninnervated cells degenerate. The relevance of the model to the initial formation of the projection, and to the formation of other nerve connections in insects, was discussed.

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The molecular basis of neural fold adhesion and fusion in closure of the mouse spinal neural tube is a crucial process, since failure may result in spina bifida. Previous studies have shown that cleavage of glycosyl phosphatidylinositol (GPl)-anchored proteins causes neural tube defects in the mouse. In this study, mouse embryos undergoing neural tube closure were treated with phosphatidylinositol specific phospholipase C (PIPLC) enzyme known to cleave GPI-anchored molecules, and then cultured for 8 hours. PIPLC treatment inhibits spinal neural tube closure, as shown by an enlarged posterior neuropore. EphrinAs are GPI-anchored cell surface proteins, and were considered as candidates for a role in adhesion. Perturbation of ephrinA ligand binding to EphA receptors by injecting EphA3 fusion protein into the amniotic sacs of cultured embryos, inhibits neural tube closure. Further microinjection experiments with EphAl fusion protein, which also inhibits closure, suggests that the specific ephrinA ligand, ephrinA 1, is required for spinal neurulation. Expression studies show localization of ephrinA 1, ephrinA3 and the EphA2 receptor in the spinal neural tube. Further work has involved RT-PCR to investigate the expression of possible ephrin binding partners and has shown that EphAl, EphA3, EphA4, EphA5, EphB2, EphB3, EphB4 and EphB6 are also expressed in the spinal neural tube. The ephrinA 1 ligand is currently being studied further by constructing a conditional knockout murine model of the gene. The EphA2 receptor shows an interesting pattern of expression in the spinal neuroepithelium and surface ectoderm during neurulation. The EphA2 receptor is expressed specifically at the point of closure and displays fluctuating amounts of expression on the tips of apposing neural folds. The fluctuating mRNA expression of EphA2 in apposing tips of neural folds may indicate a genetic role of an asymmetrical protrusion from the tip of the left neural fold as viewed via electron microscopy. EphA2 is expressed on the lamellipodia-like protrusion which emanates from the left neural fold. The findings strongly suggest that the Eph/ephrin system plays a role in determining the structure of the initial attractive properties of the tips of the apposing neural folds during adhesion and fusion of the spinal neural tube which culminates in the appearance of an apoptotic cell at the front line of closure.

The aim of this project was to investigate how the cell-cycle of both nuclear-donor and recipient donor affects the development of embryos reconstituted by nuclear transfer. Two methods of synchronising embryos were established. Embryos could be held in mitosis by culture for nine hours in 10μM nocodazole or culture for four hours in 0.1 μg/ml colcemid. Secondly, treatment with 1 μg/ml of aphidicolin of embryos previously synchronized in mitosis was able to synchronize the blastomeres at the G1/S border without any apparent effect upon development to blastocyst. A method of parthenogenetic activation of recently ovulated and preovulatory oocytes was established, involving the culture of the oocytes for 60 minutes in 25 mM strontium in a calcium magnesium free M16 medium. Spontaneous activation was almost non existent in oocytes recovered from the ovary or in those recovered very early after ovulation (14 hours after hCG). It was possible now to conduct experiments on nuclear transfer in which the timing of activation was strictly controlled. To study the effect of variations in the time of fusion in relation to activation, late 2-cell stage nuclei were fused six hours before, at the same time or 12 h after activation. After activation of enucleated oocytes, the cytoplast fragmented. This phenomenon appears to be mediated by microtubules, since culture of activated cytoplast with inhibitors of microtubules polymerisation (nocadazole) inhibited fragmentation. A "nuclear transfer" induced activation was observed, although the cytoplast donor oocytes were not activated by the procedures involved in the recovery or the enucleation methods. A greater proportion of reconstituted embryos developed to blastocyst when nuclei were transferred to preactivated cytoplasts. These results confirm the importance of controlling cell cycle during nuclear transfer and emphasise the advantage of transferring nuclei in G1-phase. In addition, they suggest that development may be achieved from later stages, provided that the nuclei are transferred into appropriate recipients.

Nanog is a divergent homeodomain protein with the capacity to direct constitutive self-renewal in the absence of otherwise obligatory cytokine stimulation. <i>Nanog</i> is expressed in the early mouse embryo and is essential for the specification of pluripotent cells. However the mechanism by which Nanog governs pluripotency is incompletely understood. In this thesis experiments are presented that further the functional characterisation of Nanog. In the mouse embryo, <i>Nanog</i> is normally down regulated in cells prior to de-lamination and ingression through the primitive streak. To address the consequence of <i>Nanog</i> over-expression <i>in vivo</i>, a revertible <i>Nanog</i> over-expressing cell line has been generated which can be tracked in the embryo. Results show that the modest 2-3 fold increase in <i>Nanog</i> expression does not cause any overt phenotype at this stage and <i>Nanog</i> over-expressing cells can be detected in the mesoderm of mouse embryos. Nanog is shown to exist in multimers in ES cells. The domain mediating multimerisation is identified as a tryptophan repeat motif and the functional consequence of deletion of this domain is investigated. To identify Nanog partner proteins, a biotinylation tagging system in ES cells has been designed, constructed, and implemented. This led to the identification of putative Nanog partner proteins via mass-spectrometry. Two Nanog partner proteins, Esrrb and HDAC2 have been confirmed by co-immunoprecipitation. In addition, the SLQQ motif within the Nanog homeodomain is shown to be the site of interaction between Nanog and Sall4. This SLQQ motif is found at a similar location in only one other homeodomain protein, Oct4.  Consistent with these observations Sall4 is also shown to bind Oct4.

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Fertilizations by diploid rabbit spermatozoa cannot be precluded on grounds of either reduced viability or abnormal functional anatomy of head structures. However, the cervix has been shown to constitute a major barrier to the progression of diploid spermatozoa to the site of fertilization. The almost total absence of double-tailed spermatozoa from the cervix indicates that the selectivity of sperm transport is most probably based on differential sperm motility. This finding is supported by the failure of detergent-killed spermatozoa to penetrate the cervix, and also by in vitro experiments using systems in which the distribution of spermatozoa is affected by their swimming abilities. There is no evidence for any active selection of spermatozoa by the female tract. Therefore those diploid spermatozoa whose only apparent fault is their larger size would seem to be potentially capable of fertilizing. So although the actual involvement of diploid spermatozoa in the production of triploid zygotes in the rabbit is still unknown, the possible incidence of such diandric triploids will be much less than the simple incidence of diploid spermatozoa in the semen. Studies of sperm transport in the human female tract have shown that thus apparent selection for spermatozoa of normal morphology can be attributed to the general principle that those spermatozoa with abnormalities which impair their motility are excluded from the cervix, whilst those with abnormalities which do not affect motility will have the same chance of reaching the site of fertilization as 'normal' spermatozoa. Again the spermatozoa select themselves, with the female tract playing an essentially passive role. Ultrastructural studies on rabbit and human spermatozoa have emphasized the stability and complexity of fine structure of the postacrosomal sheath. These findings are considered in relation to a possible role for this region in the maintenance of the structural integrity of the sperm head.

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No description available.