The ability of the oocyte to reprogram paternal chromatin is widely accepted. The oocyte cytoplasm has been reported to contain activities that alter the histone composition, demethylate and unwind highly methylated paternal DNA upon fertilization and lead to zygote formation. The same events are mimicked during the course of cell reprogramming, where somatic cells exposed to the oocyte environment acquire a pluripotent character. Amphibian oocytes have been widely used to study the reprogramming attributes of the female germ cells for over half a century. The size of amphibian oocytes renders them good candidates for technical manipulations, as well as protein biochemistry studies. Oocytes harvested from the amphibian axolotl were utilised in the present study to delineate the oocyte reprogramming machinery. Previous studies have reported the presence of key pluripotency factor orthologs, such as Nanog, in the axolotl oocyte, thus rendering it an interesting tool for the study of the chromatin remodelling and pluripotency networks as well as their interplay. To study these networks, identification of the physical interactors of the Nanog protein or the Nanog promoter was attempted. One of the key events taking place during cell and paternal chromatin reprogramming is demethylation. The removal of the methyl mark that is deposited over genetic loci during cell differentiation is linked with the transcriptional activation of the respective loci. Demethylation is therefore fundamental for the activation of pluripotency-associated factors both in fertilisation and cell reprogramming. The leading theory behind demethylation is the successive oxidation of the methyl mark to formyl and carboxyl and its subsequent removal from Thymine DNA Glycosylase (TDG). Ten Eleven Translocation (TET) family proteins catalyse methyl oxidation and therefore utilise demethylation. Additional demethylation pathways have also been supported by findings, specifically in the context of the oocyte. Base Excision Repair (BER) and Nucleotide Excision Repair (NER) factors have been associated with DNA demethylation occurring immediately after fertilisation. It is therefore fundamental to delineate the demethylation mechanism facilitated by the oocyte in the early stages of development. The identification of oocyte demethylation factors and mechanisms will greatly improve our understanding of demethylation as well as improve current cell reprogramming methodologies by using the more efficient oocyte demethylation mechanism. To delineate the demethylation mechanism employed by the oocyte and contribute to the debate of TET oxidation versus NER or BER demethylation via DNA repair in the context of the oocyte, oocyte factors preferentially binding to different cytosine modifications were analysed. The murine Nanog promoter, a genetic locus activated during cell reprogramming, harboured the different cytosine modifications in an attempt to see how each one would affect its transcriptional status. As a result, TET factors were not discovered to bind any of the cytosine modifications while both NER and BER pathway proteins were found to be significantly enriched in the case of methylcytosine. The results obtained therefore support previous findings and advocate for a TET independent DNA demethylation occurring through DNA repair. While teams researching demethylation through DNA repair have supported either NER or BER, the findings unveiled in this thesis advocate for the two pathways acting in concert in the recognition and potential removal of DNA methylation. It also has to be noted that it is the first time an experiment of this kind is carried out in the context of the oocyte and the findings offer a unique insight into oocyte demethylation dynamics. Taking into account the data obtained and previous research, a new DNA demethylation model is proposed. The model advocates for NER taking the lead in DNA demethylation, creating patchy demethylated sites that are subsequently recognised and demethylated via the BER pathway.

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Changes in the levels of expression of the prolactin receptor (PRLR) mRNA in the pituitary gland, hypothalamus, liver, pancreas, kidney and gonad from embryonic day (ED) 15 and ED21 to 1 day post-hatch, respectively, in chickens and turkeys were measured by real-time PCR. In both species, PRLR mRNA increased from low levels during the last week of ED to reach maxima at the peri-hatch period. Similarly, circulating levels of prolactin (PRL) also increased during this interval and were correlated with the observed increases in tissue content of PRLR mRNA. This suggested that PRL was up-regulating its own receptor during late embryogenesis. In support of this, in vitro stimulation of the pituitary gland of turkeys with VIP on ED24 resulted in a 4 fold and 3 fold increase in PRL and PRLR, respectively. Stimulation with VIP of either the hypothalamus or gonad had no effect on either levels of the PRLR transcript. This suggests that VIP acts indirectly through increased PRL to upregulate the number of receptors. In order to investigate the transcription of genes that may be induced/suppressed by PRL, suppressive subtractive hybridization (SSH) libraries from control or VIP stimulated ED24 turkey pituitary glands were constructed. Stimulation with VIP resulted in a 5.7 and 2.8 fold increase in media and pituitary content of PRL, respectively. The changes in PRL were consistent with endogenous levels of PRL observed just prior to hatch. Following sequence analysis of random clones (n=96) from each library, a total of 145 non-redundant putative genes were obtained. About 51% of the putative genes have as yet no assigned function, whereas, 15% were housekeeping genes and 34% had known functions within various pathways. Real-time PCR was used to confirm the differential expression of 21 of these genes in VIP treated and control pituitaries. Since the majority of these genes were expressed at levels consistent with the direction of subtraction, these data suggest that these libraries may be useful to study the direct and indirect effects of increasing levels of PRL on anterior pituitary function at about the time of hatch.

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Call number: LD2668 .T4 1967 S6 / Master of Science

The anterior visceral endoderm (AVE) is a specialised subpopulation of the visceral endoderm (VE), a single layer simple epithelium that surrounds the extra-embryonic ectoderm and epiblast of the egg cylinder stage embryo. Initially induced at the distal tip of the egg cylinder, AVE cells undergo a stereotypic migration towards the prospective anterior, stopping at the interface between the underlying epiblast and extra-embryonic ectoderm (ExE). Previous research has shown that membrane enrichment of Dvl2 is present in the VE overlying the epiblast (Epi-VE). In this thesis I confirm the presence of planar cell polarity (pep) signalling in this region by assaying the subcellular localisation of additional core pep proteins Vangl2 and Daaml. I show that null embryos of the Nodal antagonist Lefty1 exhibit ectopic membrane enrichment of Dvl2 and a previously unreported AVE over-migration phenotype. Furthermore, using pharmacological inhibition of Nodal signalling I show that the TGF~ protein Nodal modulates pep signalling in the YE. Utilising DIe and confocal microscopy I perform detailed time-lapse analyses of the VE to quantify the dynamic cell behaviour and topology. Using this assay I show that wild-type embryos exhibit dynamic cell movement, which is regionally restricted to the Epi-VE. Analysis of Leftyl-/- and ROSA26lyn-Celsr-l mutants, both of which exhibit disrupted pep signalling and AVE over-migration phenotypes, indicates that normal VE dynamics and topology are disrupted. The results of this quantitation indicate that these mutants exhibit increased cell migration and neighbour exchange across the YE. These data show that regional restriction of movement is lost and results in the AVE over-migration phenotypes observed. Together these results show that regionally restricted pep signalling in the VE acts to modulate cell behaviour and topology, which in turn determines the regional restriction and normal end-point of AVE migration.

No description available.

The causal and temporal aspects of blood tissue specification in the chick embryo were investigated in this study. The main focus was on the role of basic fibroblast growth factor (bFGF) in the determination of the erythropoietic tissue, particularly in context with its representation as a non-axial mesodermal derivative which arises in the posterior domain of the chick embryo. The initial strategy employed in this study was the use of agents that are known to block the activity of bFGF, and to determine their effects on erythropoiesis. Treatment of unincubated chick embryo explants with heparin, which binds specifically to the FGF family, was found to inhibit primitive streak formation and erythropoiesis, and also inhibited the formation of other mesodermal tissues. These initial findings suggested that one or more growth factors had become bound to the heparin, and that their activity is important for the specification of primitive streak formation and mesodermal patteming. The development of the erythropoietic tissue was assayed by a cytochemical test for haemoglobin using 0dianisidine; and by histological examination for blood islands and red blood cells in serial sections of the embryos after 48 hours incubation. Microscopic examination of the embryos at the stages of gastrulation on the first day of incubation revealed that heparin caused holes to appear in the ventral layer; and although a primitive streak did not form, a middle layer of mesenchymal cells were seen to accumulate between the ectodermal and ventral "endodermal" layers. It was significant that heparin's inhibitory effect on erythropoiesis could be reversed after the addition of a recombinant bovine bFGF to the heparin-treated embryos. However, the exogenous bFGF did not neutralize the inhibitory effect of heparin on the primitive streak and other mesodermal derivatives (Chapter Two). The inhibition of erythropoiesis by heparin was also reversed by the addition of a mesodermal-inducing factor extracted from a Xenopus embryonic cell line, namely XTC. The XTC mesodermal-inducing factor (MIF), which belongs to the transforming growth factor-B family and is a homologue of activin, could also reverse the inhibitory effect of heparin on primitive streak formation; but no recognizable axial mesodermal structures subsequently developed. Of consequence, was that both bFGF and XTC-MIF blocked heparin's effect on the ventral layer, preventing the gaps forming. Therefore, it is suggestive that the VI development of an intact ventral layer is important for the determination of the erythropoietic sequence (Chapter Three). By taking a more specific approach using antisera to bFGF (anti-FGF) and the bFGF receptor (anti-FGFR) on whole embryo explants, it was found that anti-FGP and anti-FGFR were able to inhibit erythropoiesis, but not primitive streak formation. However, these antisera caused defects in the posterior region of the embryonic axis. These embryos not only lacked posterior blood tissue, but heart and somites were missing; whereas the anterior head structures were well formed. These results therefore suggest that bFGF signalling is important for the development of the posterior body plan, which includes erythropoiesis (Chapter Four). Further evidence for the role of bFGF in the determination of the blood mesodermal tissue line was reached in an in vitro bioassay. In this part of the investigation, specific pieces of the blastoderm, namely pieces dissected from the posterior marginal zone (PMZ) and inner core of the central disc (lCD) were able to form haemoglobin under particular conditions. The PMZ components were found to have the capacity to form haemoglobin when dissected from blastoderms of stages X to xm when cultured in serum-free medium. This commitment to form haemoglobin could be blocked by treatment with anti-FGP at stages X and XI, but not at the later stages of xn and XIII. The ICD components were found to have a commitment to form haemoglobin only if this component was dissected from embryos at stage XIT and XITI, but not before. These results suggest that a determinative event for the haemoglobin differentiative pathway occurs between stages XI and XII. It was also found that the stage X central disc component could be induced to form haemoglobin if a stage xm hypoblast was added to it in tissue recombination sandwich cultures, or if bFGF (75 - 150 ng/ml) was added to the medium. These results lend further support that bFGF plays an important role in the determination of erythropoiesis; and furthermore, suggest that the hypoblastic tissue is the source of this induction (Chapter Four). Finally, immunocytochemical labelling with a polyclonal antibody to bFGF has revealed that bFGF increases significantly from stage XI in cells within the developing hypoblast layer and in the middle mesodermal layer. These cells are located predominantly in the posterior domain of the embryo. This polarized distribution of bFGF with the high value of bFGF concentration in the posterior area, is presumably responsible for inducing the overlying epiblast to form the posterior horseshoe-shaped region from which blood tissue is seen to arise. An immunocytochemical analysis of the distribution of the FGF receptor was vu assessed, as an indicator of the possible competence of the cells to respond to the bFGF signal. The bFGF receptor was found to be expressed at stage XII in cells that appeared to be in register with those immunoreactive to the bFGF ligand; therefore suggesting an autocrine function. It was interesting that at stage Xli an intense immunostaining with the anti-FGFR developed in the nuclei of cells within the epiblast layer (Chapter Five). In conclusion, this study has demonstrated that the initial determination of the erythropoietic cell lineage in the chick is at the time when the hypoblast is in the process of forming beneath the epiblast, Le. between stages XI and XII. Furthermore, it was found that an induction by an FGF-like signal from the hypoblast layer (or middle mesodermal cells that may be closely associated with the hypoblast) induces "competent" cells (Le. FGFR-positive cells) in the epiblast to form blood tissue in the posterior domain of the chick embryo. / Thesis (Ph.D.)-University of Natal, 1994.