Our current description of mouse developmental anatomy has mainly been based on studies using SEM, confocal, light or dissecting microscopy and, as the resulting picture gives an incomplete understanding of 3D morphological relationships within the embryo, it is inadequate for current molecular and genetic studies. The work reported here sets out to improve the situation by describing novel aspects of mouse developmental anatomy obtained from the analysis of 3D digital reconstructions of serial sections of mouse embryos at embryonic days 1-9 (E1-9) within which all anatomically defined tissues were delineated. These embryos were carefully selected for being representative of their developmental stage and reconstructions were checked for normal representation of tissues. The reconstructions contain histological detail accessible at any angle through the reconstruction within the reconstructions, while surface rendering software can display 3D tissue organisation. These reconstructions have been used to explain three particular aspects of mouse development: geometric relationships in the early stages of mouse development, and morphological events that accompany turning, and the relative growth rates of the key tissues during early embryogenesis and organogenesis. The geometric relationships of the reconstructions of E1-9 were analysed and compared to the existing description of mouse occurs earlier than hitherto reported, namely at E5-6.5. Two blastocysts are bilaterally symmetric but only the earlier stage shows a tilt in the inner cell mass with respect to the embryonic-abembryonic axis. The tilt in the ectoplacental cone with respect to the latter axis is not as a consistent angle, but does coincide with an asymmetry in the extraembryonic ectoderm that could be explained by the tilt of the cone. During early organogenesis, the myocardial walls, cardiac jelly and endothelial lining are congruent. The vascular system develops in isolated sacs that are inline and link up later. There is no obvious morphological asymmetry between left and right components of structures.

A primary neural tube culture assay was used to study the early stages of melanoblast specification, proliferation, and migration. This assay was used to study the roles of different cell signalling pathways known to affect melanoblast development <i>in vivo</i>. KITL significantly affected both the proportion of crest cells that were melanoblasts and the distance they migrated, while EDN3 only affected melanoblast numbers. HGF increased the migration distance of melanoblasts in culture, while MSH affected neither melanoblast migration nor numbers. Studies of adult chimaeras derived from embryos carrying different coat colour markers have suggested that total melanoblast population is derived from a small number of progenitors, each generating a discrete unilateral transverse band of colour with minimal mixing between clones. In this study, two complementary approaches were used to assess the behaviour of labelled clones during development. Firstly, aggregation chimaeras were made between <i>Dct-LacZ</i> and non-transgenic embryos. The <i>Dct</i> promoter drives expression from E10 in melanoblasts and in cells of the telencephalon. Resultant patterns of labelled melanoblasts were studied during mid-gestation. Secondly, transgenic mice were generated that carry a modified <i>LaacZ </i>reporter construct containing a 300bp duplication (<i>LaacZ</i>) under the control of the <i>Dct</i> promoter. The <i>LaacZ</i> transgene is normally inactive, but it reverts to wild type <i>LacZ</i> at low frequency, labelling a cell and all its progeny apparently at random. Together, chimaeric and mosaic embryos suggested the melanoblast population is derived from a large number of progenitors, a pool of melanoblasts may reside in the cervical region, and melanoblasts within a clone show considerable longitudinal migration, suggesting there is significant axial mixing between clones. Interestingly, radially arrayed labelled clones were also generated in the telencephalon in <i>Dct-LaacZ</i> embryos.

The related genes lin28a and lin28b code for conserved RNA-binding proteins, which contain two key RNA-binding motifs that determine their functions. Previously, our laboratory identified lin28a as a putative downstream target of FGF signalling in the early Xenopus embryo. This was found to occur at gastrulation stage, during which key signalling pathways such as the FGF pathway are active in specifying germ layer development. lin28 is a heterochronic gene in C. elegans and controls the timing of developmental events. In vertebrates, the lin28a gene shows pluripotent-specific expression, and has come to particular interest as one of four factors used to successfully re-program differentiated somatic cells into induced pluripotent stem cells. Both lin28a and lin28b have a high prevalence of ectopic expression in cancer. A well characterised target of both lin28a and lin28b is the microRNA let-7, inhibiting biogenesis to the mature microRNA form. The lin28 proteins have also been shown to potentiate translation of numerous mRNA targets. The aim of this project was to identify if lin28 has an important developmental role in vertebrates. Work in the Xenopus tropicalis embryo found that both lin28a and lin28b were targets of FGF signalling at gastrulation, and were required for correct germ layer patterning at this stage. In order to explore conservation in humans, mesenchymal stem cells were used to model the effects of FGF signalling upon the mesodermal germ layer, and whether lin28 played a role in this. Additional pluripotent cell models were investigated for their suitability in which to study lin28 function. Analysis of lin28 targets in Xenopus revealed that the miR-17-92 cluster family of miRNA may be positively regulated by the lin28 proteins, with a direct interaction possible with a member of these: pre-mir-363. These miRNAs may have further important developmental roles in response to this regulation by the lin28 proteins.

The processes which drive morphogenesis during Drosophila development are well studied. However, the mechanisms employed to preserve the structures formed as a result of these events are poorly understood. The work of this thesis examines the role of localised cell signalling in effecting the morphogenesis of epithelial tissues. The Drosophila embryonic hindgut is a curved epithelial tube which undergoes elongation and rotation to form a shepherd's crook shaped organ which breaks with the symmetry of the embryo. Work within this thesis describes how asymmetric JAK/STAT signalling, on the inside of the curve, positively regulates protein levels of the homophilic adhesion molecule Fasciclin III (FasIII). Increased levels of FasIII lead it to become asymmetrically distributed throughout the lateral cell membrane. Loss of either JAK/STAT signalling or FasIII leads to a reduction in the magnitude of the hindgut curve. Furthermore, preliminary data indicates that the spatial regulation of JAK/STAT signalling, and lateral FasIII, is also required for correct directional control of hindgut rotation. Secondly, this thesis describes a role for localised JAK/STAT signalling and lateralised FasIII in maintaining the shape of folds within the prospective hinge of the 3rd instar larval wing disc. Further investigation of these folds indicates that their development is required for correct wing posture within the adult. This thesis confirms FasIII as a mediator of cell-cell adhesion. It is therefore hypothesised that the lateralised FasIII domain increases tissue stability within the hindgut curve and the wing disc folds. Loss of this intrinsic support results in these structures being unable to maintain their form during development.

Although <i>Pax6</i> is known to be important for development of multiple eye tissues (Quinn et al., 1996) little is known of its targets or method of action in the eye. Using chimeras between small eye and wildtype mouse embryos this study has preliminary shown that retinal pigment epithelium (RPE) can be specified in mutant cells of the chimera although these cells do not differentiate to produce pigment. <i>Trp2 </i>and <i>micropthalmia (Mi)</i>, both RPE specific genes, are expressed in this mutant tissue. These preliminary data indicate a possible function for <i>Pax6 </i>in RPE differentiation. Secondly this thesis reports the identification of <i>Dach1, </i>a murine homologue of <i>dac</i>. Two domains of high sequence conservation exist, the more C-terminal of which appears to represent a novel zipper motif. Similarity to the <i>Ski</i> family of genes is also seen within these regions suggesting that <i>Dach1</i> belongs to a super-family including the <i>Ski</i> genes. <i>Dach1</i> is expressed in the eye and the limb, structures affected by the <i>Drosophila </i>loss of function mutant, and also in the CNS, ear, nasal mesenchyme, lung, gut, genital eminence and dermomyotome. <i>Pax6</i> expression overlaps but is not identical to <i>Dach1</i> expression and the data presented here suggest that <i>Dach1</i> expression is not affected in the small eye mouse brain. In addition <i>Dach1</i> maps to 14E3 in the mouse. Three zebrafish <i>dac</i> homologues were also isolated: <i>zfDachA, zfDachB </i>and <i>zfDachC. zfDachA </i>is expressed in the eye, CNS, optic vesicle, lateral mesoderm and somites. <i>zfDachB </i>is found in an extremely restricted pattern in the CNS while <i>zfDachC</i> is expressed in the CNS, gut and blood islands. At the sequence level <i>zfDachC </i>is closest to <i>Dach1</i> while <i>zfDachB</i> is the least similar. Injection of <i>zfDachA </i>RNA into 2-16 cell zebrafish embryos led to the formation of ectopic tissue in the midbrain/hindbrain region and to somite defects. Injection of a C-terminally truncated <i>zfDachA</i> also led to somite defects.

Anterior-posterior (A-P) axis specification requires signalling from a specialised extraembryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the pre-gastrulating mammalian embryo and move to the prospective anterior. This movement is essential in correctly positioning the A-P axis. Firstly, the contribution of proliferation to AVE movements has been tested. Previous studies have suggested that differential proliferation within the visceral endoderm (VE) is required to determine its direction by passive displacement. High-resolution imaging and embryo culture experiments have been used to assess the distribution and requirements of proliferation during AVE movements. Differential proliferation was not observed along the A-P axis of the VE. However, proliferation within the epiblast was found to be required for correct AVE movements in a Nodal-dependant manner. Secondly, to address the function of the AVE in vivo, this tissue was genetically ablated. Diphtheria toxin A was knocked-in to the Hex locus (an early AVE marker) by homologous recombination in embryonic stem cells. Depending on the time of ablation the AVE or its precursors could be ablated as demonstrated by AVE marker analysis. Ablation of the AVE precursors prior to 5.5 dpc resulted in embryos that displayed greatly disturbed patterning and were unable to gastrulate. AVE ablation between 5.5-6.5 dpc resulted in delayed formation of the primitive streak, epithelial to mesenchymal transition defects and a mispatterning of the anterior primitive streak. A decrease in Nodal signalling was found to be a likely cause for the defects observed in these ablated embryos, indicating that AVE maintains Nodal signalling levels in the epiblast. Together these experiments find a role for epiblast proliferation in AVE migration and identify a novel role for the AVE in patterning the primitive streak. Therefore this investigation provides a critical insight into how the A-P axis is established in mammals.

In undifferentiated ES cells, many Polycomb Repressive Complex 2 (PRC2) target genes carry not only repressive H3K27me3 but are also enriched for conventional indicators of active chromatin including methylated H3K4. This so-called bivalent domain structure is thought to silence key developmental regulators while keeping them poised for future activation (or repression). Consistent with this hypothesis, bivalent genes assemble RNAP II preferentially phosphorylated on Serine 5 residues (poised RNAP II) and are transcribed at low levels. Productive expression is, however, prevented by the action of PRC1. Here, I have focused on the pre-implantation stage of mouse development to evaluate whether bivalent or poised chromatin signatures are indeed specific attributes of emerging pluripotent cells and investigate how the fate of key developmental genes is specified while the first lineage decision event (extra-embryonic lineage formation) occurs. Using blastocyst-derived stem cells and chromatin immunoprecipitation (ChIP), I have shown that lineage-inappropriate genes retain bivalent histone marking in extra-embryonic trophoblast stem (TS). However, and in contrast to ES cells, PRC1 (Ring1B) and poised RNAP II are not recruited to these loci in TS cells, indicating that gene priming is a unique hallmark of pluripotent cells in the early embryo. To investigate the intricate relationship between lineage identity and dynamic chromatin changes, I exploited the potential to convert ES cells into trophoblast-like stem (TSL) cells using a previously established artificial system dependent on doxycycline (Dox) induced repression of an Oct4 transgene. I demonstrated that Suv39h1-mediated H3K9me3 alongside DNA methylation is targeted to PRC2-bound bivalent, lineage-inappropriate genes upon trophectoderm lineage commitment. A change in chromatin conformation was observed upon differentiation of ES cells to TSL cells comparable to that seen in TS cells derived in the traditional manner from the trophectoderm (TE) of blastocyst stage embryos. Most importantly, I have begun to explore when epigenetic differences are specified, at the locus level, from 8-cell stage embryos onwards using newly designed Carrier ChIP technology. This data validated the occurrence of bivalent chromatin domains in vivo and further support the view that alternative strategies operate in the TE to silence key developmental regulators upon blastocyst lineage segregation.

No description available.

Retinoic acid, sequestered from maternally-circulating retinol, is essential for vertebrate embryonic development. However, the mechanism underlying retinol transfer from mother to embryo is not fully understood. Preliminary experiments support the existence of a specific mechanism, such as RBP receptor-mediated uptake to transport retinol across the yolk sac. This project aimed to identify and characterize a mouse embryo homologue of the RBP receptor (RPE65). The homologue cloned was later shown to be ~,~-carotene9', 10'-dioxygenase (~,~-9',10'-CD). Amino acid sequences of embryonic ~,~-9',10'-CD showed 43% identity with mouse RPE65 and revealed a soluble protein without a distinct signal sequence. Using semi-quantitative RT-PCR, the transcript levels of ~,~9', 10'-CD were strongly detected at day 7.5 and 8.5, indicating that ~,~-9',10'-CD protein can provide an alternative pathway for synthesizing retinoic acid during early embryonic development. Disruption of ~,~-9',1O'-CDexpression using antisense oligos in whole embryo culture revealed reduced RAR~2 promoter activity and developmental anomalies compared to controls. Subsequently, RPE65 was identified in both mouse embryos and yolk sac. Whole embryo culture with Pl42 (anti-RPE65) produced defects and reduced RAR~2 promoter activity, whereas control IgM 'did not. Uptake of eH]-retinol-RBP into day 8.5 yolk sac confirmed whether the retinoic acid deficiency observed was caused by poor retinol uptake. eH]-retinol transfer by conceptuses was inhibited by P142, suggesting RPE65 may playa role in embryonic retinol uptake. Synthetic peptides were applied to examine the binding site between RBP and RPE65, but did not show a significant block of RBP binding to RPE65. Immunoprecipitation and phylogenetic tree analysis indicated RPE65 and ~,~-9',10'-CD may share motifs. Immunochemical staining using anti-~,~-9',10'-CD and anti-RPE65 revealed overlapping staining in day 10.5 embryos, notably in retinol sensitive tissues. These studies suggest that RPE65 is involved in embryonic retinol uptake and generation ofretinoic acid, working either directly as a receptor or by assisting proteins.

No description available.