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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:485114 |
| Date | January 2006 |
| Creators | Lee, Ggot-Im |
| Publisher | University of Sheffield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0012 seconds