Copper transporters in development

Abstract

Copper is an essential trace element serving as a cofactor for critical enzymes. Copper uptake occurs via two solute carriers; Copper transporter 1 (Ctr1) and Copper transporter 2 (Ctr2). The high affinity transporter, Ctr1, is crucial for embryonic development. Ctr1 null-mutant mice die in utero from a gastrulation defect. This inability to generate mesoderm has been phenocopied in in vitro differentiation cultures of Ctr1-/- embryonic stem (ES) cells. However, the role of copper transporters during development remains unknown. In this thesis we aim to elucidate the roles of Ctr1 and Ctr2 during murine development. In sperm, Ctr2 was found expressed on the acrosomal cap, whilst Ctr1 was expressed in the cytoplasmic droplet. In the preimplantation embryo, Ctr1+ particles coalesced during cleavage stages, whilst Ctr2 colocalised with Keratin-8+ trophectoderm on the surface of the morula and blastocyst. The effect of copper on development of the preimplantation embryo cultured in vitro. Addition of copper sulfate proved lethal at concentrations greater that 50 μM. Copper chelation with tetrathiomolybdate resulted developmental arrest at concentrations greater than 1 μM. To assess copper transport to the post-implantation embryo we assessed the extra-embryonic tissues, the yolk sac and the placenta. The placenta was the primary site of copper transport with Ctr2 expressed on the maternal and foetal vessels between E10.5 – E16.5, while Ctr1 was expressed at E14.5. In the yolk sac Ctr2 was expressed in the visceral endoderm from E8.5 – E18.5. We next assessed the role of Ctr1 in germ layer specification utilising in vitro differentiation of Ctr1-null embryonic stem (ES) cells. During neurectoderm differentiation, loss of Ctr1 resulted in a lineage bias towards surface ectoderm. Absence of Ctr1 did not affect the ability of Ctr1-/- ES cells to generate CXCR4+c-Kit+ definitive endoderm or PECAM- PDGRFα+ extra embryonic endoderm relative to Ctr1+/+ ES cells. In addition, the mitochondrial reactive oxygen species probe, NpFR2, revealed that decreased mitochondrial activity in the absence of Ctr1 inhibits mesoderm formation. These findings better elucidate expression of copper transporters, Ctr1 and Ctr2, in delivery mechanisms of copper to the developing embryo as well as characterise the role of Ctr1 during germ layer specification.