Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development

Abstract

The trace metal copper (Cu) plays an essential role in biology as a cofactor for many enzymes that include Cu, Zn superoxide dismutase, cytochrome oxidase, ceruloplasmin, lysyl oxidase, and dopamine beta-hydroxylase. Consequently, Cu transport at the cell surface and the delivery of Cu to intracellular compartments are critical events for a wide variety of biological processes. The components that orchestrate intracellular Cu trafficking and their roles in Cu homeostasis have been elucidated by the studies of model microorganisms and by the characterizations of molecular basis of Cu-related genetic diseases, including Menkes disease and Wilson disease. However, little is known about the mechanisms for Cu uptake at the plasma membrane and the consequences of defects in this process in mammals. Here, we show that the mouse Ctr1 gene encodes a component of the Cu transport machinery and that mice heterozygous for Ctr1 exhibit tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. Mice completely deficient for Ctr1 exhibit profound growth and developmental defects and die in utero in mid-gestation. These results demonstrate a crucial role for Cu acquisition through the Ctr1 transporter for mammalian Cu homeostasis and embryonic development.

Figure 1

Expression of mouse Ctr1 stimulates ⁶⁴Cu uptake. Human embryonic kidney cells (HEK293) were transfected with vector (●) or the mouse Ctr1 cDNA under control of the CMV promoter (■). Cu⁶⁴ (10 μM) was added to the culture medium. ⁶⁴Cu uptake was calculated at different times by using a standard curve and normalized to protein concentration of total cell lysates.

Figure 2

Targeted disruption of the mouse Ctr1 gene by homologous recombination. (A) A restriction map of the genomic locus encompassing the mouse Ctr1 coding exons is diagrammed (X, XbaI; EI, EcoRI; EV, EcoRV; S, SacI; B, BamBI). All Ctr1 coding exons were replaced by a neomycin (neo) gene expression cassette driven by the PGK promoter. The predicted locus derived from homologous recombination is shown. Hybridization probes “a” and “b” used to detect homologous recombinant allele by Southern blotting analysis are indicated. The DNA fragment sizes derived from the wild-type and recombinant Ctr1 locus, by cleavage with XbaI and BamHI, are indicated. Genomic DNA isolated from ES cells or mouse tail biopsies was genotyped by Southern blotting after restriction enzyme digestion with XbaI and hybridized with probe “a” (B) or by restriction enzyme digestion with BamHI and hybridized with probe “b” (C). (D) Primer sets specific to the Ctr1 and neo genes were used to screen Ctr1^+/+ and Ctr1^+/− mice by PCR analysis. Both Ctr1 and neo gene-specific DNA fragments were amplified from the genomic DNA isolated from Ctr1^+/− mice, but only a Ctr1-specific product was amplified from genomic DNA isolated from Ctr1^+/+ mice. (E) Ctr1 mRNA blot analysis from organs derived from wild-type and Ctr1^+/− mice. Gapdh mRNA levels were used as a loading control.

Figure 3

Brain cupro-enzyme activities in wild-type and Ctr1^+/− mice. Activities of cytochrome oxidase (Cox) (μmol/min × mg protein) and Cu, Zn SOD (units/mg protein) from whole brain homogenates were analyzed for 5–6-week-old wild-type (+/+) and Ctr1^+/− (+/−) littermates. Means of five to seven mice were compared by Student's t test. *, P < 0.01.

Figure 4

Morphological and histological analysis of littermate embryos from Ctr1^+/− intercrosses. (A) Morphology of Ctr1^+/+ and Ctr1^−/− embryos are compared. Embryos were isolated with yolk sac at E7.5, E8.5, E9.5, and E10.5 days postfertilization. Genotypes (+/+ or −/−) of Ctr1 alleles are indicated. (B and C) Embryos with decidua were dissected from the uterus at the indicated days postfertilization, sectioned, and stained with hematoxylin and eosin. (D) Four-fold magnification of the E9.5 Ctr1^−/− embryo in C for detail. A, amnionic cavity; Al, allantois; Ao, aorta; AS, amnionic sac; E, ectoplacental cavity; EC, ecocoelomic cavity; G, gut; H, heart; N, neuroepithelium; O, otic vesicle; S, somite; U, umbilical cord; YS, yolk sac; arrowhead in B, ectoderm and mesoderm layers; arrowhead in D, defective neural tube closure; MC, mesenchyme cells. (Bar = 250 μm.)