Generation and characterization of dickkopf3 mutant mice

Abstract

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

FIG. 1.

Generation of dkk3 mutant mice. (A) Schematic diagram of the dkk3 locus and targeting construct. The construct contains 4 kb of the 5′and 3′ dkk3 genomic sequence. A lacZ reporter gene followed by a floxed PGKNEO (NEO) selection marker replaces most of the coding sequence in exon 2. A counterselection cassette encoding the A subunit of diphtheria toxin (DTA) was inserted at the 5′ end of the vector. The wild-type band expected from a BamHI digestion is 15 kb. (B) Schematic diagram of the dkk3 targeted allele. After homologous recombination in ES cells, the lacZ reporter gene is maintained in frame with the initial ATG. The probes and restriction enzymes are indicated, with the size of the resulting recombined restriction fragments. (C) Southern blot analysis after digestion with BamHI and hybridization with external 5′ and 3′ probes and internal NEO probe of two positive independent ES clones. (D) Triplex PCR of mouse genomic DNA from dkk3^−/− mice (−/−), wild-type mice (+/+), and heterozygous mice (+/−). A single 199-bp fragment appears in the case of dkk3^−/− mice, a single 220-bp fragment appears in the case of wild-type mice, and both fragments appear in the case of heterozygous mice. Abbreviations: H, HindIII; B, BamHI; M, marker.

FIG. 2.

Comparative regional expression of dkk3 and p29 mRNA in wild-type and dkk3 mutant mice. Three representative autoradiographs of coronal sections organized from anterior to posterior levels are shown. Cx, neocortex; OE, olfactory epithelium; Pir, piriform cortex; VI, layer of the neocortex; Cg, cingulate cortex; LV, lateral ventricle; SFO, subfornical organ; Rc, retrosplenial cortex; CA, Ammon's horn of the hippocampus; BL, basolateral amygdaloid nucleus; 3V, third ventricle; SCO, subcommissural organ. The arrows (A and I) point to the meninges. Scale bar, 150 μm.

FIG. 3.

D2 expression in wild-type and dkk3 mutant mice. (A to L) Regional expression of D2 mRNA in wild-type (A to F) and mutant (G to L) mice. Representative coronal sections are organized from anterior to posterior levels. The cerebella were studied in sagittal sections. Pir, piriform cortex; IV, layer of the neocortex; SBF, barrel field of the somatosensory cortex; Rc, retrosplenial cortex; DGmo, molecular layer of the dentate gyrus; VPM, ventral posteromedial thalamic nucleus; 3V, third ventricle; CPu, caudate-putamen; LSD, lateral septal nucleus, dorsal part. Scale bar, 150 μm. (M and N) Micrographs showing the localization of D2 mRNA in the tanycytes of the third ventricle of wild-type (M) and mutant (N) mice. Arrows point to black silver grains. Scale bar, 50 μm.