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Mol Cell Biol. Mar 2006; 26(6): 2317–2326.
PMCID: PMC1430294

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.

The Dickkopf family of secreted proteins consists of four members, which share two conserved cysteine-rich domains (12, 24). The hallmark of Dkk proteins is that they function as Wnt antagonists or agonists by binding to and inhibiting or activating the Wnt coreceptor LRP6 (1, 31, 45). They show regionalized expression during vertebrate embryogenesis (5, 10, 13, 18, 20, 33, 46). Dkk1 is the best-characterized member of the family. It acts as an embryonic head inducer, and when overexpressed it will induce extra heads in Xenopus and zebra fish (6, 12, 18, 22, 36, 46). dkk1 mutant mice are embryonic lethal, and embryos lack anterior head structure and display fused digits (36). dkk2 mouse mutants are viable but show bone defects (28). Little is known about the biological role of dkk4.

By a number of criteria, dkk3 appears as a divergent member of the dkk family. (i) By DNA sequence similarity, vertebrate dkk1, -2, and -4 are more related to each other than they are to dkk3 (12). (ii) Hydra has two dkk genes, one related to vertebrate dkk1, -2, and -4 (16) and one related to vertebrate dkk3 (9). This suggests an ancient phylogenetic separation between these family members, where dkk1, -2, and -4 but not dkk3 arose by gene duplication from an ancestral dkk (16). (iii) Soggy is a protein of unknown function with sequence similarity to dkk3 but not to other dkk genes (24). The similarity is most pronounced outside the two conserved Dkk cysteine-rich domains, raising the possibility that the gene arose from an ancestral dkk3 precursor. (iv) Unlike Dkk1, -2, and -4, Dkk3 does not act as a Wnt modulator (24, 29, 55). While all other tested Dkk proteins bind to and modulate the Wnt receptor LRP6, as well as the Dkk coreceptor Kremen, Dkk3 has no affinity to these transmembrane proteins (7, 30, 32, 33), and no other proteins are known to interact with it.

Like other dkk members, dkk3 is expressed during vertebrate development in suggestive patterns in many organs (7, 33). Prominent expression of dkk3 is observed in the brain and in fibroblasts of adult rodents (17, 24, 34, 37, 56) and in the human adrenal cortex (50). Dkk3 has been proposed to act as a tumor suppressor, as it is downregulated in a number of tumor cells and since dkk3 overexpression suppresses cell growth (19, 25, 37, 52, 53). Hence, dkk3 is also known as REIC (for reduced expression in immortalized cells) (52). While hypermethylation of human dkk3 correlates with certain cancers (23, 43), the physiological relevance of altered dkk3 expression in tumors and its potential growth inhibitory effect are unknown.

A cDNA encoding an N-terminally truncated Dkk3 lacking the signal peptide was cloned and characterized as a presumed substrate binding subunit, “p29,” of the type II iodothyronine 5′-deiodinase (D2) in rat (26). The evidence for a role for p29 in thyroid hormone metabolism rests on the findings that p29 can be cross-linked to a thyroid hormone affinity label and that transfection of p29, directly or indirectly, enhances D2 activity in cultured astrocytes (26). Deiodinases play an important role in the local availability of brain, brown adipose tissue (BAT), and pituitary 3,5,3′-triiodothyronine (T3), which is converted from thyroxine (T4) by deiodination (2). This is different from other organs, which derive their T3 directly from plasma. All deiodinases (D1, D2, and D3) thus far characterized are selenoproteins that catalyze the removal of iodine atoms from iodoamino acids (4). The claim that an N-terminally truncated rat Dkk3 (p29) may be involved in D2 activity is controversial because (i) of the seleno nature of all other cloned deiodinases that act without substrate binding subunits and (ii) there is poor correlation between dkk3/p29 and the D2 expression patterns in rat brain (34).

In summary, despite numerous studies of dkk3, its biological role and biochemical function remain largely elusive. We have therefore generated dkk3 mutant mice by targeted disruption of the gene. Here, we present a first phenotypic characterization of these mice. Our data indicate that the gene is not essential for embryogenesis and viability, and the data do not support a role for Dkk3 in thyroid hormone metabolism. Instead, initial phenotyping indicates altered phenotypes in hematological and immunology parameters, lung ventilation, and behavior in dkk3 mutant mice.

MATERIALS AND METHODS

Generation of dkk3 mutant mice.

The targeting vector was derived from a 129/SVJ bacterial artificial chromosome clone that includes exon 2 from the dkk3 gene. The construct, which replaced most of exon 2, consisted of an in-frame-cloned lacZ cassette, followed by a loxP-flanked neomycin resistance (neo) cassette, a 4-kb 5′ homology arm, and a 4.5-kb 3′ homology arm. The A subunit of diphtheria toxin was used as a counterselection marker (Fig. (Fig.1A).1A). 129/SVJ embryonic stem (ES) cells were electroporated, and correct recombination events were verified by Southern blot analysis of genomic DNA digested with BamHI, using both internal and external probes (Fig. 1B and C). Injection of two independent positives clones generated chimeras, which transmitted the recombinant locus. No difference was evident between the two lines, and we used one for further study. Genotyping of dkk3−/− mice was performed by a triplex PCR on genomic tail DNA using oligonucleotides p1 (5′-GATAGCTTTCCGGGACACAC-3′), p2 (5′-TCCATCAGCTCCTCCACCTCT-3′), and p3 (5′-TAAGTTGGGTAACGCCAGGGT-3′) (Fig. 1A and B) to produce 220-bp and 199-bp bands from the wild-type (WT) and targeted allele, respectively (Fig. (Fig.1D).1D). dkk3 mutant mice were maintained in a C57BL/6 background. A group of 60 Dkk3 knockout animals, 30 males and 30 females, were observed during 12 months and compared to wild-type animals. No increase in mortality and no spontaneous tumor formation were observed with the Dkk3 knockout mice.

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 ...

German Mouse Clinic (GMC) screen.

General set up of the screen, husbandry, and multiparameter analysis were as previously described (11) and will be described in detail elsewhere.

Behavior screen.

Mice were analyzed with the behavior screen at the age of 9 weeks (after 2 weeks of acclimatization in the module). Three days before being tested, an object (a metal cube) was placed into the home cage and removed 1 day before testing. The modified hole board test was carried out by standardized procedures as described previously (40, 41). For each trial (5-min observation time), an unfamiliar object (a blue plastic tube lid, similar in size to the metal cube) and the familiar object (a metal cube) were placed into the test arena with a distance of 2 cm between them. Manually recorded behavioral data were analyzed using Observer 4.1 software (Noldus, Wageningen, The Netherlands), the animal's track was videotaped, and its locomotor path was analyzed with a video tracking system (Ethovision 2.3, Noldus, Wageningen, The Netherlands). Data were statistically analyzed with SPSS software (SPSS, Inc., Chicago, Ill.). The chosen level of significance was a P value of <0.05.

Clinical-chemical and hematological screen.

For the hematological investigations, 50 μl of blood per 12-week-old mouse was collected in an EDTA-coated tube (catalogue no. 078035; KABE) by puncturing the retro-orbital sinus with a nonheparinized glass capillary (0.8 mm in diameter, catalogue no. 1.28.13.1.2; Laborteam K&K, Munich, Germany) under ether anesthesia. The sample was immediately inverted several times to ensure a homogenous mixture of blood and anticoagulant and used for automated analysis with a blood analyzer, which was carefully validated for the analysis of mouse blood (ABC-Blutbild analyzer; Scil Animal Care Company GmbH, Viernheim, Germany). The influence of gender and genotype were tested by applying a two-way analysis of variance (variance equal) or general linear model (variance unequal). In the case of a significant influence of genotype on the parameter investigated, the significance of the mean differences within each sex was evaluated by the Student t test.

Lung function screen.

When mice were 14 weeks old, whole-body plethysmography (8) was applied to measure spontaneous breathing patterns of unrestrained animals (51) at different levels of activity. Automated data analysis provided tidal volumes (TV), respiratory rates (f), minute ventilation (MV), inspiratory and expiratory times (Ti, Te), and peak inspiratory and peak expiratory flow rates (PIF and PEF) at 10-s intervals. Mean inspiratory and expiratory flow rates (MEF and MIF) were calculated from the ratio of tidal volume and the respective time interval. The fraction of inspiration (Ti/TT) was determined from the ratio of inspiratory time (Ti) to total time required for the breathing cycle (TT). Specific tidal volumes and minute ventilations (sTV and sMV) were calculated by relating the absolute values to the body weight of the animal. Breathing was analyzed for the above-mentioned parameters during phases of activity and rest. Statistical analyses were performed with a commercially available statistics package (Statgraphics; Statistical Graphics Corporation, Rockville, MD). Differences between strains were evaluated by Student's t test. Statistical significance was assumed at a P value of <0.05. Data are presented as mean values ± standard error of the mean (SEM).

Immunology screen.

Blood samples were taken from 12-week-old mice. Peripheral blood leukocytes were isolated from 500 μl blood by erythrocyte lysis with NH4Cl (0.17 M)-Tris buffer (pH 7.45) directly in 96-well microtiter plates. After subsequent washing with fluorescence-activated cell sorter staining buffer (phosphate-buffered saline [PBS], 0.5% bovine serum albumin, 0.02% sodium azide, pH 7.45), peripheral blood leukocytes were incubated for 20 min with 1 μM ethidium monazide bromide (Molecular Probes, The Netherlands) and Fc block (clone 2.4G2; PharMingen, San Diego, Calif.). Ethidium monazide bromide bound to the DNA of dead cells was photo-cross-linked by brief light exposure. Cells were then stained with fluorescence-conjugated monoclonal antibodies (PharMingen). The following main cell populations were analyzed: B cells (CD19+ clone 1D3), B1 B cells (CD19+ CD5+; clone 53-7.3), B2 B cells (CD19+ CD5), T cells (CD3+; clone 145-2C11), CD4+ T cells (clone RM4-5), CD8+ T cells (CD8β; clone H35-17.2), γ/δ T cells (clone GL3), granulocytes (Gr-1+; clone RB6-8C5), and NK cells (CD49b+; clone DX5). Data were acquired on a FACScalibur (Becton Dickinson, San Diego, Calif.) and were analyzed using FlowJo software (TreeStar Inc.). All samples were acquired until a total number of 25,000 cells were reached. The plasma levels of immunoglobulin M (IgM), IgG1, IgG2a, IgG2b, IgG3, and IgA were determined by standard sandwich enzyme-linked immunosorbent assays using goat anti-mouse immunoglobulin antibodies and alkaline phosphatase conjugates (SouthernBiotech, Birmingham, Ala.).

Animals and treatments for D2 studies.

C57BL/6 animals were kept under temperature-controlled (22 ± 2°C) and light-controlled (12-h light and 12-h dark cycle; lights on at 0700 h) conditions and had free access to food and water. Animal care procedures were conducted in accordance with the guidelines set by European Community Council Directives (86/609/EEC).

For in situ hybridization studies, 11-day-old (P11) male wild-type and dkk3 mutant mice were used. This postnatal day was chosen because it is comparable to the age at which the highest levels of D2 activity and expression are found in rat brain (15, 21). For the determinations of deiodinase activities and thyroid hormones levels, four months-old mice were used from both wild-type and dkk3 mutant mice. As we observed sex-related differences in these determinations with several tissues, groups of males and females of both groups were used (7 to 12 mice per group).

cRNA probes.

A dkk3-specific probe spanning nucleotides 287 to 640 (404 bp, encompassing exons 2 to 6) from the mouse cDNA sequence (NM_015814) (33) was used. A specific rat p29 probe (nucleotides 1172 to 1499, 328 bp; AF245040) and specific D2 probe (nucleotides 535 to 901, 366 bp; U53505) were used as previously described (15, 34). The sense and antisense riboprobes for in situ hybridization were synthesized with suitable RNA polymerase (SP6 or T7) in the presence of 35S-labeled UTP (NEN Life Science Products) by in vitro transcription.

In situ hybridization histochemistry.

Mice were fixed by transcardial perfusion with 4% paraformaldehyde-0.1 M phosphate buffer, pH 7.4. Cryoprotected brains were frozen in dry ice, and 25-μm-thick sections were obtained with a cryostat.

The detection of p29, D2, and dkk-3 mRNAs with 35S-labeled riboprobes was performed with free-floating sections according to protocols previously described in detail (3). Due to the floating procedure, there are structures such as the leptomeninges and choroid plexus that were not well preserved in all sections. Briefly, the sections were pretreated in different solutions for 10 min at room temperature; each section was permeabilized with 0.05% Triton X-100 in PBS, deproteinized with 0.2 N HCl, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0), postfixed in fixative, and washed in PBS. Hybridization was performed at 55°C for 16 h, with the 35S-labeled riboprobe at 1.6 × 107 cpm/ml. The sections were mounted on coated slides, dehydrated, air dried, and exposed to Biomax MR film (Eastman Kodak, Rochester, NY) for 10 days for dkk3 and p29 expression analysis and for 2 weeks for D2 studies. For cellular resolution, the sections were dipped in Hypercoat LM-1 photographic emulsion (Amersham Pharmacia Biotech), exposed for 20 to 40 days in the cold, developed, fixed, counterstained with Richardson's blue, and dehydrated, and coverslips were applied.

The autoradiographic films were scanned with a Coolscan II slide scanner (Nikon Corp., Tokyo, Japan) at a resolution of 400 pixels/inch and printed. Optical observations and photographs were carried otu with an Eclipse E400 microscope and Dn100 digital camera (both, Nikon Corp., Tokyo, Japan). For the identification of brain structures, the atlas of Paxinos and Franklin (42) was followed.

Deiodinase activities.

Tissues were homogenized in buffer (0.32 M sucrose, 10 mM HEPES, 10 mM dithiothreitol [DTT; 1 mM DTT for liver and kidney], pH 7.0). Before each assay, 125I-labeled T4 or 125I-labeled reverse T3 (rT3) was purified from the iodide traces. The amount of iodide in the blanks was <0.5 to 1% of the total radioactivity. D2 activity was determined for tissue homogenates as previously described (39) using 125I-labeled T4, 2 nM T4, 1 μM T3, 20 mM DTT, and 1 mM propylthiouracil. Results are expressed in femtomoles per hour per milligram of protein. Detection limits were 2 to 3 fmols/h · mg of protein. D2 activity was also assayed in the neocortex and cerebellum with 125I-labeled rT3 as a substrate (2 nM); similar results were obtained (not shown). D1 activity in liver and kidney homogenates was assayed (44) using 125I-labeled rT3, 400 nM rT3, and 2 mM DTT. Results are expressed in picomoles per hour per milligram of protein. Pituitary D1 and D2 activity was assayed with 125I-labeled rT3, 2 nM rT3, and 20 mM DTT in the presence or absence of 1 mM propylthiouracil (for D2 and total deiodinase activity, respectively). Results are given in femtomoles per hour per milligram of protein.

T4 and T3 determination in tissues.

Thyroid hormones were determined by radioimmunoassays (RIAs) after extraction and purification of plasma and tissues (35, 38). Frozen tissue samples were homogenized in methanol with tracer amounts of 131I-labeled T4 and 125I-labeled T3, added to each homogenate. The iodothyronines were extracted by chloroform-methanol (2:1), back extracted into an aqueous phase, and purified through Bio-Rad AG 1X2 resin columns. The purified iodothyronines were then evaporated to dryness and dissolved in RIA buffer. Each extract was counted to determine the recovery of the 131I-labeled T4 and 125I-labeled T3 in each sample. The samples are submitted to RIAs for the determination of T4 and T3, the limits of sensitivity being 2.5 pg T4 and 1.5 pg T3. Each sample was processed in duplicate at 2 dilutions. Concentrations were calculated using the amounts of T4 and T3 found in the RIAs, the individual recovery of the 131I-labeled T4 and 125I-labeled T3 added to each sample, and the weight of the tissue sample extracted.

High-specific-activity 131I-labeled T4, 125I-labeled T3, 125I-labeled T4, and 125I-labeled rT3 (3,000 μCi/μg) were synthesized in our laboratory as previously described (35) and used for the highly sensitive T4 and T3 RIAs as recovery tracers for plasma and tissue extractions and as substrates for D1 and D2 deiodinases.

Data are presented as mean values (±SEM). One-way analysis of variance was applied after ensuring homogeneity of variance by Bartlett's test. Statistically significant differences between mean values of different groups were then identified by the least-significant-difference method. All calculations were performed as previously described (49).

RESULTS AND DISCUSSION

Generation of dkk3 mutant mice.

A targeting vector was designed to constitutively disrupt dkk3 exon 2, containing the signal sequence, by insertion of a lacZ neo cassette (Fig. (Fig.1).1). A homologous recombinant ES clone was generated and used to derive chimeras which gave rise to dkk3+/ pups. Intercrossing of these heterozygous mice produced homozygous dkk3−/−offspring, which were viable and fertile and obtained at the expected Mendelian ratio. dkk3−/−embryos and adults were examined by X-galactosidase staining but failed to produce any staining. Likewise, in situ hybridization for dkk3 confirmed downregulation of expression in mutant mice (see below). dkk3−/− mice had normal size and body weight and overall were in good general health (well-groomed coat and normal body posture and righting reflex; not shown). A gross neurological examination of animals revealed no sign of modified sensory functions, as assessed by basic tests of vision, audition, olfaction, and touch sensitivity. No enhanced tumorigenesis or major reduction in life span was observed.

Phenotypic analysis in the GMC.

dkk3−/− mice were subjected to phenotypic analysis in the GMC, an open access platform for standardized phenotyping (11). Overall, several hundred parameters, including analysis of morphology, behavior, neurology, vision and eye, clinical-chemical parameters, immunological status, nociception, lung function, and organ pathology were assessed. Four main significant differences between dkk3 mutant mice and their wild-type littermates were observed, as described below.

Behavioral phenotype.

A locomotor activity-related phenotype was observed with dkk3-deficient female mice (Table (Table1).1). dkk3−/− females were hyperactive, which was reflected by an increased number of line crossings, increased distance moved, and an increased number of turns made within the observation period. Corresponding effects in males just missed statistical significance. dkk3−/− females also reached a higher maximum velocity while exploring the arena, and additionally there was a trend toward increased mean velocity. This hyperactivity was also reflected in trends toward increased hole exploration, increased rearing on the board, and reduced grooming. There were no specific effects on anxiety-related (board entry) behavior.

TABLE 1.
Results of GMC behavior screena

Red blood cell phenotype.

Red blood cells of dkk3−/− mice showed a higher mean corpuscular volume and mean corpuscular hemoglobin (MCH) content but a reduced MCH concentration, compared to their littermate controls, resulting in an elevated hematocrit and total hemoglobin concentration in blood (Table (Table22).

TABLE 2.
Results of the GMC hematological screena

Lung phenotype.

While control and mutant mice breathed at similar respiratory rates, tidal volumes in mutants were smaller than those in control animals, both at rest and during activity (Table (Table3).3). As a result, minute ventilation was about 20% lower in mutant mice. Differences were less pronounced when body weight-specific ventilation was considered.

TABLE 3.
Results of GMC lung function screena

Immunological phenotype.

dkk3−/− mice showed a two-times-higher IgM level than littermate controls. We also detected a slight, but statistically significant increase in the frequency of natural killer (NK) cells (CD49b+) in dkk3−/− females. No significant differences were found with regard to the other cell subsets and immunoglobulins included in the screen (Table (Table44).

TABLE 4.
Results of GMC immunology screena

Expression of dkk3 and p29 in wild-type and dkk3 mutant mice.

A truncated rat Dkk3 (p29) was implicated as the thyroid hormone binding subunit of D2. To analyze the possibility that p29 and dkk3 display distinct expression patterns, e.g., due to alternative promoters, and that p29 expression may be unaffected in dkk3 mutants, we first compared the expression pattern of a mouse dkk3 5′ probe with the expression pattern representing bona fide rat p29. Rat p29 has 87% similarity to mouse dkk3 at the nucleotide level, and a p29 probe can be used for in situ hybridization in mice (34). In wild-type animals, the distribution of the hybridization signal in brain slices obtained with the p29- and dkk3-specific probes was almost identical, although the p29 probe gave weaker signals (Table (Table5),5), similar to the pattern of the same p29 probe in rat brain (34). The most prominent signals for dkk3 and p29 were observed in the Ammon's horn of the hippocampus; cingulate, retrosplenial, and piriform cortices; and layer VI of the somatosensorial cortex (Fig. 2A to C and G to I). There was also considerable expression in the internal granular layer and Purkinje cell layer of the cerebellum (not shown) and in the olfactory epithelium (Fig. 2A and G). In addition to expression in neuronal cells, dkk3 was expressed in epithelial cells lining the lateral and third ventricles with a high hybridization signal in tanycytes (Table (Table5).5). Other important sites for communication with the cerebrospinal fluid (CSF) such as the choroid plexus and leptomeninges of the blood-cerebrospinal fluid barrier and the circumventricular organs also showed dkk3 expression. Especially high levels of dkk3 expression were found in the subfornical and subcommissural organs (Fig. 2B and I).

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 ...
TABLE 5.
dkk3 and p29 mRNA levels in mouse brain at postnatal day 11a

In dkk3 mutants, hybridization signals for dkk3 and p29 were very weak (Fig. 2D to F and J to L) and limited to the regions of the cerebral cortex, which also had the highest hybridization signal in wild-type mice (Fig. 2D to F).

Taken together, the common expression pattern of mouse dkk3 and rat p29,as well as their common downregulation in dkk3 mutants, supports the notion that dkk3 and p29 represent the same gene under a common promoter, both of which are inactivated in dkk3 mutants.

Normal D2 expression and activity in dkk3 mutants.

Expression and activity of D2 in brain are homeostatically regulated by thyroid hormone status such that they increase in hypothyroidism and decrease in hyperthyroidism to maintain normal T3 concentrations (14, 27, 54). Therefore, we tested the possibility that D2 expression was altered in dkk3 mutant mice. The distribution of D2 mRNA in the brains of wild-type mice was similar to that in dkk3 mutants (Fig. 3A to L) and to that previously reported for rats (15). The hybridization signal was mainly localized in tanycytes lining the walls of the third ventricle and astrocytes in several brain regions (Fig. (Fig.3).3). There was only a slightly increased expression in the retrosplenial cortex and tanycytes of the third ventricle in dkk3−/− mice (Fig. 3J and K).

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 ...

To extend the results described above, D2 enzyme activity in different brain regions, in BAT, and pituitaries was determined. If dkk3 was required for D2 deiodinase activity, a decrease in D2 enzymatic activity in dkk3 mutants may be expected.

The results are summarized in Table Table6.6. Males and females were analyzed separately, as sex-related differences were observed in some tissues. There were no differences in D2 activity between wild-type and dkk3 mutant mice, except for a 50% decrease in the neocortex of dkk3−/− females and a 50% increase in the cerebellum of dkk3−/− males. No differences in D2 activity were observed in pituitaries. We also analyzed D1 activity, as it may compensate for loss of D2 activity, and generally found no major changes in dkk3 mutants (Table (Table6).6). Only in mutant males was a decrease in D1 activity observed, in pituitary (by 40%) and liver (by 25%). We conclude that D1 and D2 activities are unaffected in most tissues in dkk3 mutant mice.

TABLE 6.
D2 and D1 activities in several organs of adult WT and dkk3−/− micea

dkk3 mutants are euthyroid.

If a truncated rat Dkk3 (p29) was implicated as the thyroid hormone binding subunit of D2 enzyme, production of T3 should decrease in dkk3 mutant animals in those tissues in which T3 levels are dependent on the local T3 production via D2, such as brain and BAT (47, 48).

Therefore, T4 and T3 levels were measured in several tissues and in plasma of wild-type and dkk3 mutant mice (Table (Table7).7). No changes were observed in most tissues studied. T3 was decreased in the kidney of dkk3 mutant males (by 22%). T4 was elevated by 30% in the neocortex of dkk3 mutant females and T3 (by 28%) in the liver of dkk3 mutant females. These isolated changes in T4 and T3 concentrations are unlikely to be direct consequences of dkk3 deletion. We conclude that no major changes in T3 concentrations are found and that most tissues of dkk3 mutant mice remain euthyroid.

TABLE 7.
T4 and T3 concentrations in plasma and several organs of adult WT and dkk3−/− micea

Conclusions.

Our data indicate that dkk3 is not essential for embryogenesis and viability, and they neither support a role in thyroid hormone metabolism nor indicate that p29 is a naturally occurring variant of physiological relevance.

Initial phenotyping reveals abnormalities in hematological and immunological parameters, lung ventilation, and behavior. In particular, the observed hyperactivity phenotype may be correlated with the expression of dkk3 in dopaminergic neurons, abnormalities of which have been associated with alterations in locomotor activity. Recombinant Dkk3 promotes differentiation of dopaminergic neurons from undifferentiated precursors (E. Arenas, unpublished results), also hinting at a role for dkk3 in this process.

Due to their distinct functions, a compensation of dkk3 deficiency by other dkk genes appears unlikely; indeed, dkk1/dkk3 double-mutant mice do not show any synthetic phenotypes (S. Pinho and C. Niehrs, unpublished data). However, combined inactivation of dkk3 and the unique dkk3-related gene soggy (24) may reveal roles masked by redundancy.

Acknowledgments

We thank Sonia Pinho, Dana Hoppe, Socorro Duran, Maria Jesus Presas, Elena Fernandez-Duran, Maria Asuncion Navarro, and Marina Sanz-Sancristobal for excellent technical assistance and the screeners of the German Mouse Clinic for phenotypical analysis of the mice.

This work was supported by the DFG (SFB 488 A1), grants SAF2001-2243 and FIS RCMN 03/08 (M.-J.O.), BFI2001-2412 and BFU2004-05944 (A.G-F.), NGFN 01GR0430, and 01GR0434, 01GR0458, and 01GR0103 (GMC).

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