Cthrc1 gene is located adjacent to the Frizzled6 gene, which is a WNT binding receptor. The targeted replacement of the first exon of the Cthrc1 gene by a LacZ reporter gene in mice was reported to demonstrate expression of Cthrc1 in inner ear hair cells [2]. Abnormalities in inner ear development occurred when Cthrc1 mice were crossed with mice carrying one mutant allele of Vangl2, but these abnormalities were only observed when these compound mutants were on a specific mixed genetic background and not when the mutants were crossed with outbred mice [2]. With our antibodies we have been unable to detect expression of Cthrc1 in inner ear hair cells and auditory testing did not reveal any hearing abnormalities in our mutant mice (data not shown). The use of LacZ expression as a surrogate for endogenous Cthrc1 localization has obvious limitations when the endogenous protein is released into the circulation. Yamamoto et al. [2] performed most of their Cthrc1 protein interaction studies in HEK293T cells. Unlike Cthrc1 transfected CHO-K1 cells, which secrete Cthrc1 readily into the medium, Cthrc1 cannot be detected in the medium of transfected HEK293T cells (Fig. 10). This raises the question of suitability of the HEK293T cell line for studying Cthrc1 in vitro. Detection of Cthrc1 in plasma implies that it is secreted from cells. In the neurosecretory nuclei of the hypothalamus (Fig. 6A–C) and the paraventricular cells shown in Fig. 6M, we observed immunoreactivity with granular appearance restricted to the cytoplasm and axon of the cell. This is suggestive of Cthrc1 packaging in secretory granules, a characteristic of a regulated release mechanism that has been described for the release of neuropeptides from neuroendocrine cells [12]. A role for carboxypeptidase E (CPE) in the transport of peptide hormone- containing vesicles to the site of release has been demonstrated in this process [13]. An interaction of Cthrc1 with CPE still needs to be demonstrated. The presence of a C terminal lysine residue in the Cthrc1 sequence would be compatible with an interaction with CPE. We examined the entire midbrain region with the attached pituitary by serial sectioning and Cthrc1 immunohistochemistry both in mice and pigs but we were unable to obtain evidence for transport of Cthrc1 expressed in the hypothalamus along axons to the pituitary gland. CPE also cleaves off C terminal lysine or arginine residues and if this is the case for Cthrc1, it could affect the ability of the C terminal specific antibody (Vli-55) to detect Cthrc1 by immunohistochemistry. Thus, if the C terminal lysine gets cleaved during the transport of the protein along axons to the posterior pituitary we might be unable to detected it with the Vli- 55 antibody. Finding Cthrc1 in small vessels near sites of Cthrc1 expression (Fig. 6N) provides indirect evidence for the release of Cthrc1 into the circulation. Here we generated a novel Cthrc1 mutant mouse and focused on the characterization of its phenotype in adulthood. Unlike the two other reported targeted Cthrc1 mutants [2,3] we replaced 3 of the 4 exons with a neomycin cassette and confirmed that Cthrc1 mRNA was not expressed. Using both N terminal and C terminal specific antibodies no Cthrc1 protein was detectable, ruling out the possibility of a hypomorph phenotype. In agreement with the published Cthrc1 mutants, our Cthrc1 mice were also viable and showed no obvious developmental abnormalities. In summary, our study identifies Cthrc1 as a novel circulating hormone with metabolic effects. Expression in the hypothalamus, pituitary gland and remodeling tissues are likely to contribute to Cthrc1 plasma levels. While Cthrc1 expression was not detectable in the liver and skeletal muscle of the wild type, examination of these organs in our Cthrc1 mutant mice on the C57BL/6J background revealed excessively fatty livers and increased glycogen levels in skeletal muscle and livers of Cthrc1 mice on the 129S6/SvEv Hormonal Functions of Cthrc1 Figure 10. Cthrc1 secretion is cell type dependent. Detection of Cthrc1 in conditioned medium (CM) and cell lysate (CL) of CHO-K1 or HEK293T cells 72 h after transfection with a Cthrc1 expression vector. Note the absence of Cthrc1 in the CM of HEK293T cells. doi:10.1371/journal.pone.0047142.g010 background. This ultimately led us to consider the possibility that Cthrc1 functions as a hormone. We have currently no data related to the mechanism how Cthrc1 affects these organs but our findings do suggest that hepatocytes and myocytes in vivo may express a receptor for Cthrc1 and the binding of 125I-Cthrc1 to the liver supports this concept. The Cthrc1 mutant mice examined here were derived from matings of homozygous mice. Therefore, we cannot rule out the possibility that some of the metabolic abnormalities seen in the mutants were due to maternal influences. However, litter sizes of the wild type and the homozygous matings were comparable and we did not see any evidence of early postnatal failure to thrive on any genotype. With a sensitive monoclonal anti-Cthrc1 antibody suitable for detection by ELISA we succeeded in demonstrating the presence of Cthrc1 in plasma. Working with plasma we deliberately avoided the use of secondary anti-IgG antibodies because even minimal cross-reactivity with human immunoglobulins could make dis- crimination between Cthrc1 and the similarly migrating band of the immunoglobulin light chain difficult on a Western blot. As shown in Fig. 2A and Fig. 9, no non-specific bands are detected with HRP-conjugated monoclonal anti-Cthrc1 antibodies in plasma samples by Western blotting. Detection of Cthrc1 in plasma of Cthrc1 transgenic mice and a half-life of approximately 2.5 hours in circulation provide additional support for Cthrc1 as a circulating factor. Our magnetic bead-based pull-down assay was designed to provide a proof of principle and a double antibody sandwich ELISA obviously needs to be developed for a high throughput quantitative screening assay for Cthrc1 in plasma. Our PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47142