Hormonal Functions of Cthrc1 Figure 2. Characterization of rabbit monoclonal anti-Cthrc1 (Vli-55) antibody. (A) Western blot analysis of plasma from Cthrc1 transgenic mice (TG) and their negative wild type littermates (WT). (B) Cthrc1 immunohistochemistry shows expression in adventitial cells of the remodeling renal artery following angiotensin II infusion, (C) dermal cells in 7 day old skin wounds, and (D) embryonic cartilage (E17.5). (E) Cthrc1 expression is restricted to the midbrain in wild type 129S6/SvEv mice and (F) no expression is detected in the midbrain of a corresponding Cthrc1 mouse. doi:10.1371/journal.pone.0047142.g002 where it is normally expressed, such as bone and brain (Fig. 1C). Mice were backcrossed more than eleven generations to obtain the mutant allele on a 129S6/SvEv or C57BL/6J genetic background. Cthrc1 mice were viable and displayed no obvious de- velopmental abnormalities. To verify that our Cthrc1 allele is a mutation, we generated and characterized anti-Cthrc1 antibodies. We succeeded in developing highly sensitive and specific rabbit monoclonal antibodies against the conserved C terminus of Cthrc1. These antibodies allowed us to detect Cthrc1 by immunoblotting and in formalin fixed, paraffin embedded tissue sections by immunohis- tochemistry (Fig. 2). The average litter size was slightly lower for Cthrc1 mice, however, this did not reach a statistically significant level (Fig. 3A). Growth curves were established for Cthrc1 mice on the 129S6/SvEv background and they were similar for Cthrc1 mice and wild type mice (Fig. 3B, C). The percentage of fat of total body weight was measured in eight week old female mice by dual energy x-ray absorptiometry, and this analysis detected no differences between Cthrc1 and wild type mice (Fig. 3D, E). In addition, blood triglyceride levels were also similar comparing Cthrc1 and wild type mice (Fig. 3F). Metabolic Alterations in Adult Cthrc1 Null Mice Liver histology with staining of sections for carbohydrates with the Periodic Acid Schiff (PAS) stain suggested increased carbohy- drate storage in hepatocytes of Cthrc1 mice on the 129S6/ SvEv background. Quantification of the glycogen content in liver tissue with biochemical methods confirmed increased glycogen content in livers from Cthrc1 mice (Fig. 3G). Measurements of glycogen content in skeletal muscle also consistently showed elevated levels of glycogen in Cthrc1 mice compared to wild types (Fig. 3H). A glucose tolerance test revealed normal glucose handling with similar fasting blood glucose levels in Cthrc1 and wild type mice (Fig. 3I). However, insulin sensitivity was slightly reduced in the mutants with significantly higher blood glucose levels at 20 minutes after insulin administration (Fig. 3J). Routine histology of livers from seven month old Cthrc1 mice on the C57BL/6J background revealed strikingly larger hepatocytes compared to wild type controls. Overall this resulted in a significantly lower cell density (Fig. 4A). The liver to body weight ratio was similar for Cthrc1 mutants and wild type mice (Fig. 4B). We performed oil red O staining to determine whether lipid content was increased in livers from mutants and this revealed increased lipid storage in Cthrc1 mice resulting in macro- and micro-vesicular steatosis (Fig. 4C–G). The abnormal- ities observed in liver and skeletal muscle of Cthrc1 mice raised the question whether Cthrc1 was expressed in these organs. We were unable to detect Cthrc1 in normal adult livers and skeletal muscle at the protein level (data not shown) or by RT- PCR (Fig. 5). This led to the consideration whether Cthrc1 may be a circulating factor acting as a hormone. Expression of Cthrc1 in the Central Nervous System and Pituitary Gland We undertook a comprehensive immunoblot-based and immu- nohistochemistry-based survey of Cthrc1 expression in tissues from adult mice, rats and pigs. Expression of Cthrc1 in two month old C57BL/6J mice was detected in the supraoptic nucleus (son, Fig. 6A, B) and paraventricular nucleus (pvn, Fig. 6A, C) of the hypothalamus, both of which contain neurosecretory cells with axon terminals in the posterior pituitary. Serial sectioning of the entire midbrain region, however, did not indicate that any of Cthrc1 immunoreactivity of the hypothalamus was transported to the posterior pituitary for secretion (data not shown). We did not detect Cthrc1 expression anywhere in the pituitary of mice at this age nor did we find colloid-filled follicles in the anterior pituitary in mice at two months of age. Similarly, neurons of the supraoptic nucleus in pigs also expressed Cthrc1 but no Cthrc1 was found in the posterior pituitary (data not shown). PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47142