Hormonal Functions of Cthrc1 Figure 4. Fatty liver phenotype in C57BL/6J Cthrc1 mice. (A) Cell density in livers of wild type and Cthrc1 mice (n = 4 per group). (B) Liver to body weight ratio was similar among groups. (C) Lipid content in liver was determined by image analysis on oil red O stained sectins of 7 month old mixed gender Cthrc1 and wild type mice (n = 6 per group). (D) Oil red O stain of a liver section from a Cthrc1 mouse shows macrovesicular and microvesicular steatosis and (E) only minor lipid accumulations in a wild type mouse. (F) Hematoxylin & eosin stain of a liver section from a Cthrc1 mouse and (G) a wild type mouse. doi:10.1371/journal.pone.0047142.g004 In wild-type mice older than six months of age, we occasionally observed small accumulations of Cthrc1 immunoreactivity in the anterior pituitary (Fig. 6D). In all pig pituitaries that we obtained, Cthrc1 immunoreactivity was variable in extent, but was consistently localized in the anterior lobe (Fig. 6E, G). Some of the Cthrc1 was found inside the cytoplasm of cells suggesting synthesis by those cells (Fig. 6G). Often Cthrc1 was observed in the Figure 5. Cthrc1 expression in the pig pituitary but absence in adult mouse liver and skeletal muscle. RT-PCR with Cthrc1 specific primer pair was performed on the indicated mouse and pig RNA samples. doi:10.1371/journal.pone.0047142.g005 lumen of colloid-filled follicles that were lined by folliculostellate cells (Fig. 6H–I). Preincubation of the antibody with the peptide antigen completely abolished the immunoreactivity, demonstrat- ing specificity of the antibody (Fig. 6F, I). Colloid-filled follicles of the anterior pituitary, increasing in number and size with age, have been reported in numerous vertebrates including humans [5,6]. Colloid material has also been reported in the pituitary cleft with adenohypophysial canaliculi connecting to it [8]. Cthrc1 accumulations were found close to the cleft (Fig. 6J) and also inside the pituitary cleft (Fig. 6K), often with small canaliculi carrying Cthrc1 connecting to the cleft (Fig. 6L). The significance of pituitary colloid follicles has yet to be determined, as none of the known pituitary hormones have been localized to them. Here we demonstrate that these follicles store Cthrc1. As shown above, certain areas of the brain constitutively express Cthrc1 but we currently have no evidence that Cthrc1 from those sites enters the circulation. In brains from pigs, we found foci of paraventricular cells of the lateral ventricles that expressed Cthrc1 (Fig. 6M) and these sites are known to harbor neuronal stem cells. PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47142