Cthrc1 immunohistochemistry of the brain and pituitary gland from mouse and pig.

<p>Sections of mouse (A–D) and pig (E–N) brain and pituitary were immunostained for Cthrc1 expression: (A) Low power view of a coronal section of the hypothalamus from a C57BL/6J mouse showing Cthrc1 immunoreactive cells in the paraventricular nucleus (pvn) and the supraoptic nucleus (son), ot = optic tract, v = third ventricle. (B) High power view of supra optic nucleus and (C) paraventricular nucleus. (D) Cthrc1 positive follicle in the anterior lobe of a 7 month old C57BL6/J male, (E) extensive accumulations of Cthrc1 in the anterior lobe of a pig pituitary, and (F) pre-absorption of antibody completely eliminates staining on an adjacent section. (G) Cytoplasmic localization of Cthrc1 in cells of the anterior lobe indicates expression. (H–I) are serial sections of a typical colloid-filled follicle of the anterior pituitary with (H) showing extensive immunoreactivity, (I) which is completely eliminated by pre-absorbing the antibody with peptide antigen (H). Also note the encapsulation of the follicle by folliculostellate cells. (J, K) Cthrc1 localization in the pituitary cleft (c) and (L) canaliculi connecting to the cleft. (M) An isolated area of cells in the paraventricular zone of the lateral ventricle expresses Cthrc1 (note granular appearance), and (N) nearby small vessels (arrows) contain Cthrc1. (O) Cthrc1 is expressed by some osteocytes (arrows) and osteoblasts in adult mouse bone. Scale bar = 50 µm.</p

Isolated cells in the rat pituitary express Cthrc1.

<p>(A) Cthrc1 immunohistochemistry on pituitary glands from three month old male Sprague Dawley rats identified Cthrc1 expression by isolated cells. Cytoplasmic immunoreactivity is clearly detectable in cells adjacent to extracellular accumulations of Cthrc1 (arrows), suggesting Cthrc1 synthesis by these cells. (B) Pre-absorption of antibody with peptide antigen completely eliminates staining on an adjacent section. Scale bar = 50 µm.</p

Fatty liver phenotype in C57BL/6J Cthrc1 null mice.

<p>(A) Cell density in livers of wild type and Cthrc1 null 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 null and wild type mice (n = 6 per group). (D) Oil red O stain of a liver section from a Cthrc1 null 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 null mouse and (G) a wild type mouse.</p

Normal growth but increased glycogen storage in liver and skeletal muscle of 129S6/SvEv Cthrc1 null mice.

<p>(A) Differences in litter size were not significantly different (n.s.). (B) Growth curves for females and (C) males were similar (n = 5–33 mice per time point). (D, E) Total body fat expressed as a percentage of body weight was similar in 8 week old female mice among mutants and wild type mice. (F) Blood triglyceride levels in 3–4 month old male and female mice were similar. (G) Glycogen storage in skeletal muscle and (H) liver is significantly increased in Cthrc1 null mice on the 129S6/SvEv background (males and females, 3–5 months of age). (I) Glucose handling is normal in Cthrc1 null mice (n = 8 per group, mixed gender, 3–4 months old). (J) Reduced insulin sensitivity was observed in Cthrc1 null mice (n = 8 per group, mixed gender, 3–4 months old).</p

Characterization of rabbit monoclonal anti-Cthrc1 (Vli-55) antibody.

<p>(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 null mouse.</p

Cthrc1 detection in human plasma.

<p>A pull-down assay with monoclonal Cthrc1 antibody coupled to magnetic beads was performed on human plasma samples (#1–#4). (A) The eluate was analyzed by Western blotting with HRP-conjugated anti-Cthrc1 antibody, conditioned medium from hCthrc1 expressing CHO-K1 cells was used as a positive control. No Cthrc1 was detectable in the unbound plasma fraction (note absence of non-specific bands). (B) The eluates from plasma samples #2–#4 were immunoblotted with biotin-conjugated anti-Cthrc1 antibody followed by HRP-conjugated streptavidin.</p

Cthrc1 secretion is cell type dependent.

<p>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.</p

Cthrc1 accumulation in areas surrounded by chromophobe cells of the anterior pig pituitary.

<p>Adjacent sections were stained for Cthrc1 (A) and morphology (C, H&E). (B) and (D) are enlarged images of the boxed area shown in (A). Arrows indicate areas of Cthrc1 accumulation. Scale bar = 50 µm.</p

Generation of Cthrc1 null mice.

<p>(A) Cthrc1 gene targeting schematic showing replacement of exons 2–4 with a PGK-neo cassette. (B) Embryonic stem cell targeting with Southern blot analysis of genomic DNA from ES cell clones shows a targeted clone. (C) RT-PCR shows lack of Cthrc1 transcripts in RNA preparations of skull bone and whole brain of adult Cthrc1 mutants.</p

Cthrc1 expression in the pig pituitary but absence in adult mouse liver and skeletal muscle.

<p>RT-PCR with Cthrc1 specific primer pair was performed on the indicated mouse and pig RNA samples.</p