Expression analysis of mouse Rhobtb3 using a LacZ reporter and preliminary characterization of a knockout strain

Julia Lutz, Eva-Maria Grimm-Günter, Pooja Joshi, and Francisco Rivero

Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, United Kingdom

Corresponding author: Francisco Rivero E-mail: francisco.rivero@hyms.ac.uk

Histochemistry and Cell Biology

Supplementary figures and tables

Suppl. Fig. 1 LacZ staining of wildtype mouse tissues. LacZ and Nuclear Fast Red staining of 18 µm thick cryosections of E19 embryos and isolated adult tissues. No blue staining is apparent in any of the tissues. Ac, acinus; Adv, adventitia; Bo, bone; Cap, capsule; Ca, cartilage; Cor, cortex; De, dermis; Diaph, diaphragm; Epid, epidermis; Epit, epithelium; Fol, hair follicle; Glo, glomerulus; IEE, inner enamel epithelium; Len, lens; Muc, mucosa; Musc, muscularis; OCo, organ of Corti; Ret, retina; SemT, seminiferous tubule; SubM, submucosa; Scle, sclera. Scale bar in panel o represents 25 µm and applies to panels a-o. Scale bar in panel t represents 100 µm and applies to panels p-t.

Suppl. Fig. 2 Rhobtb3-LacZ expression in nervous tissue of E19 mouse embryos. LacZ and Nuclear Fast Red staining of 18 µm thick cryosections of E19 embryos. Boxes show the position of higher magnification panels as indicated. In the cortex of the cerebellum (a) a very weak expression is apparent in few scattered cells and appears slightly stronger in the meninges (Men). In the intermediate part of the choroid plexus (b) a moderate expression is visible. In the superior colliculus (c) a weak expression is apparent in few scattered cells (arrows) and is even less in the inferior colliculus (d). In the medulla (e) strong localized expression is apparent in isolated areas, probably nuclei. In the pons (f) very weak expression is apparent in few scattered cells (arrows). No expression is visible in the neocortex (g). A fairly strong expression is apparent in the cortical area of the hippocampus (h). In the thalamus (i) weak expression is visible in localized areas. Weak expression occurs in the choroid plexus (arrows) and hardly any expression is seen in the striatum (j). Relatively strong expression is visible in all parts of the pituitary gland (k). The expression is especially strong in the intermediate lobe (k’) compared to the anterior (k’’’) and posterior (k’’) lobes. Scale bars represent 100 µm in overview panels (columns one and three) and 25 µm in high magnification panels (columns two and four).

Suppl. Fig. 3 Normal localization of the apical markers β-actin, plastin-1, keratin-19, ezrin and the lateral marker β-catenin in Rhobtb3 knockout and wildtype mice. 10 µm thick paraffin sections of colon and jejunum of knockout and wildtype mice were stained with indicated primary antibodies and Alexa568-secondary antibodies (green). The DAPI staining of nuclei (red) served as a control. Pictures were taken with a Zeiss LSM710 confocal microscope, overlaid and pseudocolored for better contrast. Scale bar, 100 µm. An antibody against β-actin was used to stain the apical region of epithelial cells. Plastin-1 (also known as fimbrin) is an actin bundling protein of the microvilli. Keratin-19 is an intermediate filament protein only found in the intestine, which localizes predominantly at the apical region of epithelial cells. Ezrin is a membrane anchoring protein that is expressed in microvilli. The lateral marker β-catenin is localizes to adherent junctions and the antibody stains the lateral walls of intestinal epithelial cells. Overall no significant differences were observed between knockout and wildtype in the localisation of those markers in both small and large intestine. Western blots for quantification of the above mentioned proteins in lysates of intestinal epithelial cells did also not reveal any differences in their abundance (not shown).

Suppl. Fig. 4 Normal cell proliferation in the intestinal mucosa of Rhobtb3 knockout mice. 10 µm thick paraffin sections of jejunum were incubated with anti-Ki67 or anti phosphohistone 3 primary and Alexa568-secondary antibodies (green). The DAPI staining of nuclei (red) served as a control. Pictures were taken with a Nikon Eclipse 801 microscope and a CoolSnap ES camera, overlaid and pseudocolored for better contrast. Scale bar, 100 µm. Ki67 is a nuclear protein that is expressed in the active phases of the cell cycle, namely the late G1, S, G2 and M phases. In resting cells in the G0 phase the antigen cannot be detected. Phosphohistone-3 identifies cells in the M phase. Comparable results were obtained in sections of the large intestine.

Suppl. Table 1. Percentage of intraepithelial lymphocytes and number of goblet cells per millimeter of villus in Rhobtb3 knockout and wildtype mice. 20 villi of each wildtype (WT) and knockout (KO) animal were scored. Data are means ± standard deviation of 3 animals of each genotype. Differences are not significant.

Duodenum Jejunum Ileum

Goblet cells

Rhobtb3 KO 93.7 ± 11.7 77.6 ± 10.1 127.0 ± 26

Rhobtb3 WT 88.5 ± 22.5 113.4 ± 24.3 112.1 ± 20.6

Student’s t-test p = 0.46 p = 0.06 p = 0.29

Lymphocytes

Rhobtb3 KO 7.4 ± 2.9 5.4 ± 2.1 7.4 ± 2.1

Rhobtb3 WT 6.0 ± 1.7 5.5 ± 1.7 8.2 ± 2.9

Student’s t-test p = 0.11 p = 0.85 p = 0.40