INVESTIGATION The First Scube3 Mutant Mouse Line with Pleiotropic Phenotypic Alterations Helmut Fuchs,*,†,1 Sibylle Sabrautzki,*,‡,1 Gerhard K. H. Przemeck,*,†,1 Stefanie Leuchtenberger,* Bettina Lorenz-Depiereux,§ Lore Becker,* Birgit Rathkolb,*,†,** Marion Horsch,* Lillian Garrett,*,†† Manuela A. Östereicher,* Wolfgang Hans,* Koichiro Abe,‡‡ Nobuho Sagawa,‡‡ Jan Rozman,*,† Ingrid L. Vargas-Panesso,*,§§,*** Michael Sandholzer,* Thomas S. Lisse,*,2 Thure Adler,*,††† Juan Antonio Aguilar-Pimentel,* Julia Calzada-Wack,*,‡‡‡ Nicole Ehrhard,*,§§§,3 Ralf Elvert,*,4 Christine Gau,* Sabine M. Hölter,*,†† Katja Micklich,*,** Kristin Moreth,* Cornelia Prehn,* Oliver Puk,*,††,5 Ildiko Racz,**** Claudia Stoeger,* Alexandra Vernaleken,*,§§,*** Dian Michel,*,6 Susanne Diener,§ Thomas Wieland,§ Jerzy Adamski,*,† Raffi Bekeredjian,†††† Dirk H. Busch,‡‡‡‡ John Favor,§ Jochen Graw,†† Martin Klingenspor,§§§§,***** Christoph Lengger,* Holger Maier,* Frauke Neff,*,‡‡‡ Markus Ollert,†††††,‡‡‡‡‡,§§§§§ Tobias Stoeger,****** Ali Önder Yildirim,****** Tim M. Strom,§ Andreas Zimmer,**** Eckhard Wolf,** Wolfgang Wurst,††,††††††,‡‡‡‡‡‡,§§§§§§ Thomas Klopstock,§§,***,††††††,§§§§§§ Johannes Beckers,*,†,******* Valerie Gailus-Durner,*,† and Martin Hrabé de Angelis*,†,***,*******,7 ORCID IDs: 0000-0002-5143-2677 (H.F.); 0000-0002-3435-921X (S.S.); 0000-0003-3730-3454 (G.K.H.P.); 0000-0003-3730-3454 (S.L.); 0000-0002-6890-4984 (L.B.); 0000-0003-1239-0547 (B.R.); 0000-0003-4333-2366 (M.A.Ö.); 0000-0002-1221-4108 (W.H.); 0000-0002-8035-8904 (J.R.); 0000-0001-5682-8454 (I.L.V.-P.); 0000-0001-5434-9521 (T.A.); 0000-0002-4694-7107 (J.A.A.-P.); 0000-0002-4263-3071 (C.G.); 0000-0003-4878-5241 (S.M.H.); 0000-0001-7170-5284 (K.M.); 0000-0002-1274-4715 (C.P.); 0000-0002-4302-6513 (O.P.); 0000-0001-7329-0641 (I.R.); 0000-0001-8361-0274 (C.S.); 0000-0001-9259-0199 (J.A.); 0000-0001-8713-093X (D.H.B.); 0000-0003-0298-9660 (J.G.); 0000-0002-4502-6664 (M.K.); 0000-0002-2207-4153 (C.L.); 0000-0003-2514-8290 (H.M.); 0000-0002-2790-0389 (T.S.); 0000-0003-1969-480X (A.Ö.Y.); 0000-0003-3127-287X (A.Z.); 0000-0002-0430-9510 (E.W.); 0000-0003-2805-4652 (T.K.); 0000-0001-7874-3822 (J.B.); 0000-0002-7898-2353 (M.H.d.A.) ABSTRACT The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family KEYWORDS consists of three independent members, Scube1–3, which encode secreted cell surface-associated membrane SCUBE3 glycoproteins. Limited information about the general function of this gene family is available, and their roles systemic during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly phenotype shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our un- pleitropy derstanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Paget disease of Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of bone (PDB) parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings mouse model correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3. In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function. The vertebrate Scube (Signal peptide, CUB and EGF-like domain- Human SCUBE3 was originally identified following transcriptional containing protein) family consists of three independent members, profiling of vascular endothelial cells and demonstrated significant Scube1–3. These encode secreted cell surface-associated glycoproteins enrichment in primary osteoblasts and long bones (Wu et al. 2004). that share a domain, organization of at least five recognizable motifs SCUBE3 is a signal protein that is expressed during embryonic de- and the ability to both homo- and heterodimerize (Xavier et al. 2013). velopment in several tissues (Xavier et al. 2013). In mice, Scube3 is Volume 6 | December 2016 | 4035 *German Mouse Clinic, Institute of Experimental Genetics, ‡Research Unit Comparative Medicine, §Institute of Human Genetics, ††Institute of Developmental Genetics, †††Institute of Stem Cell Research, ‡‡‡Institute of Pathology and ÃÃÃÃÃÃInstitute of Lung Biology and Diseases Member of the German Center for Lung Research (DZL), Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany, †German Center for Diabetes Research, Munich, Germany, ÃÃMolecular Animal Breeding and Biotechnology, Gene Center of the Ludwig-Maximilians-Universität München, 81377, Germany, ‡‡Department of Life Science, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan, §§Department of Neurology, Friedrich-Baur-Institut and §§§§§§Munich Cluster for Systems Neurology (SyNergy), Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität München, 80336, Germany, ÃÃÃGerman Center for Vertigo and Balance Disor- ders, Munich, 81377 Germany, §§§Cedars-Sinai Medical Genetics Research Institute, Los Angeles, California 90048, ÃÃÃÃInstitute of Molecular Psychiatry, Medical Faculty, University of Bonn, 53127, Germany, ††††Division of Cardiology, University of Heidelberg, 69120, Germany, ‡‡‡‡Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, 81675, Germany, §§§§Else Kröner Fresenius Center for Nutritional Medicine and ÃÃÃÃÃZIEL – Institute for Food and Health, Technische Universität München, 85350 Freising-Weihenstephan, Germany, †††††Department of Dermatology and Allergy, Biederstein, Klinikum Rechts der Isar, Technische Universität München, 80802, Germany, ‡‡‡‡‡Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, L-4354 Luxembourg, §§§§§Department of Dermatology and Allergy Center, Odense Research Center for Anaphylaxis, University of Southern Denmark, DK-5000 Denmark, ††††††German Center for Neurodegenerative Diseases, 80336 Munich, Germany, ‡‡‡‡‡‡Lehrstuhl für Entwicklungsgenetik, Technical University München- Weihenstephan, c/o Helmholtz Zentrum München, 85764 Neuherberg, Germany, and ÃÃÃÃÃÃÃLehrstuhl für Experimentelle Genetik, Technische Universität München, 85350 Freising-Weihenstephan, Germany expressed in ectodermal, endodermal, and mesodermal derivatives, with phenotypic alterations: Scube3N294K/N294K. The mutant mouse line as are other members of the Scube gene family (Haworth et al. 2007). carries a recessive point mutation in Scube3 and was derived from the Expression of these genes has been shown to be dynamic, and both Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project reciprocal and complementary to each other (Xavier et al. 2013; (MEP, Hrabé de Angelis et al. 2000; Sabrautzki et al. 2012). A Haworth et al. 2007). systemic phenotypic characterization (Hrabé de Angelis et al. 2015) Although our understanding of the function of SCUBE3 in embry- of this new mutant mouse line annotates Scube3 gene function in mice onic development as well as during adulthood is still marginal, one to bone metabolism and morphology, renal function, and hearing, as major role appears to be in bone development and homeostasis, with well as neurological and behavioral functions and energy metabolism. another one in neurological functions. Interestingly, human SCUBE3 maps to chromosome 6p21.3, a region that has been linked to Paget MATERIALS AND METHODS disease of bone 1 (PDB1) (Fotino et al. 1977; Tilyard et al. 1982), which N294K/N294K is characterized by focal areas of increased bone turnover (Ralston et al. Generation of Scube3 mutants 2008). SCUBE3 function is also associated with other tissues, for exam- ENU mutagenesis and breeding were performed as described on a pure ple, Scube3 overexpression in transgenic mice induced cardiac hyper- C3HeB/FeJ (C3H) background (Hrabé de Angelis et al. 2000; Sabrautzki trophy (Yang et al. 2007), and zebrafish Scube3 was recently identified et al. 2012; Aigner et al. 2011). Briefly,C3Hmicewereoriginallypur- as a key regulator of fast muscle development by modulating fibroblast chased from the Jackson Laboratory (Bar Harbor, ME) and ENU (Serva growth factor signaling (Tu et al. 2014). Further associations of Scube3 Electrophoresis, Heidelberg, Germany) was applied in three weekly in- have been reported with hedgehog signal transduction (Johnson et al. tervals by intraperitoneal injections of 90 mg/kg body weight to 10–12 wk 2012), angiogenesis (Yang et al. 2013), and the immune system (Luo old male mice (G0). G0 mice were mated with wild-type C3H females to et al. 2012). In addition, deregulation of SCUBE3 has been found in produce F1 offspring. F1 males not showing any obvious phenotypic different tumor tissues such as lung cancer (Wu et al. 2011; Zhao et al. alterations were mated with wild-type C3H females to obtain the G2 2013) or renal carcinomas (Morris et al. 2011). generation. We either choose 6–8 female G2 mice for matings with their Although SCUBE3 seems to be involved in many different organ F1 father or performed intercross matings of G2 mice to produce at least systems and diseases, there is no suitable mouse model so far for the 20 mice (G3 families). Phenotyping for dysmorphological alterations was study of functional alterations. Recent publications on mice lacking performed according to a standardized protocol (Fuchs