Genetics: Published Articles Ahead of Print, published on May 27, 2009 as 10.1534/genetics.109.104562 Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small eye and belly spotting phenotypes Jack Favor*, Alan Bradley†, Nathalie Conte†, Dirk Janik‡, Walter Pretsch*, Peter Reitmeir§, Michael Rosemann**, Wolfgang Schmahl‡, Johannes Wienberg†† and Irmgard Zaus* Institute of Human Genetics*, Institute of Health Management§, Institute of Radiation Biology**, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg D- 85764, Germany † Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK ‡ Lehrstuhl für Allgemeine Pathologie und Neuropathologie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, München D-80539, Germany †† Chrombios GmbH, Raubling D-83064, Germany The mutant allele symbols Del(2)Pax611Neu/1Neu, Del(2)Pax612Neu/2Neu and Del(2)Pax613Neu/3Neu were submitted to and approved by the Mouse Genetic Nomenclature Committee, and assigned the MGI accession ID numbers 3698295, 3698296 and 3710946, respectively. 1 Running head: Mouse Pax6 contiguous gene deletions Key words: Mouse, Pax6, contiguous gene deletions, microphthalmia, belly spotting Corresponding author: Jack Favor Institute of Human Genetics Helmholtz Zentrum München German Research Center for Environmental Health Ingolstädter Lanstr. 1 D-85764 Neuherberg Germany Telephone No. +49-89-3187-2395 FAX No. +49-89-3187-3297 e-mail [email protected] 2 ABSTRACT In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were previously shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different than intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca+2-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of 3 Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions. 4 INTRODUCTION Contiguous gene deletions account for a significant portion of human genetic syndromes. The application of fluorescence in situ hybridization (FISH) cytogenetics and array comparative genome hybridization (array-CGH) technologies has enabled more accurate localization of deletion breakpoints. This deletion information combined with the annotation of the human genome structure provides critical information to identify genes responsible for particular phenotypes within an array of phenotypes which define a particular syndrome. For example, the 11p11p12 and 11p13 regions on the short arm of human chromosome (Chr) 11 have been associated with the Potocki- Shaffer syndrome (SHAFFER et al. 1993; BARTSCH et al. 1996; POTOCKI and SHAFFER 1996) and the Wilm’s tumor- aniridia- genitourinary abnormalities- mental retardation (WAGR) syndrome (RICCARDI et al. 1978; FRANCKE et al. 1979; HITTNER et al. 1979; FRYNS et al. 1981), respectively. Deletion analyses were important in identifying genes associated with clinical features of the syndromes: EXT2 for multiple exostoses and ALX4 for parietal foramina in Potocki-Shaffer syndrome (LIGON et al. 1998; WU et al. 2000; WAKUI et al. 2005); WT1 for Wilm’s tumor and PAX6 for aniridia in WAGR syndrome (VAN HEYNINGEN et al. 1985; GLASER et al. 1986, 1992; FANTES et al. 1992). Deletion analyses have also defined the extent of the deleted region in 5 patients with combined Potocki-Shaffer and WAGR syndromes (MCGAUGHRAN et al. 1995; BRÉMOND-GIGNAC et al. 2005) as well as microdeletions 3’ to PAX6 which prevent expression of PAX6 and cause aniridia (LAUDERDALE et al. 2000; D'ELIA et al. 2007; DAVIS et al. 2008). The mouse Chr 2 region homologous to the human WAGR region contains the genes Wt1, Rcn1, Pax6 and Elp4, and has been intensively studied. An extensive allelic series at Pax6 has been identified (BULT et al. 2008). Heterozygote Pax6 intragenic null mutants express microphthalmia, iris anomalies, corneal opacities, lens opacities and lens-corneal adhesions. Homozygote mutants are anophthalmic and die shortly after birth (ROBERTS 1967; HOGAN et al. 1986). Five deletions in the region have been identified; Pax6Sey-Dey, Pax6Sey-H, Pax6Sey-2H, Pax6Sey-3H, Pax6Sey-4H of which two, Pax6Sey-H (HOGAN et al. 1986; KENT et al. 1997; KLEINJAN et al. 2002; WEBB et al. 2008) and Pax6Sey-Dey (THEILER et al. 1978; HOGAN et al. 1987; GLASER et al. 1990), have been well characterized. Heterozygotes for both deletions express belly spotting and a more extreme eye phenotype than that observed for heterozygotes of intragenic Pax6 null mutations. Homozygotes for both deletions are lethal at an early embryonic stage. We were particularly interested in the extreme eye phenotype associated with the Pax6 deletions and considered two alternative hypotheses. Either Pax6 deletions are functionally different from Pax6 intragenic null mutations or, deletion of 6 a region linked to but distinct from the Pax6 structural gene affects the eye phenotype. In the present study we identify three new deletions encompassing the Pax6 region of the mouse. They have been assigned the mutant allele symbols Del(2)Pax611Neu/1Neu, Del(2)Pax612Neu/2Neu and Del(2)Pax613Neu/3Neu and will be referred to throughout this publication as Pax611Neu, Pax612Neu , and Pax613Neu, respectively. All three deletions are homozygous lethal at an early embryonic stage. The deletions differentiate for the extent of the eye abnormality expressed by heterozygotes: Pax611Neu heterozygotes express extreme microphthalmia similar to that observed in the Pax6Sey-Dey and Pax6Sey-H deletions. Pax612Neu and Pax613Neu heterozygotes express the milder eye abnormality seen in heterozygous intragenic null mutants. For all three deletions, heterozygotes do not express belly spotting. Genetic, phenotypic and molecular characterization of the deletions allowed us to identify regions associated with the array of phenotypes in these contiguous gene deletions. 7 MATERIALS AND METHODS Mutations, animals and mapping: The original Pax611Neu and Pax613Neu mutants were found in our breeding colonies. The original Pax612Neu mutant was recovered in a mutagenesis experiment. Ophthalmological examinations were done as previously described (FAVOR 1983). Congenic C3H/HeJ mutant lines were constructed prior to initiating the studies. The mapping of the mutations followed our standard laboratory protocol (FAVOR et al. 1997). For timed pregnancies, females were mated and checked daily for the presence of a vaginal plug. The day at which a vaginal plug was observed was defined as day 0 p.c. (E0). In matings which were set up to generate offspring, females were checked daily for new born litters and the day of birth was defined as post-natal day 0 (P0). Animals were bred and maintained in our animal facilities according to the German law for the protection of animals. All inbred strain C3H/HeJ and C57BL/6El animals used in the present study were obtained from breeding colonies maintained by the Department of Animal Resources at Neuherberg. Histology, gross embryo morphology, and slit lamp photography: Pregnant females were sacrificed by cervical dislocation. Embryos were carefully freed from placentae and embryonic membranes in room temperature PBS, phenotyped under a dissecting microscope (MZ APO; Leica, Bensheim, Germany), and 8 photographed. Post-natal day 1 (P1) mice were sacrificed by decapitation and phenotyped after carefully dissecting away the skin overlying the eyes. P21 mice were phenotyped by slit lamp examination and sacrificed by CO2 asphixiation. Embryos and heads of P1 or P21 mice were fixed in 10% buffered formalin. The heads from P21 mice were demineralised in EDTA. All fixed materials were embedded in paraffin, and serially sectioned (coronal) at 5 µm. Sections were stained with hematoxylin and eosin, and evaluated by light microscopy (Axioplan; Carl Zeiss, Hallbergmoos, Germany). Digital photos were acquired (Axiocam and Axiovision; Carl Zeiss, Hallbergmoos, Germany) and imported into Adobe Photoshop CS (Adobe Systems, Unterschleissheim, Germany). P35 mice were anesthetized with 137 mg ketamine and 6.6 mg xylazine per kg body weight and quickly photographed with a slit lamp microscope (Zeiss SL 120) equipped with a compact video camera. Images were captured in Axiovision (Zeiss) and imported into Adobe Photoshop CS. After photography ophthalmic salve (Regepithel, Alcon) was applied to the eyes of the anesthetized mice to prevent eye