Copyright Ó 2009 by the Genetics Society of America DOI: 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,*,1 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 Mu¨nchen, German Research Center for Environmental Health, Neuherberg D-85764, Germany, †Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom, ‡Lehrstuhl fu¨r Allgemeine Pathologie und Neuropathologie, Tiera¨rztliche Fakulta¨t, Ludwig-Maximilians-Universita¨t, Mu¨nchen D-80539, Germany and ††Chrombios GmbH, Raubling D-83064, Germany Manuscript received April 30, 2009 Accepted for publication May 16, 2009 ABSTRACT In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were 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 from 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 Ca12-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions. ONTIGUOUS gene deletions account for a et al. 1993; Bartsch et al. 1996; Potocki and Shaffer C significant portion of human genetic syndromes. 1996) and the Wilm’s tumor- aniridia- genitourinary The application of fluorescence in situ hybridization abnormalities- mental retardation (WAGR) syndrome (FISH) cytogenetics and array comparative genome (Riccardi et al. 1978; Francke et al. 1979; Hittner hybridization (array-CGH) technologies have enabled et al. 1979; Fryns et al. 1981), respectively. Deletion more accurate localization of deletion breakpoints. analyses were important in identifying genes associated This deletion information combined with the annota- with clinical features of the syndromes: EXT2 for tion of the human genome structure provides critical multiple exostoses and ALX4 for parietal foramina in information to identify genes responsible for particular Potocki–Shaffer syndrome (Ligon et al. 1998; Wu et al. phenotypes associated with a syndrome. For example, 2000; Wakui et al. 2005), WT1 for Wilm’s tumor, and deletions of the 11p11p12 and 11p13 regions on the PAX6 for aniridia in WAGR syndrome (van Heyningen short arm of human chromosome (Chr) 11 have been et al. 1985; Glaser et al. 1986, 1992; Fantes et al. 1992). identified in the Potocki–Shaffer syndrome (Shaffer Deletion analyses have also defined the extent of the deleted region in patients with combined Potocki– Shaffer and WAGR syndromes (McGaughran et al. The mutant allele symbols Del(2)Pax611Neu/1Neu, Del(2)Pax612Neu/ 1995; Bre´mond-Gignac et al. 2005) as well as micro- 13Neu 2Neu and Del(2)Pax6 /3Neu were submitted to and approved by the deletions 39 to PAX6, which prevent expression of PAX6 Mouse Genetic Nomenclature Committee, and assigned the MGI auderdale ‘elia accession ID nos. 3698295, 3698297, and 3710946, respectively. and cause aniridia (L et al. 2000; D et al. Supporting information is available online at http://www.genetics.org/ 2007; Davis et al. 2008). cgi/content/full/genetics.109.104562/DC1. The mouse Chr 2 region homologous to the human 1Corresponding author: Institute of Human Genetics, Helmholtz Zen- WAGR region contains the genes Wt1, Rcn1, Pax6, and trum Mu¨nchen German Research Center for Environmental Health, Ingolsta¨dter Lanstrasse 1 D-85764, Neuherberg, Germany. Elp4. An extensive allelic series at Pax6 has been E-mail: [email protected] identified (Bult et al. 2008). Heterozygote Pax6 in- Genetics 182: 1077–1088 (August 2009) 1078 J. Favor et al. tragenic null mutants express microphthalmia, iris were obtained from breeding colonies maintained by the anomalies, corneal opacities, lens opacities, and lens- Department of Animal Resources at Neuherberg. corneal adhesions. Homozygotes are anophthalmic and Histology, gross embryo morphology, and slit lamp oberts ogan photography: Pregnant females were killed by cervical dislo- die shortly after birth (R 1967; H et al. cation. Embryos were freed from placentae and embryonic 1986). Five deletions in the region have been identified: membranes in room temperature PBS, phenotyped under a Pax6Sey-Dey, Pax6Sey-H, Pax6Sey-2H, Pax6Sey-3H, Pax6Sey-4H of which dissecting microscope (MZ APO; Leica, Bensheim, Germany), two, Pax6Sey-H (Hogan et al. 1986; Kent et al. 1997; and photographed. Postnatal day 1 (P1) mice were killed by Kleinjan et al. 2002; Webb et al. 2008) and Pax6Sey-Dey decapitation and phenotyped after dissecting away the skin overlying the eyes. P21 mice were phenotyped by slit lamp (Theiler et al. 1978; Hogan et al. 1987; Glaser et al. examination and killed by CO2 asphyxiation. Embryos and 1990), have been well characterized. Heterozygotes for heads of P1 or P21 mice were fixed in 10% buffered formalin. both deletions express belly spotting and a more The heads from P21 mice were demineralized in EDTA. Fixed extreme eye phenotype than that observed for hetero- materials were embedded in paraffin, and serially sectioned zygotes of intragenic Pax6 null mutations. Homozygotes (coronal) at 5 mm. Sections were stained with hematoxylin and eosin, and evaluated by light microscopy (Axioplan; Carl Zeiss, for both deletions are lethal at an early embryonic stage. Hallbergmoos, Germany). Digital photos were acquired (Ax- We were particularly interested in the extreme eye iocam and Axiovision; Carl Zeiss, Hallbergmoos, Germany) phenotype associated with the Pax6 deletions and and imported into Adobe Photoshop CS (Adobe Systems, considered two alternative hypotheses. Either Pax6 Unterschleissheim, Germany). deletions are functionally different from Pax6 intra- P35 mice were anesthetized with 137 mg ketamine and 6.6 mg xylazine per kg body weight and quickly photographed genic null mutations or deletion of a region linked to with a slit lamp microscope (Zeiss SL 120) equipped with a but distinct from the Pax6 structural gene affects the eye compact video camera. Images were captured in Axiovision phenotype. (Zeiss) and imported into Adobe Photoshop CS. After photog- In the present study we identify three new deletions raphy ophthalmic salve (Regepithel, Alcon) was applied to the encompassing the Pax6 region of the mouse. They have eyes of the anesthetized mice to prevent eye injury due to 11Neu dehydration and the animals were caged individually until fully been assigned the mutant allele symbols Del(2)Pax6 / recuperated. 12Neu 13Neu 1Neu, Del(2)Pax6 /2Neu, and Del(2)Pax6 /3Neu Segregation analysis of embryos: Pregnant females were and will be referred to throughout this publication as killed as above between days 14 and 16 p.c. (E14 and E16 Pax611Neu, Pax612Neu,andPax613Neu, respectively. All three stages) of pregnancy. The entire uterus was removed, opened, deletions are homozygous lethal at an early embryonic and the uterine contents classified for live embryos, dead implants (implantation site with an obvious placenta, extra- stage. The deletions differentiate for the extent of the embryonic membranes, and necrotic embryonic tissue), and 11Neu eye abnormality expressed: Pax6 heterozygotes ex- decidua (resorption sites consisting of the remnants of the press extreme microphthalmia similar to that observed decidual reaction tissue due to implantation but subsequent in the Pax6Sey-Dey and Pax6Sey-H deletions. Pax612Neu and early embryonic death). The live embryos were freed from the Pax613Neu heterozygotes express the milder eye abnor- placentae and embryonic membranes and phenotyped. mality seen in heterozygous intragenic null mutants. For Body weight and gross eye morphology: Eye morphology was assessed as previously described (Favor et al. 2001, 2008). all three deletions, heterozygotes do not express belly P35 heterozygous mutant and wild-type littermates were oph- spotting. Genetic, phenotypic, and molecular charac- thalmologically examined by slit lamp microscopy and catego- terization of the deletions allowed us to identify regions rized for the degree of lens/corneal opacity and extreme associated with the array of phenotypes in these contig- microphthalmia. The animals were weighed and killed by uous gene deletions. cervical dislocation. Eyes were