Cecr2 mutant mice as a model for human cat eye syndrome

Cat eye syndrome (CES), a human genetic disorder caused by the inverted duplication of a region on chromosome 22, has been known since the late 1890s. Despite the significant impact this disorder has on affected individuals, models for CES have not been produced due to the difficulty of effectively duplicating the corresponding chromosome region in an animal model. However, the study of phenotypes associated with individual genes in this region such as CECR2 may shed light on the etiology of CES. In this study we have shown that deleterious loss of function mutations in mouse Cecr2 effectively demonstrate many of the abnormal features present in human patients with CES, including coloboma and specific skeletal, kidney and heart defects. Beyond phenotypic analyses we have demonstrated the importance of utilizing multiple genetic backgrounds to study disease models, as we see major differences in penetrance of Cecr2-related abnormal phenotype between mouse strains, reminiscent of the variability in the human syndrome. These findings suggest that Cecr2 is involved in the abnormal features of CES and that Cecr2 mice can be used as a model system to study the wide range of phenotypes present in CES.

However, since the syndrome is so variable, it is not possible to extrapolate to whether other CES phenotypes may be associated with these genes.
There is no animal model currently available with the equivalent duplication of the CES critical region, but candidate gene mutations can provide insight into the function of these genes. Gene mutations in mice are usually loss of function, which would seem to be inappropriate for the study of a duplication syndrome. However, in humans the loss or gain of a chromosomal region has been shown to sometimes produce similar phenotypes. For instance, the loss or gain of one copy of a region of chromosome 22q11.2, adjacent to but not associated with the CES region, produces a microdeletion or microduplication syndrome with many similar symptoms to each other. In fact, the similarities to the 22q11.2 microdeletion syndrome (DiGeorge syndrome/velocardiofacial syndrome) led in part to the identification of the 22q11.2 microduplication syndrome 12 . One candidate gene for CES is CECR2, a gene in the original and modified CES critical regions. CECR2 is a chromatin remodelling protein and part of the CERF complex 13 . In mice, loss of CECR2 results in the misregulation of several mesenchymal/ectodermal transcription factors 14 , and this role in developmental regulation would support CECR2 as a gene of interest in CES. Cecr2 mutations in mice result in the lethal neural tube defect exencephaly, equivalent to human anencephaly 13 . Although neural tube defects have never been associated with CES, we hypothesized that there may be other more subtle features in this loss of function mouse that resemble CES clinical features and suggest the involvement of the gene in CES when duplicated.
Here we show that mice homozygous for mutations in Cecr2 show coloboma, microphthalmia, and skeletal, heart, and kidney defects at variable penetrance in a strain specific manner, recapitulating many of the features of human CES associated with the gain of CECR2 copies. This suggests the involvement of CECR2 in multiple features of human CES, making the Cecr2 mouse lines a useful model for understanding these features in CES.

Materials and methods
Mice. All experiments involving mice were approved by the Animal Care and Use Committee of the University of Alberta (University of Alberta AUP 00000094). All methods were performed in accordance with the relevant guidelines and regulations (Canadian Council on Animal Care). The mice were housed in individually ventilated cages (Tecniplast IVC blue line) with a 14 h light/10 h dark cycle and an ambient temperature of 22 ± 2 °C. Mice were fed ad libitum LabDiet Laboratory Rodent Diet 5001 except breeders, who were fed LabDiet Mouse Diet 9F 5020. Breeding females were housed with males and checked each morning for the presence of a copulatory plug, then separated and considered day 0.5 of pregnancy. Embryo ages are listed as E followed by the day of gestation.
Mutations and strains. This study used four different alleles of the Cecr2 gene: the wild-type allele (Cecr2 + ), a genetrap which partially disrupts Cecr2 (Cecr2 Gt(pGT1)1Hemc or Cecr2 GT ) 13 , a presumptive null deletion of the first exon (Cecr2 tm. 1.1Hemc or Cecr2 Del ) 14 and a second presumptive null genetrap (the Cecr2 tm2b(EUCOMM)HMGU with floxed exon 4 removed, or Cecr2 tm2b ) (Fig. 1a). In each case the simplified symbol is used in this report. These alleles were on three different backgrounds: C57Bl/6N, BALB/cCrlAlt and FVB/NJ. The BALB/cCrlAlt strain originated from Charles River Laboratories but was maintained independently since ~ 1988 at the University of Alberta and now differs in some respects from the original BALB/cCrl line. Each Cecr2 mutation results in the lethal neural tube defect exencephaly at a variable penetrance, depending on the mutation and the strain (see Fig. 1b). The Cecr2 GT and Cecr2 Del alleles were congenic on BALB/cCrlAlt and FVB/NJ backgrounds. The Cecr2 Del allele was moved onto the C57Bl/6N background through successive matings to C57Bl/6N wild types and analyzed from generation 3 to 5 (N3-N5). The Cecr2 tm2b allele was congenic on a C57Bl/6N background and was moved to BALB/cCrlAlt background and analyzed from N4-N5.
Histology. Tissues and embryos were fixed in 10% formalin immediately upon dissection. Embryos were cut at the neck and abdomen to allow fixative to enter. Eyes, hearts and kidneys were later dissected from the embryos. Tissues were processed and embedded in paraffin, serially sectioned at 5 or 7 μm and stained in hematoxylin and eosin (H&E). X-Gal staining was done on embryos and isolated kidneys as previously described in Banting et al. 2005 13 . For immunostaining, antigen retrieval was done on paraffin sections using 95 °C 1 mM EDTA (pH 8), then sections were blocked in 10% normal goat serum for 2 h. They were then incubated overnight at 4 °C with 1:20,000 anti-CECR2 antibody 15 , followed by AlexaFluor-488 goat anti-rabbit secondary antibody (Life Technologies) at 1:200 for 2 h at room temperature. All rinses were done with PBS at 2 × concentration. After counterstaining with DAPI, Fluoromount-G was used for mounting and imaging was accomplished using a Nikon Eclipse 80i confocal microscope.
Analysis of cartilage and bone in developing embryo. Alcian blue and alizarin red whole mount skeletal staining was performed on E17.5-E18.5 embryos as described by 16 . In brief, embryos were dissected from the uterus and scalded at 65℃. Embryos were then skinned and washed in 95% ethanol. Embryos were stained for at least 24 h in Alcian Blue stain, rinsed, and cleared with 1% KOH solution. After clearing, embryos were counterstained in 0.005% Alizarin Red in 2% KOH. Images were obtained by immersing embryos in 1:1 glycerol and 2% KOH in agar coated dishes, and then photographed. www.nature.com/scientificreports/ removed from the cast through immersion in Maceration Fluid from several hours to overnight, followed by washing in distilled water. Casts were dissected to reveal the pulmonary arteries and veins on the dorsal side of the heart, then photographed using an Olympus SZ61 microscope and SeBaCam5C camera. Casts were injected with blue-dyed resin in the right ventricle and red-dyed resin in the left ventricle, but the dyes mixed unpredictably due to the presence of the foramen ovale connecting the atria. Because of this, meaningless colour variation in the vessels can be seen even in the greyscale images shown.
Micro-CT imaging. Mouse tails were fixed in Bouin's fixative for up to one week and incrementally washed into 100% ethanol. The skeletal structures were serially X-rayed in a Skyscan 1076 at 35 μm resolution. Computer tomography rendered the X-rays into a 3D skeletal model using programs CTAn, CTVol, CTVox, and DataViewer. The rendered models were visually inspected for skeletal defects.
Statistical analysis. Phenotype penetrance was compared using a two-tailed Fisher's Exact test.
To observe colobomas at a more detailed resolution, C57Bl/6N embryo eyes with either the Cecr2 Del or Cecr2 Tm2b mutation were examined via histology at E18.5 ( Fig. 2i-j'). For both mutations the coloboma fissure was clearly visible on the inferior aspect of the eye.
These results strongly indicate that the presence of coloboma in Cecr2 mutant mice is strain dependent, being present on C57Bl/6N but not BALB/cCrlAlt backgrounds with both Cecr2 mutations tested.
Furthermore, X-gal staining of an FVB/NJ Cecr2 GT/GT embryo at E12.5 suggested that Cecr2 is expressed in the forming retina and lens around the time of optic fissure closure (Fig. 2m). Table 1. Penetrance of defects in late stage embryos, with 3 different Cecr2 mutations on 3 different genetic backgrounds. Numbers represents the number of embryos (ND = not done). a Exencephaly penetrance applies only to the coloboma/micropthalmia/polydactyly datasets. b At least one duplex kidney present in the embryo. c In addition 2/22 showed hypoplastic RV, which was not seen in wildtype controls. d 1/7 showed a defect of the great vessels, and 2/7 showed abnormal vessels from the right lung to the RSVC. e 1/27 had one kidney with a central cavity which may have been hydronephrotic and duplex. f 168 adults were also examined: 16 13 . Specifically, a CECR2/LacZ-fusion protein was seen in the skeletal structure of the fore and hind limbs and this expression resolved to the fore and hind paws later in development. However, no structural abnormalities were seen in the limbs of Cecr2 GT/GT embryos. Nevertheless, we analyzed the Cecr2 Tm2b/Tm2b and Cecr2 Del/Del embryos on both C57Bl/6N and BALB/ cCrlAlt backgrounds during late gestation (E15.5-E18.5) for limb abnormalities. We observed 2 major polydactyly phenotypes in E15.5-18.5 embryos. Mice have 5 digits on both their foreand hindlimbs. Post-axial polydactyly manifested as a small piece of tissue protruding from the ulnar side of the forelimb (Fig. 3a,e). This post-axial protrusion was never seen on the hindlimb. Limbs that were observed to have this phenotype were then assayed with Alcian blue and Alizarin red for cartilaginous or skeletal changes. Limbs with post-axial polydactyly showed no underlying cartilaginous or skeletal changes (Fig. 3b,f).
The other polydactyly phenotype was pre-axial polydactyly, where the extra digit was on the tibial side of the hindlimb, and was never seen on the forelimb. The outermost/first normal digit appeared bifurcated, resulting in an extra digit (Fig. 3c,d). When stained with Alcian blue and Alizarin red, the pre-axial digit was shown to have a cartilaginous component that split to create two digits with bone components on the end of each (Fig. 3e,f). (a-d) and Cecr2 Del/Del (e-h) embryos on a C57Bl/6N background show the variation in the severity of the defects. Each image has an inset at higher magnification. In (c) and (d) insets the eye was photographed after removal. The additional defect of microphthalmia is seen in (h). For the 2 mutations on a BALB/cCrlAlt background, which have unpigmented eyes, identification of the presence of coloboma was determined partially or completely by histology. Eyes were serially sectioned in the frontal plane and H&E stained. Colobomas were present on a C57Bl/6N background (i,j, with i' and j' at higher magnification) but not on a BALB/cCrlAlt background (k,l). X-gal staining of an eye from an FVB/NJ Cecr2 GT/GT embryo at E12.5 suggested that Cecr2 is expressed in the forming retina and lens around the time of optic fissure closure. L = lens, R = forming retina. All embryos pictured also had exencephaly.  Tables 1 and 2). In total, 36/72 incidences of polydactyly were observed in Cecr2 Tm2b/Tm2b embryos (50%). Pre-axial polydactyly was found in 27/72 Cecr2 Tm2b/Tm2b embryos (38%), while 6/72 Cecr2 Tm2b/Tm2b embryos had post-axial polydactyly (8.3%) and 3/72 Cecr2 Tm2b/Tm2b embryos had both (4.2%). Cecr2 Tm2b/+ heterozygotes had 14/197 embryos with polydactyly (7.1%,), with 5/197 pre-axial (2.5%), 9/197 post-axial (4.6%) and 0/197 with both. In the BALB/cCrlAlt background with the Cecr2 Tm2b mutation, only post-axial polydactyly was observed (Fig. 3a). In Cecr2 Tm2b/Tm2b embryos 32/48 showed polydactyly (67%). We also observed post-axial polydactyly in 13/131 Cecr2 Tm2b/+ heterozygotes (9.9%).
The Cecr2 Del mutation on both the BALB/cCrlAlt and C57Bl/6N backgrounds showed similar results, although fewer embryos were examined (Tables 1, 2 BALB/cCrlAlt Cecr2 GT/GT and Cecr2 Del/Del mice also have tail kinks, a defect commonly associated with neural tube defects. Tail kinks ranged in severity from a single small kink to multiple large kinks (Fig. 3g,h respectively). Tail kinks are common in mutants, and were quantified at 37/72 (51%) in a study of BALB/cCrlAlt Cecr2 GT/Del compound heterozygotes. They were also seen at a lower penetrance in heterozygotes (although not quantified), but were never or rarely seen in BALB/cCrlAlt wildtype mice. Micro-CT imaging was used to examine the tails of a Cecr2 GT/+ heterozygote without tail kinks and 2 homozygotes with tail kinks (Fig. 3i,j, respectively). The homozygotes showed occasional shortened, wedge and hemi-vertebrae at one or multiple sites along the tail. We did not examine the upper spine for vertebral defects.
Cecr2 mutant mouse embryos show multiple heart defects. CES is characterized by heart defects in 50-63% of human patients 2,3 . Common abnormalities include septal defects, both ventricular (VSDs, 36%) and atrial (ASDs, 30%) 3 . Total anomalous pulmonary venous return (TAPVR), occurring in 30-43% of patients 2,3 , is a defect of the patterning of the pulmonary veins such that they drain directly or indirectly into the right rather than left atrium. To see if mice with a loss of CECR2 have similar heart defects, we examined E15.5-18.5 mutant and wild-type embryos using serial sections to detect VSDs and embryonic heart resin casting in E17.5-18.5 embryos to detect abnormalities of the pulmonary veins (Table 1).
In order to determine if there was aberrant patterning of the major blood vessels of the heart, and particularly the pulmonary veins (PV), we examined the resin casting of E17.5-E18.5 hearts ( Table 1). The majority of mutant embryos showed a subtle abnormality of the PV organization, suggesting that Cecr2 is involved in PV patterning. Mice normally have 3 PVs (left, right and central, or alternatively left, right inferior and right superior) that join in a duct to the left atrium 19 (Fig. 5a), unlike humans who have 2 left and 2 right PVs. In mice the central vein then branches and drains both lungs (although primarily the right). On the C57Bl/6N background 5/7  (Fig. 5b,c), or a small RPV with a novel rightward branch from the central PV (Fig. 5d). The patterning of PVs in wild-type littermates was normal in 7/7 embryos (p = 0.021). Interestingly, 1 of the mutants with 2 RPVs (Fig. 5b) did not have exencephaly (a rare occurrence in mutants), which suggests that the aberrant PV patterning was not an indirect consequence of the neural tube defect. We also examined 2 C57Bl/6N Cecr2 Del/Del embryos, both of which had 2 RPVs (Fig. 5e). Preliminary evidence indicates that abnormal RPV patterning is also typical for both mutations on a BALB/cCrlAlt background. Three mutants for each mutation were examined, and for both Cecr2 Tm2b/Tm2b and Cecr2 Del/Del embryos, 2 had 2 RVs and 1 had 3 ( Fig. 5f-i). The 3 Cecr2 +/+ wild-type littermates examined had normal PV patterning. While examining PV patterning, we also saw other vessel abnormalities. An unusual abnormality of the great vessels was seen in 1 C57Bl/6 Cecr2 Tm2b/Tm2b embryo (Fig. 6b,d) compared to a control littermate (Fig. 6a,c). The placement of the great vessels suggests a right aortic arch with isolated origin of the left subclavian artery, where the latter appeared to originate from the pulmonary trunk or ductus arteriosus rather than the aortic arch. X-gal staining of E9.5 embryos showed Cecr2 expression in the outflow tract of the heart, which becomes Figure 3. Loss of CECR2 results in skeletal defects. Both post-axial polydactyly of the forelimb and pre-axial polydactyly of the hindlimb were seen in Cecr2 Tm2b/Tm2b embryos on a C57Bl/6N background (a,c respectively), whereas only post-axial polydactyly of the forelimb was seen in Cecr2 Tm2b/Tm2b embryos on a BALB/cCrlAlt background (e). Representative limbs, with defects similar to those pictured in (a) (c) and (e), were stained with Alcian blue and Alizarin red to identify forming cartilage (light blue) and bone (dark staining) respectively (b,d,f). Post-axial extra digits did not contain cartilage or bone, but pre-axial extra digits did, formed by bifurcation of the first normal digit (c,d). BALB/cCrlAlt Cecr2 GT/GT adults also can show skeletal defects of the tail (arrows), with minor (g) and severe kinks (h). Compared to a heterozygote with no tail kinks (i), micro-CT scans of two mutant tails (j) showed intermittent shortened, wedge or hemi-vertebrae (arrows). www.nature.com/scientificreports/ the ascending aorta and pulmonary trunk, but little expression in the rest of the heart (Fig. 6e). In addition, 2/7 C57Bl/6 Cecr2 Tm2b/Tm2b embryos had an abnormal vessel coming from the right lung and draining into the right superior vena cava (RSVC) rather than the RPV (Fig. 6f,g). One of these ( Fig. 6f) was the mutant that did not have exencephaly. A vessel draining from the lung into the RSVC is reminiscent of defects seen in human partial anomalous pulmonary venous return, where 1 or 2 of the PVs drain into the left atrium or dorsal vena cava 20 . None of these defects were seen in any wild-type embryo heart casts.
Cecr2 mutant mouse embryos and adults show kidney defects. Kidney defects are a common feature of human CES, particularly unilateral renal agenesis (38%, 29/77 kidney defects) and hydronephrosis (34%, 26/77 kidney defects) 2,3 . Duplex kidney has also been reported 21,22 . We found all 3 defects in Cecr2 homozygous mice on the FVB/NJ background (Table 1). Kidneys from E18.5 FVB/NJ Cecr2 Del/Del embryos were examined by serial sectioning and H&E staining for structural defects. At least one duplex kidney, with 2 renal pelvises and two ureters, was found in 13/27 mutants (48%) (Fig. 7b,c), compared to a control kidney (Fig. 7a). Four of the 13 mutants showed the duplex phenotype in both kidneys. An additional mutant had an embryonic kidney with evidence of hydronephrosis and appeared to be a duplex (Fig. 7d, sectioning could not be done). Of 9 wild-type littermates examined, all had normal kidneys with 1 renal pelvis (p = 0.0136). The kidneys of FVB/NJ Cecr2 GT/GT embryos were also examined, with 8/20 (40%) showing at least 1 duplex kidney (no significant difference, p > 0.05, compared to FVB/NJ Cecr2 Del/Del embryos).
The role of Cecr2 in kidney development is further supported by its expression. X-Gal staining shows Cecr2 is expressed in the tubules of the forming kidney as early as the mesonephros (Fig. 7i). In later development at E18.5 Cecr2 is expressed in condensing mesenchyme as well as comma and S-shaped bodies of the forming glomeruli ( Fig. 7j-l). CECR2 protein expression in forming glomeruli at E18.5 was confirmed using immunofluorescence with a CECR2-specific antibody (Fig. 7m-p).

Discussion
CES is a rare human disorder with a highly complex and variable phenotype. Because it is usually caused by a 1.5 Mb duplication, it is difficult to study with mouse models. Prior to this report, there was little definitive evidence to suggest which of the at least 14 genes in this duplication contributes to the phenotype, alone or in combination, or whether the smaller duplication of 600 kb containing 3 genes 11 represents all of the phenotypic Figure 5. Loss of CECR2 results in abnormal patterning of the right pulmonary vein (RPV). Representative pictures of the PVs are shown for different mutations and genetic backgrounds. The asterisks label abnormal RPVs, either 2 (a-f,h) or 3 (g,i), as opposed to the normal single RPV in a control (a). Casts were made by injecting resin into the hearts of E17.5-18.5 embryos and then dissecting away extraneous tissues and vessels to reveal the dorsal aspect of the heart. Cpv-central pulmonary vein, Lpa-Left pulmonary artery, Lpv-left pulmonary vein, Rpa-right pulmonary artery, Rpv-right pulmonary vein. www.nature.com/scientificreports/ abnormalities. Creating a chromosomal duplication in mice is further complicated by the fact that one gene in the human CES critical region, Cecr1 (aka Ada2), is not present in mice 23 . This report now shows that mouse lines with the loss of a single gene in the CES critical region, Cecr2, show multiple examples in multiple organs of phenotypic abnormalities reminiscent of those found in human CES, therefore suggesting that Cecr2 is critical in the pathways leading to those defects in CES. CES is named after the eye defect coloboma, although only 55-61% of CES patients show this feature 2,3 . We showed that C57Bl/6N Cecr2 Tm2b/Tm2b and Cecr2 Del/Del embryos have a high penetrance of coloboma similar to patients with CES. Microphthalmia, seen in 19-39% of patients with CES 2,3 , was also seen with both presumptive null Cecr2 mutations on this background, but only associated with coloboma and at a lower penetrance than in humans (8.3-10%). X-gal staining in mice showed that Cecr2 is expressed at the time of optic fissure closure, supporting its role in this process. We further showed that the presence of this defect in Cecr2 mutants is dependent on genetic background, with 59-82% penetrance on the C57Bl/6N background but 0% on the BALB/ cCrlAlt background. The importance of genetic background mirrors the variability seen in CES. In this study penetrance also appears to be dependent on the mutation, with Cecr2 Tm2b/Tm2b embryos showing significantly Figure 6. Other heart defects resulting from loss of CECR2. Embryonic resin heart casts revealed that 1/7 C57Bl/6N Cecr2 Tm2b/Tm2b embryos (b dorsal, d ventral) showed what appears to be right aortic arch with isolated left subclavian artery (Lsc) compared to a normal embryo (a dorsal, c ventral). The Lsc arises from the pulmonary tract/ductus arteriosus rather than the aorta. X-gal staining of a Cecr2 GT/GT E9.5 embryo (e) shows Cecr2 expression in the outflow tract (Oft) of the heart, which becomes the ascending aorta and pulmonary tract. In 2/7 C57Bl/6N Cecr2 Tm2b/Tm2b embryos (f,g), an aberrant vessel (marked along the length with asterisks) was seen coming from the right lung and draining into the right superior vena cava. A-aorta, Bc-brachiocephalic artery, Cpv-central pulmonary vein, Cvc-common ventricular chamber, Da-ductus arteriosus, Lc-left carotid artery, Lpa-left pulmonary artery, Lpv-left pulmonary vein, Lsc-left subclavian artery, Nt-neural tube, Oftoutflow tract, Pt-pulmonary tract, Rpa-right pulmonary artery, Rsvc-right superior vena cava. www.nature.com/scientificreports/ higher penetrance (82%) than Cecr2 Del/Del embryos (59%). Although both alleles are presumptive nulls, one possible complication is that the Cecr2 Tm2b mutation was congenic on the C57Bl/6N background (at least 10 generations) while our Cecr2 Del mutation had only been moved from the non-penetrant BALB/cCrlAlt background to the C57Bl/6N background for 3-5 generations. It is possible that remaining BALB/cCrlAlt modifiers affected the coloboma penetrance. A difference in penetrance between the 2 null mutations is not seen with exencephaly (94 vs. 93%), microphthalmia (8.3 vs. 10%) or polydactyly (50 vs. 41%). Skeletal defects are found in 29-73% of CES cases, depending on the review study 2,3 . We show that Cecr2 homozygotes can have post-and pre-axial polydactyly, of which only pre-axial involves cartilage or bone. Only one relevant case of CES has been reported with right upper limb post-axial polydactyly 24 . A more common skeletal defect in CES is scoliosis 2,3 . BALB/cCrlAlt Cecr2 GT and Cecr2 Del homozygotes and heterozygotes often show tail kinks, which we showed evidence of being due to malformed vertebrae. These wedge and hemi-vertebrae resemble defects found in human cases of congenital scoliosis 25,26 . It would be interesting to examine Cecr2 mutant spines to look for subtle changes in the vertebrae.
Heart defects are a major feature of CES (50-63% penetrance) 2,3 , of which 30% of heart defects are total anomalous pulmonary venous drainage (TAPVR) involving abnormal patterning of the pulmonary veins. Although no Cecr2 homozygous embryos showed classic TAPVR, a high penetrance of a subtle RPV patterning abnormality was seen in mutants but not in controls, regardless of the two genetic backgrounds tested or the Cecr2 mutation. Two embryos also showed anomalous vessels draining from the right lung into the right superior vena cava, suggestive of partial anomalous pulmonary venous return 20 . These results suggest that Cecr2 is involved in the patterning of PVs, which are commonly mispatterned in CES. A further 36% of CES heart defects are ventricular septal defects (VSDs) 3 , which were present in 23% C57Bl/6N Cecr2 Tm2b/Tm2b embryos. However, this defect showed strain dependence, since none were present in BALB/cCrlAlt Cecr2 Tm2b/Tm2b embryos. We also observed what appears to be a right aortic arch with isolated origin of the left subclavian artery (LSA) from either the pulmonary trunk or ductus arteriosus. This a rare but previously reported finding in humans 27 . Although it has not been seen in CES, other abnormalities of the great vessels have been. Intriguingly, we have shown that Cecr2 is expressed in the outflow tract of the heart, which forms the root of the great vessels. Taken together, our data suggests a role for Cecr2 in the development of the heart, particularly the pulmonary veins.
Kidney defects are seen in 31% of cases with CES 3 . The most common defects are unilateral renal agenesis and hydronephrosis, which are present, although relatively rare, in the FVB/NJ Cecr2 GT/GT adults studied. In embryos there was a high penetrance of duplex kidneys, which have been reported in CES 21,22 . Duplex kidneys are predisposed to hydronephrosis 28 . Unilateral renal agenesis was seen in a significant number of FVB/NJ Cecr2 GT/GT adults (16/168) yet was never seen in E18.5 embryos. It is possible that in a small percentage of mutants, one kidney involutes over time and is no longer detectable in adulthood 29,30 , perhaps due to perinatal hydronephrosis.
Our study shows that Cecr2 is involved in the development of the eyes, skeleton, heart and kidneys, and that loss of CECR2 results in defects in the organs similar to those seen in CES. We did not study anal anomalies in mice, which are present in 73-81% of cases with CES 2,3 . This would be a useful future study. Furthermore, in order for Cecr2 to cause these defects through both loss of function and duplication, one would expect the gene to be dosage-sensitive. It was known that Cecr2 heterozygous embryos have a low penetrance of exencephaly 13 . We have now shown that Cecr2 heterozygotes also have a low penetrance of coloboma, microphthalmia, tail kinks and polydactyly. This suggests that Cecr2 is indeed dose-sensitive.
While chromatin remodelling proteins are involved in many processes, including transcription, DNA repair, DNA replication, homologous recombination and chromatin assembly 31 , the specific function of CECR2 and its CERF complex are currently unclear. In human HEK-293T cells, CECR2 has been shown to be involved in DNA double-strand break (DSB) repair 32,33 . However, we recently showed that loss of CECR2 in Cecr2 Del/Del neurospheres does not affect DSB repair 15 . Therefore, while it is unlikely that exencephaly in Cecr2 mutants is due to a defect in DSB repair, other tissues would need to be tested to determine if the CES-like features involve a loss of DSB repair. We have also previously shown misregulation of mesenchymal/ectodermal transcription factors in Cecr2 GT/GT and Cecr2 Del/Del embryos (Fairbridge et al. 2010). Experiments such as ChIP-Seq would be required to determine whether CECR2 directly or indirectly affects the transcription of specific genes during embryogenesis. Since it is established that CECR2 can be involved in DNA repair in one cell type but not another, determining  13 , the Cecr2 mouse has been used to study the lethal neural tube defect exencephaly as a loss of function. The coloboma phenotype that suggests a role for Cecr2 in CES only appeared when the mutations were crossed onto a C57Bl/6N background, highlighting the importance of strain differences when studying a complex phenotype. Further investigation has revealed skeletal, heart, and kidney defects reminiscent of CES defects, all but polydactyly showing strain dependence. This strain dependency mirrors the variability of CES phenotypes in humans. CES is highly variable, even within families, and this must partially be due to genetic background differences. To see the effect of varying genetic background, one must look at multiple inbred mouse lines, as we see here with Cecr2 lines. This may be true of other mouse models for other disorders-to see the full phenotype, different backgrounds are needed to mimic the variability in the human population. However, even within inbred strains there is only partial penetrance of the abnormal phenotypes that we saw, suggesting stochastic developmental factors are affecting the outcome as well.
Human reciprocal microdeletion and microduplication syndromes sometimes show some similar phenotypes 34,35 , indicating a need for a tightly controlled dosage of specific gene products to allow normal development. Outside this range, whether the dosage is abnormally high or low, abnormal development may go down similar paths. For instance, the reciprocal 22q11 microdeletion/microduplication syndromes (which do not overlap with the CES region), share features including heart defects, velopharyngeal insufficiency, cleft palate, hearing loss and cognitive deficits 12 , although the penetrance is likely lower in the microduplication syndrome 36 . Reciprocal microdeletion and microduplication of 7q11.23 (the former being Willliams-Beuren syndrome) share features including abnormal brain imaging, heart defects, joint laxity, ADHD, autism and mild cognitive defects 37 . PMP22 is an example of a single gene that produces similar but distinguishable peripheral neuropathies in both a 1.5 Mb microdeletion (Charcot-Marie-Tooth syndrome type 1A, CMT1A) and a reciprocal microduplication (hereditary neuropathy with liability to pressure palsies, HNPP) due to disruption of peripheral nerve myelination 38 .
Based on our observations, we suggest that CECR2 is involved in many of the abnormal features seen in CES in humans. While a duplication of this gene in mice could give much insight into the syndrome, the Cecr2 loss of function mutations can also be used to study the features of CES. Figure 7. Loss of CECR2 is associated with kidney defects. Coronal sections of E18.5 kidneys show a single renal pelvis (arrow) in a FVB/NJ wild-type control (a) and representative duplex kidneys with two renal pelvises in FVB/NJ Cecr2 Del/Del embryos (b,c). The duplex kidneys ranged from compact (b) to elongated (c), the latter section which clearly showed two ureters (U). An additional E18.5 FVB/NJ Cecr2 Del/Del embryo showed hydronephrosis, and appeared duplex (arrows) through the fluid-filled cavity in the lower image (d). The only kidney defect seen in 27 BALB/cCrlAlt Cecr2 Tm2b/Tm2b embryos was one kidney with a large central cavity, which was likely early hydronephrosis secondary to a duplex kidney (e), the latter suggested by what appears to be two pelvises (arrows) in the lower image. Most FVB/NJ Cecr2 GT/GT adults had two normal kidneys (f), but 16/168 showed unilateral agenesis (g, asterisk) with compensatory hypertrophy of the other kidney. Additionally, 3/168 showed hydronephrosis (h, the hydronephrotic right kidney (Hyd) deflated during dissection). The expression of CECR2 in the kidney was determined using X-gal staining of coronal kidney sections from FVB/NJ Cecr2 GT/GT embryos, which contain a LacZ genetrap. Expression was seen as early as E10.5 (i) in the mesonephric tubules (Mt). At E18.5 X-gal staining showed non-specific endogenous staining of the collecting ducts (Cd) in a FVB/NJ wild-type control (j). In FVB/NJ Cecr2 GT/GT embryos specific X-gal staining was seen in the cortex in the tubules of the forming nephrons: comma bodies (Cb) and S-shaped bodies (S) (l is a higher magnification of k). The endogenous staining in the collecting ducts was faintly visible. CECR2 protein expression was confirmed by immunofluorescence using a CECR2-specific antibody counterstained with DAPI (m-p). An E18.5 wild-type BALB/cCrlAlt embryo showed expression in the kidney cortex, specifically in the C and S-shaped bodies and some diffuse staining in the collecting ducts (m-CECR2 antibody, n-DAPI, o-merge). A CECR2 and DAPI merge of a Cecr2 Del/Del kidney showed only non-specific diffuse staining of the collecting ducts. B-bladder, C-kidney cortex, Cb-comma bodies, Cd-collecting tubules, G-gut, Hyd-hydronephrotic kidney, M-kidney medulla, S-shaped bodies, U-ureter.