To understand the in vivo role of Wdr13 gene, we have created a mouse strain lacking this gene and show that these mice have higher pancreatic islet mass as a result of higher beta cell proliferation, develop hyperinsulinemia and mild obesity. Results Generation of Wdr13 knockout mice Wdr13 is a single copy gene located on the X-chromosome [8]. The targeting strategy was designed to substitute exon 2, intron 2 and exon 3 (partial) of the endogenous gene with neomycin gene cassette containing polyA (Figure 1A). Targeted ES cell clones were identified by southern blot (Figure 1B). Chimaeras were generated from one of the targeted ES cell clones after injecting these cells into C57BL/6 blastocysts. Male chimaeras were bred with CD-1 females and germ line transmission from the ES cell component was identified by the presence of agouti progeny. The mutant allele in these progeny was further confirmed by southern blot. Northern blot analysis using Wdr13 cDNA probe revealed the lack of 4 kb and 2 kb transcripts from brain and testis of the knockout mice, respectively as compared to that from the wild type mice (Figure 1C). Further, western blot using anti WDR13 antibody showed the absence of WDR13 protein in various tissues of the knockout mice (Figure 1D, E). These results confirmed that targeting of Wdr13 gene had led to the generation of a allele. Wdr13 knockout mice were viable and fertile Wdr132/0 male and Wdr132/2 female knockout mice were viable and fertile. Given the comparatively high level of expression of Wdr13 gene in spermatogonia and spermatocytes, and the presence of a different size transcript in testis [8], we analysed the litter size from various matings involving the mutant and wild type mice. The genotypes of the parents had no effects on the litter size (Table S1). The sperm number from Wdr132/0 males (40.665.49 million/ml) was similar to that of Wdr13+/0 (37.963.05 million/ ml). Increased body weight of Wdr13 knockout mice Wdr13 deficient mice differentiated in body weight from their wild type littermates around nine months of age when fed normal chow. At 11 months the mutant male and female mice had 13% and 11% higher body weights (P#0.05), respectively than their littermates (Figure 2A, B). The weight of epididymal fat pad was 2.5-fold more in Wdr132/0 mice in comparison to that of wild type, whereas the weight of ovarian fat pad was 2- fold more in Wdr132/2 in comparison to that of the wild type (Figure 2D). Histological examination of epididymal fat pad revealed adipocyte hypertrophy in Wdr13 knockout mice (Figure 2C). Adipocyte hypertrophy was also noticed in skin sections of the mutant mice. Various organs including, brain, lung, heart, liver, pancreas, spleen and testis of the mutant and wild type mice were weighed at 12 months. The mutant mice had significantly higher pancreatic weight. There was no difference in the weights of other organs (Table S2). To understand the effect of diet on the higher body weight of the mutant mice, one month old mice were kept on a high fat (60%) diet and their body weights were followed. Interestingly, Wdr132/0 weighed significantly more at five months (Figure 2I) indicating the advancement of the obesity phenotype of the mutant mice. Glucose and insulin levels in Wdr13 knockout mice Given the age-dependent obesity phenotype, we measured fasting and random blood glucose levels at different age points in mice fed with normal chow (Figure 2E, F). There was no effect of Role of Wdr13 Gene in Beta Cell Proliferation Wdr13 genotype on fasting glucose levels. Similarly, the random glucose levels at 2 months did not differ between the mutant and wild type mice. However, the mutant mice showed lower random glucose at 12 months on normal chow. In the light of lower random glucose at 12 months, we estimated the serum insulin levels. The fasting insulin levels were similar in the wild type and mutant mice at both age points (Figure 2G). While random insulin levels in Wdr13 deficient and wild type mice at 2 months were similar, interestingly, at 12 months the mutant mice had 2.13 fold more random insulin level on normal chow (Figure 2H). Similarly, the mutant mice had 1.6-fold more random insulin levels at six months when fed on a high fat diet (Figure 2J). Increased glucose clearance but unaltered insulin sensitivity in Wdr13 knockout mice Given the high level of random insulin in Wdr13 deficient mice, we challenged these mice with glucose to determine their glucose clearance at 2 months and 12 months on normal chow, and at 6 months on a high fat diet. The mutant and wild type mice showed similar glucose clearance (Figure 3A) at 2 months. At 12 months the mutant mice appeared to have marginally better glucose clearance; however, the difference was statistically non-significant (Figure 3B). However, the mutant mice showed consistently better glucose clearance at 6 months on a high fat diet (Figure 3C). Insulin tolerance test (ITT) at 2 months (Figure 3D) and 12 months (Figure 3E) did not reveal any difference between the mutant and wild type mice fed on normal chow, indicating that insulin sensitivity was similar in these two groups. Consistent with this, insulin sensitivity in the mutant mice at 6 months (Figure 3F) and 9 months (Figure 3G) was similar to that of wild type mice when fed on a high fat diet. Increased islet mass and beta cell proliferation in Wdr13 knockout mice Since Wdr13 deficient mice were hyperinsulinemic and mildly obese, we analyzed the pancreatic histology at 6 months in these mice when fed on a high fat diet. Interestingly, total islet mass was significantly more in Wdr13 knockout mice at 6 months (Figure 4A, B). To understand the increased islet mass, we measured beta cell proliferation by in-situ BrdU labeling. Briefly, one month old mice were kept on a high fat diet for three weeks before infusion of BrdU and we observed a 2-fold more proliferation of beta cells in Wdr13 mutant mice as compared to their wild type littermates (Figure 4C, D). Higher insulin level in knockout mice is due to the increased islet mass rather than insulin secretion per unit islet Given the better glucose clearance in Wdr132/0 mice, glucose was injected into 6 month old (on high fat diet) fasting mice and the insulin secretion was monitored at different time points (Figure 4E). Insulin secretion after 30 minutes of glucose injection was 1.8 fold more in Wdr132/0 mice. The higher insulin levels in the mutant mice might result from the increased islet mass observed by us in these mice (Figure 4A, B) or might from higher insulin secretion per unit beta cell in response to glucose stimulation. To rule out the latter possibility, we isolated islets from 3 month old Wdr132/0 mice and their wild type littermates (n = 3). Twenty-five equal sized islets from each mouse were induced with 16 mM glucose and insulin secretion was measured after 60 minutes. There was no difference in the in vitro insulin secretion between the mutant and wild type islets (Figure 4F). PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38685