another Fto knockout mouse model [31,36]. The death mostly happened within a few days after birth (Table S2). It is unclear what causes the death at present. However, near term FtoD/D embryos were recovered at the Mendelian ratio (Table S3), indicating that Fto is not required for embryogenesis in mice and the death mostly likely happened after the birth. Surviving FtoD/D mice displayed immediate growth retardation in both males and females (Figure 3A). Within the first few days after the birth, the FtoD/D mice were already significantly lighter in weight comparing to their wildtype and heterozygous littermates, and the difference increased over time (Figure 3B). This is unlikely a result of that the mutant pups were born smaller than normal, because there was no weight difference among embryos of all three genotypes at E18.5 (Figure 3C). At the time of weaning, both male and female FtoD/D mice were about 65% the weight of wildtype and heterozygous littermates (Figure 3D). While the male FtoD/D mice remained behind throughout adulthood, the females showed a trend of catching up in weight later on (Figure 3D). FtoD/D mice were also significantly shorter in body length throughout lifetime (Figure 3E), and they had much lower bone mineral density (Figure 3F). Consistent with the growth retardation phenotype, FtoD/D mice had significantly lower serum levels of IGF-1 (insulin-like growth factor 1) than the controls at the time of growth spurt (Figure 3G). FtoD/D mice do not display a lean phenotype To determine whether Fto-deficiency affects body fat mass, the body compositions of 16-week-old mice fed on normal chow were measured by DEXA (dual energy X-ray absorptiometry). Despite the general decrease in size, FtoD/D mice did not show a specific reduction in fat. Among the 16-week-old males, FtoD/D mice had about 25% less lean mass than Fto+/+ or Fto+/D mice (Figure 4A) and slightly less fat mass (statistically not significant) (Figure 4B). When normalized to the total tissue weight (lean mass plus fat mass) (Figure 4C), male mice of all three genotypes had similar fat content (fat mass/total tissue mass %) (Figure 4D). On the other hand, at the age of 16 weeks, female FtoD/D mice had even more fat mass on average than their wildtype and heterozygous littermates (Figure 4B), while their lean mass were still less than the controls (about 12% less, not as dramatic as the difference in males) (Figure 4A). As a result, female FtoD/D mice contained higher proportion of fat (fat mass/total tissue mass %) than wildtype or heterozygous mice (Figure 4D). The increased fat mass in mutant females compensated for their slight deficit in lean mass, and made it the total tissue mass even (Figure 4C), which also explains the trend of catching-up in whole body weight as the mice got older (Figure 3D). FtoD/D mice are susceptible to diet-induced-obesity To determine if the status of Fto has an effect on the response to diet, both male and female mice of three genotypes (Fto+/+, Fto+/D and FtoD/D) were fed on high fat diet (60 kcal % fat) from 4-week-old on for 10 weeks. Unexpectedly, about half of the FtoD/D mice on high fat diet developed dermatitis (Figure S2A, Table S4) at the late stage of the diet regimen, and they became very lean due to the illness (Figure S2B). The reason of this illness is unclear. The dermatitis-free FtoD/D mice did respond to high fat diet and developed DIO (diet-induced-obesity) (Figure S2B). At the end of the diet regimen, all the mice free of dermatitis were measured by DEXA (dual energy X-ray absorptiometry). The FtoD/D mice still had significantly less lean mass than wildtype and heterozygotes (Figure 5A), while they had the similar amount of fat mass (Figure 5B). When normalized to total tissue mass (Figure 5C), the FtoD/D mice had relatively higher percentage of fat mass than controls, though not statistically significant in females (Figure 5D). Fto and Postnatal Growth At the same time, the FtoD/D mice on high fat diet retained other aspects of the growth retardation phenotype, such as shorter body length (Figure 5E) and lower bone mineral density (Figure 5F). FtoD/D mice have higher metabolic rates To determine whether the deletion of Fto would affect the metabolism in mice, the O2 consumption and CO2 production of 16,17-week-old male mice were measured using indirect calor- iometry. As there was no phenotypic difference in wildtype and heterozygous mice, only Fto+/D and FtoD/D mice were used here due to the availability of mice at the time of experiment. When the absolute amount of O2 consumption (VO2) and CO2 production (VCO2) were measured, FtoD/D mice showed relatively lower rates of O2 consumption and CO2 production than Fto+/D mice (Figure 6A, C), during both light and dark periods (though the difference of VO2 during light period was not statistically significant). When divided by lean mass (conventional normaliza- tion), the FtoD/D mice displayed significantly higher metabolic rates (Figure 6B, D), possibly due to their much-reduced lean mass. When the data was analyzed by ANCOVA using lean mass as a covariant, the FtoD/D mice still showed higher O2 consumption and CO2 production rates during both light and dark period (Figure S3A–D), suggesting the deletion of Fto increases the metabolic rate, even after its effect on body mass has been taken into account. While the oxygen consumption was measured, we also measured the food intake of Fto mutant mice. In a period of 72 hours, Fto+/D and FtoD/D mice consumed similar amount of food (Figure 6E). However, FtoD/D mice appeared to have consumed more food when the amount of food intake was normalized to lean mass (Figure 6F). Taken together, these data suggest that Fto affects the food intake and energy expenditure at the same time. In its absence, both are increased. Neural-specific Fto knockout mice are phenotypically similar to the complete knockout Before we generated the Fto conditional knockout line, we had made another genetrap mouse line (Figure S1A, B) which still had residual expression of wildtype FTO (Figure S1C). It is interesting that homozygous genetrap mice (Ftogt/gt) displayed none of the phenotypes (Figure S1D–F) of FtoD/D mice. It is possible the residual FTO in the peripheral tissues may be enough for its function, considering Fto heterozygous knockout mice, as well as humans heterozygous for the FTO loss of function mutations are generally normal [37]. On the other hand, since there was only minimal reduction of FTO in the brain tissues of the gene-trap mice comparing to other tissues (Figure S1C), we reasoned that Fto might function in the brain, To explore this possibility, we deleted Fto in the nervous system by crossing Ftoflox allele with the Nestin- Cre transgenic mice [38] (Figure 7A). Western blotting analysis of Ftoflox/flox/Nestin-Cre (will be referred to as FtoND hereafter, N denotes neural) mice confirmed the efficient reduction of FTO protein in the brain, but not the peripheral tissues (Figure 7B). These mice displayed growth retardation, similar to the complete knockout mice (Figure 7C, D). Both male and female FtoND mice had shorter body length (Figure 7E) and lower bone mineral density (Figure 7F). FtoND mice also had much lower serum levels of IGF-1 during growth spurt (Figure 7G). Neural-specific deletion of Fto affects body composition of mice in a similar way as the whole body knockout Similar to the whole body knockout, the body composition of 16-week old FtoND mice didn’t show any deficits in fat PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e14005