Cdkn1c Boosts the Development of Brown Adipose Tissue in a Murine Model of Silver Russell Syndrome.

The accurate diagnosis and clinical management of the growth restriction disorder Silver Russell Syndrome (SRS) has confounded researchers and clinicians for many years due to the myriad of genetic and epigenetic alterations reported in these patients and the lack of suitable animal models to test the contribution of specific gene alterations. Some genetic alterations suggest a role for increased dosage of the imprinted CYCLIN DEPENDENT KINASE INHIBITOR 1C (CDKN1C) gene, often mutated in IMAGe Syndrome and Beckwith-Wiedemann Syndrome (BWS). Cdkn1c encodes a potent negative regulator of fetal growth that also regulates placental development, consistent with a proposed role for CDKN1C in these complex childhood growth disorders. Here, we report that a mouse modelling the rare microduplications present in some SRS patients exhibited phenotypes including low birth weight with relative head sparing, neonatal hypoglycemia, absence of catch-up growth and significantly reduced adiposity as adults, all defining features of SRS. Further investigation revealed the presence of substantially more brown adipose tissue in very young mice, of both the classical or canonical type exemplified by interscapular-type brown fat depot in mice (iBAT) and a second type of non-classic BAT that develops postnatally within white adipose tissue (WAT), genetically attributable to a double dose of Cdkn1c in vivo and ex-vivo. Conversely, loss-of-function of Cdkn1c resulted in the complete developmental failure of the brown adipocyte lineage with a loss of markers of both brown adipose fate and function. We further show that Cdkn1c is required for post-transcriptional accumulation of the brown fat determinant PR domain containing 16 (PRDM16) and that CDKN1C and PRDM16 co-localise to the nucleus of rare label-retaining cell within iBAT. This study reveals a key requirement for Cdkn1c in the early development of the brown adipose lineages. Importantly, active BAT consumes high amounts of energy to generate body heat, providing a valid explanation for the persistence of thinness in our model and supporting a major role for elevated CDKN1C in SRS

<i>Cdkn1c</i> is expressed and imprinted in rpWAT and iBAT.

<p>(A) QPCR of <i>Cdkn1c</i> in P7 rpWAT, subcutaneous (sc) WAT, and iBAT relative to mesenteric (mes) WAT (n = 4 each depot taken from two litters). Data expres sed as mean ± SEM, <i>t</i> test. ** <i>P</i> <0.01.(B) E16.5 transverse sections through IBAT depots stained for <i>Cdkn1c</i> mRNA and protein. (C) WT, BACx1 and BACx2 P7 iBAT sections stained for <i>Cdkn1c</i>. -galactosidase staining of P7 BAC-lacZ iBAT depot (far right panel). <i>Cdkn1c</i>-positive cells indicated by arrows. (D) WT, BACx1 and BACx2 P7 rpWAT sections stained for <i>Cdkn1c</i>. -galactosidase staining of P7 BAC-lacZ rpWAT depot (far right panel). <i>Cdkn1c</i>-positive cells indicated by arrows. (E) <i>Cdkn1c</i> maternal allele-specific expression in P7 and adult iBAT and rpWAT from hybrid offspring from BL6 female mated with a BL6^spretus-chr7 male assessed by the presence (BL6; B) or absence (<i>spretus</i>; S) of an <i>AvaI</i> restriction enzyme site within the <i>Cdkn1c</i> PCR product. (F) Average methylated CpGs per sample with examples of differential methylation of <i>Cdkn1cDMR</i> in P7 and adult iBAT and rpWAT. Each row corresponds to an individual sequenced DNA clone. Each circle represents a CpG on the strand, filled circles and open circles indicate methylated and unmethylated sites, respectively. Percentage values given for n = 3 of each condition.</p

CDKN1C and PRDM16 co-localise to the nucleus of rare BrdU label-retaining cells in iBAT.

<p>(A) Confocal imaging of P7 iBAT co-stained for CDKN1C and PRDM16. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). (B) Western analysis of PRDM16 protein after siRNA-induced knock-down of <i>Cdkn1c</i> in the undifferentiated brown fat preadipocyte cell line HIB1.1. (C) Immunohistochemistry for CDKN1C (green), PRDM16 (red) and BRDU (purple) in WT iBAT 8 weeks after <i>in utero</i> pulsed exposure to BrdU. DNA (DAPI, blue).</p

<i>Cdkn1c</i> promotes the browning of WAT in young mice.

<p>(A) H&E of P7 WT, BACx1 and BACx2 rpWAT (WT from line BACx1). (B) Electron micrograph of WT and BACx1 P7 rpWAT (4000X). Mitochondria indicated by m and lipid by l. (C) QPCR analysis of <i>Cdkn1c</i> mRNA levels in P7 rpWAT from BACx1 and BACx2 relative to wild type controls. (D) QPCR of BAT-selective genes in WT, BACx1 and BACx2 P7 rpWAT. (E) Western blot analysis of UCP1, PRDM16 and β-ACTIN in P7 rpWAT from single litters of WT and BACx1 pups. Data expressed as mean ± SEM, <i>t</i> test. * <i>P</i> <0.05; ** <i>P</i> <0.01; *** <i>P</i> <0.005.</p

Elevated <i>Cdkn1c</i> drives thinness in adult mice.

<p>(A) Weights of WT and BAC transgenic male and female mice at 10 weeks. (B) Dissection of WT and BAC transgenic mice at 10 weeks to reveal adipose depots <i>in situ</i>. (C) Weights of adipose depots relative to body weights. (D) Food consumption per day, measurements taken over 5 days. (E) Rectal body temperature. (F) H&E sections of 10 week rpWAT depots from WT, BACx1 and BACx2 and BAClacZ (WT from line BACx1). (G) QPCR analysis of <i>Cdkn1c</i>, <i>Ucp1</i> and <i>Elovl3</i> in BACx1 female 10 week rpWAT depots (n = 4 per genotype). Data expressed as mean ± SEM, <i>t</i> test.</p

<i>Cdkn1c</i> induces a BAT-like gene program <i>ex-vivo</i>.

<p>(A) QPCR of analysis <i>Cdkn1c</i> mRNA levels in WT and BACx1 MEFs over 8 days of adipocyte induction relative to WT day (D) 0. (B) QPCR analysis of <i>Cdkn1c</i> expression in WT, BACx1, KO^MAT and BACx1+KO MEFs after 8 days of directed differentiation. (C) Oil Red O (ORO) staining of D8 adipocyte-differentiated WT, BACx1, KO^MAT and BACx1+KO MEFs. All genotypes produced lipid filled adipocytes. (D) QPCR analysis of BAT marker genes <i>Ppargc1a</i>, <i>Cidea</i>, <i>Ucp1</i>, and <i>Elovl3</i> in WT, BACx1, KO^MAT and BACx1+KO D8 adipocyte-differentiated MEFs. As <i>in vivo</i>, key markers of BAT fate and function were elevated. Critically BACx1+KO MEFs(<i>Cdkn1c</i> expressed at WT levels) expressed the BAT markers at WT levels confirming that induction was in response to the transgenic elevation of <i>Cdkn1c</i>. (E) QPCR analysis of <i>Cdkn1c</i> and BAT marker genes <i>Ppargc1a</i>, <i>Cidea</i>, <i>Ucp1</i>, and <i>Elovl3</i> in WT and BACx2 D8 adipocyte-differentiated MEFs illustrating further elevation of BAT markers driven by increased <i>Cdkn1c</i> dosage. (F) Confocal images of D8 adipocyte-differentiated WT, BACx1 and KO^MAT MEFs. Membranes stained with Cell mask Deep Red plasma (633nm; red), nuclei stained with Hoechst 366243 (450nm; blue) and mitochondria stained with Rhodamine-123 (540nm; green). Fields shown were visualised under fluorescence microscope at appropriate wavelengths. Mitochondria indicated by m, lipid by l and nucleus by n. Scale bar = 19 d. (G) Western analysis of UCP1 and β-ACTIN in D8 adipocyte-differentiated WT and BACx1 MEFs and after addition of 1 mM 9-<i>cis</i>-retinoic acid for 48 hours. UCP1 protein detectable by Western analysis in transgenic but not WT samples, an effect amplified by exposure to the positive regulator of <i>Ucp1</i> gene transcription, retinoic acid. For each QPCR analysis n = 4 genotypes per group taken from two independent litters; error bars represent ± s.e.m. * <i>P</i> <0.05; ** <i>P</i> <0.01; *** <i>P</i> <0.005.</p

<i>Cdkn1c</i> is required for the proper formation of iBAT.

<p>(A) Photograph of E18.5 WT and <i>Cdkn1c</i>^<i>-/+</i> (KO^MAT) fetuses with position of iBAT depot highlighted by dotted black line. (B) H&E staining of iBAT sections of E18.5 WT and KO^MAT iBAT. (C) QPCR of <i>Cdkn1c</i> and the adipocyte regulators <i>Rb1</i>, <i>PPARγ</i>, <i>C/EBPα</i> and <i>C/EBPβ</i>, pan-adipocyte genes <i>Fabp4</i>, <i>Cidec</i> and <i>Plin1</i>, BAT-selective genes <i>Ppargc1a</i>, <i>Cidea</i>, <i>Prdm16</i>, <i>Ucp1</i> and <i>Elovl3</i>, mitochondrial genes <i>Cycs</i> and <i>Cox2</i>, and the skeletal muscle-selective genes <i>Myf5</i> and <i>Myod1</i> in E18.5 KO^MAT iBAT relative to WT (n = 4 per genotype). (D) Mitochondrial DNA content of iBAT from E18.5 KO^MAT iBAT relative to WT (n = 6 per genotype). (E) Western blot analysis of UCP1 and β-ACTIN in E18.5 iBAT isolated from two WT and two KO^MAT fetuses. Within litter comparison. (F) Western blot analysis of CDKN1C, PRDM16 and β-ACTIN in E18.5 iBAT isolated from two WT and two KO^MAT fetuses. Within litter comparison. (G) Western blot analysis of MYOD and GAPDH in E18.5 E18.5 iBAT isolated from two WT and two KO^MAT fetuses. Within litter comparison. Data expressed as mean ± SEM, <i>t</i> test. * <i>P</i> <0.05; ** <i>P</i> <0.01; *** <i>P</i> <0.005.</p

Bacterial artificial chromosome (BAC) spanning the intact <i>Cdkn1c</i> locus in mice models minimal microduplication reported in Silver Russell Syndrome.

<p>(A) Genomic map of human 11p15 imprinted region. Line below indicates extent of minimal region duplicated in SRS. (B) Genomic map of mouse distal chromosome 7 imprinted region. Below is the map of the 85 kb BAC transgene (BAC144D14). Inset: Image of WT and BACx1 pups carrying one copy of the BACx1 examined on a mixed 129/BL6 genetic background on postnatal day (P) 2. (C) Weights of WT and BAC transgenic pups at birth (P0) after breeding onto BL6 genetic background for >12 generations. (D) Brain weight to body weight ratio. (E) Blood glucose levels (mmol/l). NS = not significant. Data expressed as mean ± SEM, <i>t</i> test. Numbers given in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005916#pgen.1005916.s001" target="_blank">S1 Fig</a></b>.</p

Two-fold <i>Cdkn1c</i> expression results in fetal growth restriction with characteristic features of Silver Russell Syndrome whereas loss-of function of <i>Cdkn1c</i> results in fetal overgrowth with characteristic features of Beckwith Weidemann Syndrome.

<p>Two-fold <i>Cdkn1c</i> expression results in fetal growth restriction with characteristic features of Silver Russell Syndrome whereas loss-of function of <i>Cdkn1c</i> results in fetal overgrowth with characteristic features of Beckwith Weidemann Syndrome.</p

Elevated <i>Cdkn1c</i> boosts the formation of classic BAT in young mice.

<p>(A) Weights of WT, BACx1 and BACx2 iBAT relative to total body weight (WT n = 20, BACx1 n = 18, BACx2 n = 8). (B) H&E staining of P7 iBAT depot sections from WT, BACx1 and BACx2 pups (WT from line BACx1) and cell counting data (n = 6 per genotype). (C) QPCR of <i>Cdkn1c</i>, adipogenesis regulators, thermogenic, BAT-selective and mitochondrial genes in BACx1 and BACx2 P7 iBAT relative to WT (n = 4 per genotype). (D) Quantitation of mitochondrial genomic DNA of BACx1 and BACx2 iBAT relative to WT (n = 6). (E) Surface body temperature of BACx2 P2 pups relative to WT littermates was assessed by thermal imaging (WT n = 19, BACx2 n = 8) within 1 minute of removal from nest temperature (33°C; approaching thermoneutrality) and after 20 minutes at room temperature (22°C). Data expressed as mean ± SEM, <i>t</i> test. * <i>P</i> <0.05; ** <i>P</i> <0.01; *** <i>P</i> <0.005.</p