Abstract

Objective

   Cyclin C (CCNC) is the most conserved subunit of the Mediator complex,
   which is an important transcription cofactor. Recently, we have found
   that CCNC facilitates brown adipogenesis in vitro by activating
   C/EBPα-dependent transcription. However, the role of CCNC in brown
   adipose tissue (BAT) in vivo remains unclear.

Methods

   We generated conditional knock-out mice by crossing Ccnc^flox/flox mice
   with Myf5^Cre, Ucp1^Cre or Adipoq^Cre transgenic mice to investigate
   the role of CCNC in BAT development and function. We applied glucose
   and insulin tolerance test, cold exposure and indirect calorimetry to
   capture the physiological phenotypes and used immunostaining,
   immunoblotting, qRT-PCR, RNA-seq and cell culture to elucidate the
   underlying mechanisms.

Results

   Here, we show that deletion of CCNC in Myf5^+ progenitor cells caused
   BAT paucity, despite the fact that there was significant neonatal
   lethality. Mechanistically different from in vitro, CCNC deficiency
   impaired the proliferation of embryonic brown fat progenitor cells
   without affecting brown adipogenesis or cell death. Interestingly, CCNC
   deficiency robustly reduced age-dependent lipid accumulation in
   differentiated brown adipocytes in all three mouse models.
   Mechanistically, CCNC in brown adipocytes is required for lipogenic
   gene expression through the activation of the C/EBPα/GLUT4/ChREBP axis.
   Consistent with the importance of de novo lipogenesis under
   carbohydrate-rich diets, high-fat diet (HFD) feeding abolished CCNC
   deficiency -caused defects of lipid accumulation in BAT. Although
   insulin sensitivity and response to acute cold exposure were not
   affected, CCNC deficiency in Ucp1^+ cells enhanced the browning of
   white adipose tissue (beiging) upon prolonged cold exposure.

Conclusions

   Together, these data indicate an important role of CCNC-Mediator in the
   regulation of BAT development and lipid accumulation in brown
   adipocytes.

   Keywords: CCNC, brown fat, Lipid droplet, Progenitor, Proliferation,
   Lipogenesis

   Abbreviations: CCNC, cyclin C; BAT, brown adipose tissue; UCP1,
   uncoupling protein 1; C/EBP, CCAAT/enhancer-binding protein; ChREBP,
   carbohydrate response element-binding protein; SREBP, sterol regulatory
   element-binding protein

Highlights

     * •
       CCNC plays different roles at different developmental stages in
       BAT.
     * •
       CCNC controls the prenatal BAT development by regulating the
       proliferation of brown progenitors.
     * •
       CCNC critically supports lipid accumulation in adult BAT through
       the C/EBPα/GLUT4/ChREBP axis.

1. Introduction

   The current epidemic of obesity and type 2 diabetes has increased the
   need for novel approaches to reduce adiposity. Obesity is caused by
   prolonged periods of positive energy balance in which energy intake
   exceeds energy expenditure. Brown adipose tissue (BAT) is specialized
   to dissipate energy through uncoupling protein 1 (UCP1) and thus may
   counteract obesity [[37]1]. Rodents also displays an inducible
   thermogenic adipose tissue termed beige adipocytes that arises within
   white adipose tissue (WAT) depots, that can use both UCP1 and possibly
   other futile cycles for heat production [[38]2,[39]3]. In humans, BAT
   is abundant at birth but is rapidly replaced by WAT through unknown
   mechanisms and is relatively scarce in adults as an unidentifiable
   tissue [[40]4]. However, studies with ^18fluoro-labelled
   2-deoxy-glucose positron emission tomography (^18FDG-PET) scanning
   demonstrated that adult humans could have active thermogenic adipose
   tissue deposits and the amount of this thermogenic tissue is inversely
   correlated with adiposity and body mass index (BMI) [[41]5,[42]6].
   Whether human thermogenic adipose tissues are more reflective of rodent
   brown or beige adipocytes has not been fully resolved. In any case,
   activation of BAT thermogenesis by cold exposure, or by β3-adrenergic
   receptor agonist, has been linked to increased energy expenditure,
   reduced adiposity and lower plasma lipids [[43][7], [44][8], [45][9]],
   indicating that BAT plays an important role in energy homeostasis both
   in animal models and humans. Therefore, a better understanding of the
   molecular control of BAT development and function may lead to new
   therapeutic avenues to combat obesity and metabolic disorders.

   The classical brown fat is localized in the interscapular region in
   rodents and arises from a Myf5-positive lineage [[46]10].
   PR-domain-containing protein 16 (PRDM16), a BAT-enriched transcription
   cofactor, acts as a fate switch to control brown adipocyte versus
   myocyte between days 9–12 of gestation in mice [[47]10,[48]11].
   Mechanistically, PRDM16 forms a transcriptional complex with the active
   form of CCAAT/enhancer-binding protein-beta (C/EBPβ) in the myogenic
   precursors to activate the brown adipogenic gene program by inducing
   peroxisome proliferator-activated receptor-gamma (PPARγ) expression
   [[49]12]. Meanwhile, it has been shown that euchromatic histone-lysine
   N-methyltransferase 1 (EHMT1) is a BAT-enriched lysine
   methyltransferase in complex with PRDM16 and regulates brown adipocyte
   fate by stabilizing PRDM16 proteins [[50]13]. Extracellularly, BMP7
   signaling promotes brown adipocyte differentiation and thermogenesis
   through the induction of PRDM16 and PPARγ coactivator-1a (PGC-1a),
   which increase the expression of the brown fat marker UCP1 and
   adipogenic transcription factors PPARγ and C/EBPs as well as
   mitochondria biogenesis [[51]14]. However, the regulatory mechanisms
   for BAT development are not completely understood.

   Unlike white adipocytes, differentiated brown adipocytes are
   characterized by multilocular lipid droplets, in which triglycerides
   are increasingly accumulated with age. Although the physiological
   functions remain less clear, lipolysis and lipogenesis in BAT are
   dynamic and highly active in response to environmental cues. The
   regulation of lipolysis in BAT has been well documented
   [[52]15,[53]16], but the regulation of lipogenesis and triglyceride
   biosynthesis in BAT is less studied. Recently, one study has shown that
   AKT2, a cold-induced kinase, promotes de novo lipogenesis (DNL) in BAT
   through carbohydrate response element-binding protein (ChREBP) but not
   sterol regulatory element-binding protein-1 (SREBP-1) [[54]17].
   Moreover, DNL in BAT may play a role in optimizing fuel storage and
   thermogenesis in mice, as inhibition of DNL in BAT causes compensatory
   increase of WAT browning under prolonged cold exposure [[55]17].
   Importantly, brown fat DNL in human is positively correlated with Ucp1
   expression, and negatively correlated with BMI [[56]17,[57]18].
   Although there have been significant insights into the signaling
   pathways and transcription factors that regulate DNL in BAT, the
   regulation of lipid metabolism in BAT remains largely unclear.

   The Mediator complex is a conserved transcription cofactor that
   primarily regulates activator-dependent transcription [[58][19],
   [59][20], [60][21], [61][22]]. In mammalian cells, the Mediator complex
   is comprised of up to 30 subunits [[62][19], [63][20], [64][21],
   [65][22]]. It has been shown that the Mediator subunit MED1 regulates
   white adipogenesis in vitro through PPARγ [[66]23,[67]24]. MED14
   [[68]25], MED19 [[69]26], MED23 [[70]27,[71]28] and MED31 [[72]29] are
   also involved in the regulation of white adipogenesis in vitro and in
   mouse models through PPARγ and/or other aspects of insulin signaling
   pathway. Moreover, MED1 regulates the brown adipocyte transcriptional
   program by interacting with PRDM16 [[73]30,[74]31]. More recently,
   using tissue specific MED1 knockout mouse models, two studies have
   demonstrated a critical role of MED1 in adipose tissue formation
   in vivo [[75]32,[76]33]. On the other hand, the Mediator subunits MED1
   [[77]33], MED15 [[78]34] and MED17 [[79]35,[80]36] are directly or
   indirectly involved in the induction of lipogenesis. Previously, we
   have shown that the Mediator subunit CDK8 together with its activating
   partner CCNC negatively regulates DNL in hepatocytes and Drosophila fat
   bodies by promoting nuclear SREBP-1a/c phosphorylation and subsequent
   degradation [[81]37]. Although initially identified as a potential cell
   cycle regulator [[82]38,[83]39], CCNC functions to regulate gene
   transcription in the context of the Mediator complex. As a highly
   conserved Mediator subunit, CCNC has been shown to play multiple roles
   in various biological processes including cell proliferation [[84][38],
   [85][39], [86][40]], cell apoptosis [[87]41,[88]42], and cellular lipid
   metabolism [[89]37,[90]43]. Recently, we have identified CCNC as an
   essential regulator for brown adipogenesis in vitro [[91]43].
   Mechanistically, CCNC-Mediator physically interacts with C/EBPα and
   activates the C/EBPα transactivation domain activity to facilitate
   brown preadipocyte differentiation [[92]43]. Interestingly, the CCNC
   level is decreased during brown adipogenesis and during aging in mice
   [[93]43], suggesting that CCNC may play a role in BAT development and
   function. Human individuals with mutations in the genomic locus of
   Cdk8, one major effector of CCNC, are prone to develop type 2 diabetes
   [[94]44]. Moreover, the GWAS Central data indicate that human Ccnc gene
   is significantly associated with BMI. However, the underlying mechanism
   remains unknown. Based on these findings, we hypothesize that CCNC
   plays an important role in BAT development and function in vivo.

   Here, we show that CCNC deletion in Myf5^+ cells caused a partial
   neonatal lethality in mice likely due to the developmental defects in
   rib cartilage. The survived CCNC-deficient mice displayed a paucity in
   BAT due to the impaired proliferation of brown adipocyte progenitor
   cells. In contrast to tissue culture, CCNC deficiency did not affect
   brown adipogenesis in vivo. Interestingly, CCNC-deficient BAT displayed
   a reduced ability of accumulating lipids over time when mice were fed
   with a carbohydrate-rich normal chow diet. This phenotype was
   recapitulated when CCNC was deleted in either Ucp1^+ or Adipoq^+ cells.
   RNA-seq analyses revealed that CCNC deficiency reduced the expression
   of some key enzymes for fatty acid and triglyceride biosynthesis that
   are critically regulated by the C/EBPα/GLUT4/ChREBP pathway. Consistent
   with the regulation of DNL, CCNC depletion -caused lipid accumulation
   defects in BAT was abolished when mice were fed with a high fat diet
   (HFD). Moreover, deletion of CCNC in Ucp1^+ cells also enhanced WAT
   beiging upon chronic cold exposure. Thus, our results indicate that
   CCNC plays a pivotal role in BAT prenatal development and postnatal
   lipid metabolism.

2. Materials and methods

2.1. Animals and diets

   All mouse experiments conformed to the protocols approved by the Animal
   Care and Use Committees of the Albert Einstein College of Medicine in
   accordance with the National Institutes of Health guidelines. Myf5^Cre
   (Stock #010529), Ucp1^Cre (Stock #024670) and Adipoq^Cre (Stock
   #028020) transgenic mice were obtained from the Jackson Laboratory.
   Ccnc-floxed mice was generated and backcrossed for ten generations into
   the C57BL/6J strain. Mice were housed in a pathogen-free animal
   facility at 22 °C with a 12 h light/dark cycle (7:00 am - 7:00 pm) with
   free access to water and the normal chow diet (#5053, LabDiet; 13 kcal%
   fat and 62 kcal% carbohydrate) or the high-fat diet (no. D12492,
   Research Diets; 60 kcal% fat and 20 kcal% carbohydrate), which started
   at the age of six weeks.

2.2. Body composition and indirect calorimetry

   Fat/lean mass was measured by quantitative nuclear magnetic resonance
   (NMR) noninvasive imaging (EchoMRI, TX). Metabolic measurement was
   performed using an Oxymax indirect calorimetry system (Columbus
   Instruments, Columbus, OH). Mice were individually housed in metabolic
   chambers at 22 °C with a 12 h light/dark cycle and free access to food
   and water. After acclimation in metabolic chambers for 48 h, energy
   expenditure, oxygen consumption (VO[2]), carbon dioxide production
   (VCO[2]), and spontaneous locomotor activity were measured as
   previously described [[95]45]. VO[2], VCO[2], and energy expenditure of
   each mouse were normalized by its lean mass.

2.3. Glucose and insulin tolerance test (GTT and ITT)

   For GTT, mice were fasted for 14 h and then received an intraperitoneal
   injection of glucose (1 g/kg body weight). Two weeks later, ITT was
   performed by an intraperitoneal injection of insulin (1.5 U/kg body
   weight) after 4 h fasting. Blood was collected at consecutive time
   points after the injection of glucose or insulin by tail bleeding, and
   blood glucose levels were measured using the AlphaTRAK 2 Blood Glucose
   Monitoring System.

2.4. Cold exposure

   To assess cold tolerance, 8-month-old control and Ccnc^Ucp1(KO) mice
   were fasted for 4 h in the morning, and then placed at 6 °C
   individually in pre-chilled cages with free access to pre-chilled water
   and food. Intrarectal temperatures were monitored and recorded with a
   TH-8 Thermalert Monitoring Thermometer (Physitemp Instruments, Clifton,
   NJ) at consecutive time points up to 14 days.

2.5. Measurement of triglycerides

   Triglycerides in BAT were measured using the Triglyceride
   Quantification Colorimetric Fluorometric Kit (Biovision, Milpitas, CA)
   according to the manufacturer's instructions. Briefly, total lipids
   were extracted from homogenized BAT (∼20 mg each), and triglycerides
   were converted to free fatty acids and glycerol by the addition of
   lipases. Glycerol was detected by an enzyme-coupled reaction and using
   a VersaMax spectrometry microplate reader (Molecular Devices,
   Sunnyvale, CA). The triglyceride levels were normalized by genomic DNA.

2.6. Alcian Blue-Alizarin Red staining

   Dead Ccnc^Myf5(KO) newborns and littermate controls (alive) were fixed
   in 4% paraformaldehyde for Alcian Blue-Alizarin Red staining as
   previously described with minor modifications [[96]46]. Briefly, skins
   and tissues were carefully removed, and fat was depleted by washing
   with 95% ethanol followed by overnight incubation in acetone. The
   skeletons were immersed in 0.03% alcian blue solution for 24 h to stain
   cartilage, washed with 70% ethanol, and incubated in 1% potassium
   hydroxide until clear. Bone was counterstained with 0.03% alizarin red
   solution until sharply delineated. The stained samples were cleaned in
   1% potassium hydroxide/glycerol (1:1) for a week and kept in glycerol
   at the end. Images were captured by a Nikon Digital Camera and
   processed using the Photoshop CS6 software.

2.7. TUNEL assay, immunofluorescence, immunohistochemistry and histological
analyses

   An 8-μm thick, 4% neutral buffered paraformaldehyde-fixed cryosections
   of tissues were embedded in an optimal cutting temperature compound.
   TUNEL assay was performed using the ApopTag Red in situ Apoptosis
   Detection Kit (EMD Millipore, Burlington, MA) according to the
   manufacturer's instructions. Briefly, sections were treated with the
   Equilibration Buffer and the Working Strength TdT Enzyme sequentially,
   and counterstained with an anti-digoxigenin conjugate antibody and
   mounted with the prolong antifade reagent with DAPI (Invitrogen) before
   imaging. For Ki67 immunofluorescent staining, slides were permeabilized
   with 0.3% Triton X-100 (Sigma–Aldrich), washed with 1xPBS, and blocked
   with 5% normal donkey serum (ab166643, Abcam) before adding anti-Ki67
   primary antibody (ab16667, Abcam, 1:500) that was diluted in blocking
   solution. After wash, Alexa Flour 488-labelled secondary antibody
   (Invitrogen, 1:500) was added. The slides were then washed,
   counterstained, and mounted with the prolong antifade reagent with DAPI
   (Invitrogen) before imaging. For UCP1 immunohistochemical staining,
   5 μm thick paraffin sections were deparaffinized, rehydrated, and
   epitopes unmasked by boiling with the Citra buffer (BioGenex, Fremont,
   CA). Tissues were then blocked with 5% normal goat serum solution
   before incubation with anti-UCP1 primary antibody (PA1-24894, Pierce,
   1:200). Vectashield DAB Peroxidase Substrate Kit was used to detect the
   primary antibody. Sections were counterstained with hematoxylin,
   mounted, and imaged. For histological analyses, samples were embedded,
   cut, and performed Hematoxylin and Eosin (H&E) staining by the Albert
   Einstein College of Medicine Histology Core facility.

2.8. Western blotting

   Cultured cells were washed with cold 1xPBS, scraped, and homogenized by
   pipetting in a lysis buffer containing 50 mM Tris–HCl (pH 8.0), 420 mM
   NaCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 0.05% SDS, 10% Glycerol, and a
   Protease and Phosphatase Inhibitor Cocktail (78442, Thermo Fisher
   Scientific). Tissues were homogenized in the lysis buffer using Ceria
   Stabilized Zirconium Oxide Beads. The cell or tissue homogenates were
   centrifuged for 30 min at 21,000×g at 4 °C, and supernatants were
   collected for protein assays. Twenty μg of total proteins for each
   sample were loaded and separated by 4–12% SDS-PAGE and transferred to a
   nitrocellulose membrane using the iBlot Blotting System (Thermo Fisher
   Scientific). The membrane was blocked with 5% non-fat dry milk in 1x
   Tris-buffered saline containing Tween-20 (TBST) and incubated with a
   primary antibody in the blocking buffer. The membranes were washed with
   1xTBST and incubated with HRP-conjugated secondary antibody in blocking
   buffer. The membrane was then washed with 1xTBST, visualized through
   ECL (Pierce, Thermo Fisher Scientific), and exposure to X-ray films.
   The following primary antibodies and dilutions were used: Anti-CCNC
   (558903,1:500) from BD Pharmingen; Anti-Actin (A5060, 1:200) from
   sigma; Anti-β-tubulin (1235662A, 1:2000) from Invitrogen; Anti-ChREBP
   (NBP2-44307, 1:500) from NOVUS; Anti-C/EBPa (8178, 1:250), Anti-FAS
   (3180, 1:1000) and Anti-ACC1 (3676, 1:1000) from Cell Signaling
   Technology; Anti-GLUT4 (ab654, 1:500) from Abcam; Anti-SREBP1
   (sc-13551, 1:200) and Anti-CPT1B (sc-393070, 1:500) from Santa Cruz
   Biotechnology.

2.9. RNA extraction and real-time RT-PCR

   Total RNA was prepared from tissue or cell samples using the Trizol
   Reagent (Invitrogen) according to the manufacturer's instructions.
   Genomic DNA contamination was removed by RNase-free DNase I (Thermo
   Scientific, USA). The quantity and quality of total RNA were determined
   by a spectrophotometer (Nanodrop 2000, Thermo Fisher). Total cDNA was
   synthesized from 1 μg of total RNA using iScript cDNA Synthesis Kit
   (BIO-RAD). Real-time PCR was performed using the PowerUp SYBR Green
   Master Mix (Thermo Fisher, USA). Specific primers for each gene are
   listed in [97]Table S1. Tbp or Rpl7 was used as the invariant control.

2.10. RNA sequencing (RNA-seq)

   After RNA extraction from BAT of 6-month-old Ccnc^Ucp1(KO) and control
   mice (three biological replicates each), RNA-seq libraries were
   prepared by the Einstein Genomics Core Facility. The library quality
   was analyzed by a Bioanalyzer (Agilent Technologies). Deep sequencing
   was performed using a HiSeq 2500 instrument (Illumina). Raw reads for
   each library were mapped using TopHat version 2.0.8 against the indexed
   mouse (mm9) genome, and transcripts were assembled using Cufflinks.
   Genes that were not expressed in all samples (FPKM<1) were filtered
   out. Differentially expressed genes (DEGs) were identified using a
   RNA-seq processing tool DESeq2, selected by adjusted_p value < 0.05 and
   fold change >1.4 or < 0.5, and visualized using R tools ggplot2 and
   pheatmap, respectively. Kyoto encyclopedia of genes and genomes (KEGG)
   pathway enrichment analysis of DEGs was obtained from DAVID database
   (version 6.8). The heat map for the eight down-regulated genes in the
   insulin signaling pathway was based on the normalized reads, and they
   were listed according to their p values.

2.11. Tissue culture

   Immortalized wildtype and CCNC-knockout brown preadipocytes were
   generated as shown previously [[98]43]. These cells were maintained in
   DMEM with 25 mM glucose plus 10% FBS, 1% penicillin-streptomycin (P/S)
   and 1% L-glutamine. For differentiation, cells were cultured to
   confuent and then treated with the differentiation medium of DMEM
   containing dexamethasone (0.5 mM), insulin (20 nM),
   isobutylmethylxanthine (0.5 mM), indomethacin (0.125 mM), T3 (1 nM) and
   rosiglitozone (0.5 μM). After two days of culture, cells were
   maintained in DMEM containing 1 nM T3 and 20 nM insulin. Medium was
   replaced every other day. Full differentiation was achieved in about
   seven days.

2.12. Retrovirus transduction

   Mouse ChREBPβ was amplified by PCR from pCMV-Flag-ChREBPα, which was a
   gift from Dr. Donald K. Scott of Icahn School of Medicine at Mount
   Sinai, and subcloned into the EcoR1 site of the retroviral vector
   pMSCV-PIG using an In-Fusion Cloning kit (Clontech) to generate a
   ChREBPβ-expressing plasmid, termed pMSCV-PIG-ChREBPβ, which was
   verified by DNA sequencing. Retroviral packaging plasmids, pUMVC
   (#8449) and pCMV-VSV-G (#8454), were obtained from Addgene. To generate
   pseudotyped virus, pUMVC, pCMV-VSV-G and pMSCV-PIG vectors were
   co-transfected into sub-confluent HEK293T cells using Lipofectamine
   3000 Transfection Reagent (Invitrogen) according to the manufacturer's
   instructions. The virus stocks were collected at 48 h and 72 h after
   transfection, filtered through a 0.45 μm filter, and frozen at −80 °C.
   Brown preadipocytes (20–30% confluent) were infected with retrovirus
   stocks containing 8 μg/ml polybrene for 12 h, washed and cultured in
   DMEM.

2.13. Luciferase reporter assays

   The constructs pcDNA3-Flag-C/EBPα (#66978) and 3xPPRE-TK-luc (#1015)
   were obtained from Addgene. The Slc2a4 promoter was amplified from
   mouse genomic DNA by PCR [[99]47], and subcloned into 3xPPRE-TK-luc by
   replacing the 3xPPRE sequence, which is located between Hind III and
   BamH I sites, using In-Fusion Cloning kit (Clontech). The resulting
   plasmid, Slc2a4-TK-luc, was verified by DNA sequencing. DNA
   transfection and luciferase reporter assays were conducted as described
   previously [[100]43]. Briefly, brown preadipocytes were seeded at a
   density of 1 × 10^5 per well in 24-well plates. Next day, cells were
   transfected with a mix of 250 ng of pcDNA3-Flag-C/EBPα (or control),
   250 ng of Slc2a4-TK-luc and 50 ng of renilla luciferase reporter using
   the Lipofectamine 3000 Transfection Reagent. After incubation for two
   days, cells were lysed and analyzed using the Dual-Luciferase System
   (Promega) according to the manufacturer's instructions. The firefly
   luciferase activity was normalized by the corresponding renilla
   luciferase activity.

2.14. Statistical analyses

   All numerical results were expressed as Mean ± SD. Statistical
   difference was determined by unpaired Student's t-test at a
   significance level of p < 0.05. The statistical analyses and figure
   preparation were performed using GraphPad Prism 9 (GraphPad software).

3. Results

3.1. CCNC deficiency in the Myf5^+ lineage causes partial neonatal lethality
and reduction of brown adipose tissues

   To understand the role of CCNC in BAT development, we deleted CCNC in
   Myf5^+ cells by crossing Ccnc^flox/flox mice with Myf5^Cre transgenic
   mice, as Myf5 is expressed specifically in the progenitor cells that
   give rise to brown adipocytes and skeletal muscle cells [[101]10]. As
   shown in [102]Figure 1A, genotyping analyses revealed that
   approximately 10% of mice were homozygous CCNC knockout (Ccnc^Myf5(KO):
   Ccnc^flox/flox; Myf5^Cre) at the time of weaning. While the
   distribution of CCNC heterozygous or wildtype mice was closer to the
   expected 25% Mendelian ratio, the percentage of Ccnc^Myf5(KO) mice was
   significantly lower than 25%, suggesting that many Ccnc^Myf5(KO) mice
   may have died before weaning. Although a previous study has shown that
   germline CCNC ablation causes embryonic lethality at E10.5 [[103]40],
   partial lethality of Ccnc^Myf5(KO) pups was not expected. After closely
   monitoring the breeding pairs, we discovered an unusual number of dead
   newborns, and genotyping analyses showed that the majority of those
   dead pups (25 out of 30) were Ccnc^Myf5(KO). However, genotyping
   analyses of embryos showed normal Mendelian ratios for all genotypes
   ([104]Figure 1A), suggesting that the death of those Ccnc^Myf5(KO) mice
   may have occurred shortly after birth.

Figure 1.

   [105]Figure 1
   [106]Open in a new tab

   CCNC deficiency in the Myf5^+ lineage causes partial neonatal lethality
   and prenatal brown fat paucity. (A) Summary of the genotypes of mice at
   E18.5 and P1 stages. (B) Representative pictures of Alcian
   Blue-Alizarin Red Skeletal Staining of the live control and dead
   Ccnc^Myf5(KO) mice at P1. The abnormal ribs are indicated by red arrow.
   (C) Macroscopic examination of BAT of the control and Ccnc^Myf5(KO)
   mice at P1. BAT is highlighted by dashed lines. (D) H&E staining of
   cervical transverse sections of the control and Ccnc^Myf5(KO) mice at
   E18.5. BAT is highlighted by dashed lines. Scale bar, 200 μm. (E)
   Representative images of BAT isolated from the live control and
   Ccnc^Myf5(KO) mice at P1. Scale bar, 1 mm. (F) Body weight of the
   control and Ccnc^Myf5(KO) mice at P1. (G) Percentage of BAT weight over
   body weight of the control and Ccnc^Myf5(KO) mice at P1. (H) qRT-PCR
   analysis of Ccnc, Cdk8 and Cdk19 expression in BAT of the control and
   Ccnc^Myf5(KO) mice at E18.5. ∗∗∗ p < 0.001 vs control. (For
   interpretation of the references to color in this figure legend, the