BACKGROUND
two types of adipose tissue exist based on morphological appearance and biological function.
white adipose tissue  stores energy in the form of triacylglycerol  for later release and use by other tissues, whereas brown adipose tissue  metabolizes fatty acids and glucose for heat production.
thermogenesis through uncoupled respiration in bat depends on a high mitochondrial density and expression of uncoupling protein  <dig>   <cit> .
larger mammals such as primates and ruminants are born fully developed and able to thermoregulate minutes after birth due to the presence of relatively large amounts of functional bat, which gets activated at birth.
the majority of this innate bat disappears after birth and is replaced by wat  <cit> .
besides brown adipocytes in bat, another type of thermogenic adipocytes exists: the brite  or beige adipocytes, which are known to be recruited in subcutaneous wat of rodents in response to cold exposure or stimulation with β-adrenergic agonists  <cit> .
the developmental origin of brite/beige adipocytes and their contribution to human bat are still a matter of debate  <cit> .
rodents as opposed to larger mammals are born with immature bat which matures only postnatally and is largely retained throughout life  <cit> .
it is relevant to understand the brown to white adipose tissue remodeling in large mammals, as it is likely to mimic the transition occurring in human infants.
the most frequently studied adipose tissue transition in a large mammal is the postnatal transformation of the perirenal adipose tissue in sheep.
around the time of birth, all visceral adipose depots in lambs are brown of nature  <cit> .
lambs are normally born with approximately 30 g perirenal adipose tissue constituting 80% of all their adipose tissue.
the brown characteristics of the perirenal adipose depot change dramatically to a white adipose phenotype within a few weeks after birth  <cit> .
although some gene expression details have been reported  <cit> , relatively little is known about this transition at the molecular level.
here we report a comprehensive time course analysis of the postnatal bat to wat transformation process of the perirenal adipose depot in lambs, including histological, biochemical and molecular examination as well as analyses of global gene expression profiles.
we provide evidence for dramatic changes in mitochondrial function and fatty acid metabolism during the adipose remodeling and we identified a number of transcriptional components linked to this adipose tissue transformation process.
methods
animals and tissues
experimental procedures were in compliance with guidelines laid down by the danish inspectorate of animal experimentation.
lambs from cross-bred ewes  in their second or third parturition sired by purebred texel ram, born and raised at a commercial farm in denmark, were used.
during gestation ewes were fed hay ad libitum, 200 g barley and 200 g commercial concentrate per day.
ewes were housed in groups of  <dig> until lambing.
after lambing they were housed individually for 2 days and subsequently housed in groups of  <dig> until they were transferred to pasture approximately one week after lambing.
the ewe-reared lambs were kept on pasture being a mixture of 70% ray grass and 30% white clover.
lambs were killed by bolt pistol and bled by licensed staff.
perirenal adipose tissue was carefully dissected and frozen in liquid nitrogen for biochemical or molecular analyses or fixed for histology as described below.
lambs at the following ages  were used: − <dig> ,  <dig> ,  <dig>  ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig> .
live weights of the lambs were kept similar within groups.
the ratio of females to males was similar  in the three phases of the transformation.
hematoxylin-eosin  staining
samples were fixed in 4% neutral buffered formaldehyde  at room temperature for 24 h and subsequently at 4°c until preparation.
the tissue was processed to paraffin and sectioned in 4 μm sections.
he staining was performed according to standard procedures.
transmission electron microscopy
samples were fixed in karnowsky’s fixative  for 3– <dig> days at room temperature and subsequently stored in  <dig>  m cacodylate buffer at 4°c until further processing.
the samples were rinsed three times in  <dig>  m sorensen’s phosphate buffer  and subsequently post fixed in 1% oso <dig> in  <dig>  m sodium cacodylate buffer  for 2 h. the specimens were dehydrated in graded series of ethanol, transferred to propylene oxide and embedded in epon according to standard procedures.
ultra-thin sections were cut with a reichert-jung ultracut e microtome and collected on single slot copper grids with formvar supporting membranes.
sections were stained with uranyl acetate and lead citrate and examined with a philips cm- <dig> transmission electron microscope, operated at an accelerating voltage of 80 kv.
digital images were recorded with a sis megaview <dig> camera and the analysis software package.
reverse transcription-quantitative polymerase chain reaction
tissues were homogenized in trizol  using a dispomix  and total rna was purified.
reverse transcriptions were performed in 25 μl reactions containing 1st strand buffer , 2 μg random hexamers ,  <dig>  mm of each dntp ,  <dig> units of rnaseout , 1 μg of total rq <dig> dnase -treated rna and  <dig> units of moloney murine leukemia virus reverse transcriptase .
reactions were left for 10 min at room temperature, followed by incubation at 37°c for 1 h. after cdna synthesis, reactions were diluted with 50 μl of water and frozen at −80°c.
the cdna was analyzed by rt-qpcr using the stratagene mx3000p qpcr system.
each pcr mixture contained, in a final volume of 20 μl,  <dig>  μl of 1st strand cdna, 10 μl of sensifast™ sybr lo-rox kit  and  <dig> pmol of each primer .
all reactions were run using the following cycling conditions: 95°c for 10 min, then 40 cycles of 95°c for 15 s, 55°c for 30 s and 72°c for 15 s. pcr was carried out in 96-well plates and each sample was run in duplicate.
target gene mrna expression was normalized to the stably expressed β-actin  mrna.
protein extracts and immunoblotting
tissues were homogenized in a gg-buffer  containing 25 mm glycyl-glycin, 150 mm kcl, 5 mm mgso <dig> and 5 mm ethylenediaminetetraacetic acid  as well as freshly added dithiothreitol , bovine serum albumin  and triton x- <dig> .
homogenization was performed with a tissuelyser  using 5 mm stainless steel beads, and homogenates were subsequently frozen in liquid nitrogen.
protein concentrations were determined by the lowry method  <cit>  and equal amounts of protein from each animal were pooled according to age and diluted in a buffer containing  <dig> % sds and 10% glycerol.
proteins were separated on 4-12% bis-tris gradient gels , blotted onto immobilon pvdf membranes  and stained with amido black 10b .
membranes were blocked in tris-buffered saline  or phosphate-buffered saline  with 5% nonfat dry milk and  <dig> % tween  <dig>  and subsequently probed with antibodies.
primary antibodies used were against transcription factor iib   , atp synthase β    and ucp <dig>  .
secondary antibodies were horseradish peroxidase-conjugated .
enhanced chemiluminescence  was used for detection.
quantification of relative mitochondrial dna  copy numbers
relative mtdna amount  was measured as the ratio between mtdna and nuclear dna .
tissues were homogenized using a tissuelyser  in lysis buffer containing 100 mm tris-base , 5 mm edta ,  <dig> % sodium dodecyl sulphate, 200 mm nacl and 100 mg/ml proteinase k and incubated overnight at 55°c with rotation.
dna was precipitated with two volumes of 99% ethanol and fished out with inoculation loops, washed in 70% ethanol and dissolved in tris-edta buffer containing 10 mg/ml rnase a at 55°c overnight.
dna concentrations were determined on the eppendorf biophotometer at 260 nm and 50 ng dna was used for qpcr.
pcr reactions and cycling conditions were as described above, and primers were against cytochrome c oxidase i   and suppression of tumorigenicity  <dig>   .
citrate synthase  activity
tissue homogenates  were generated in gg-buffer  as described above.
homogenates were thawed on ice and centrifuged at 4°c at  <dig>  g for 2 min.
supernatants were used for activity measurements.
cs activity was measured spectrophotometrically at 25°c and 412 nm in cs buffer containing 100 mm tris-base , 10 mm  <dig> ′-dithiobis, 5 mm acetyl-coa and 50 mm oxaloacetic acid and activity was measured as described  <cit> .
each sample was measured in duplicate and the mean was used for subsequent calculations.
activities were normalized to the amount of total protein determined by the lowry method  <cit> .
statistical analyses of qpcr data
the time course study was analyzed for statistical significance using one-way anova and student’s t-test with bonferroni correction for multiple testing as post hoc test.
a p-value <  <dig>  was considered statistically significant.
targeted rna-sequencing and data analysis
isolation of mrna and synthesis of first strand cdna
equal amounts of total rna from perirenal adipose tissue from lambs at the same age  were pooled.
mrna was isolated from 4 μg of total rna by magnetic oligo beads, which was used to synthesize bead-bound cdna, according to the instructions of the manufacturer .
tag library construction
the library for digital gene expression analysis was constructed according to the instructions of the manufacturer .
bead-bound cdna was digested with nlaiii, followed by ligation of the gex adapter  <dig> to the bead-bound nlaiii-digested cdna.
this was then digested with mmei, releasing the gex adaptor  <dig> linked to 17 bp cdna from the beads.
the released fragment was ligated to gex adapter  <dig>  the 17 bp tags of cdna were pcr amplified using two primers that anneal to the two adapters.
the resultant tag library was used for illumina sequencing.
data analysis for rna-seq data
quality control, trimming and adapter removal was performed using fastqc  <cit>  and fastx_clipper from the fastx-toolkit  <cit> .
the  <dig> bases catg were added to the 5’ end of reads to increase the specificity of mapping.
bwa  <cit>  was employed for the alignment and mapping of reads to the sheep genome.
the october  <dig> release of the sheep genome v <dig>  <cit>  was used as reference for the alignment.
mapped reads were sorted and indexed with samtools  <cit> .
since the rna-seq data was used for expression quantification, duplicated reads were not removed.
ht-seq  <cit>  was used for counting mapped reads per annotated gene, using the annotation file provided for the sheep genome v <dig> release.
deseq  <cit>  and r  <cit>  were used for the post processing and statistical analysis of these read counts.
principal component analysis  and hierarchical clustering of time points
a two-dimensional pca plot was employed to visualize the overall effect of experimental covariates.
hierarchical clustering of the total gene expression was performed using a distance matrix to assess the relationship between the samples and identify clusters amongst the time points.
grouping of time points
based on the pca and hierarchical clustering of the total gene expression, days − <dig> and  <dig> were used as replicates of the “brown adipose state”, days  <dig> ,  <dig>   <dig> and  <dig> as replicates of the “transition state” and day  <dig> as the “white adipose state”.
using the deseq package of bioconductor  <cit> , differentially expressed genes were found between the brown adipose and the transition state as well as between the transition and the white adipose state.
for the differential gene expression between “transition state” and “white adipose state”, the partial replicate function of deseq for calculation of dispersion of the genes was used.
this method ignores the sample from day  <dig> when estimating dispersion for the genes and would use only days  <dig> ,  <dig>   <dig>   <dig> for calculating the dispersion per gene, which is an indicator of variability in that gene, and is used to fit the negative binomial model on the count data.
heatmaps representing clustering for the differentially expressed genes were created using the ggplots  <cit>  package in r. the sheep proteins were queried against the human non-redundant protein database using blast  <cit>  to find human homologous genes for further functional analysis.
reciprocal blast, a computation method used to countercheck the blast results, was employed to filter the correct mapping between the sheep and human proteins.
the biomart tool on ensemble version  <dig>  <cit>  was used for gene identification and conversion, and for obtaining human genome organization  gene nomenclature committee  approved gene names for the human homologous proteins.
uniprot  <cit>  was used to annotate the proteins for function, transcriptional activity and subcellular localization.
go term enrichment was done using explain™  <cit>  from biobase corporation.
the explain tool uses false discovery rate  correction for finding significantly enriched go classes.
for the two sets of differentially expressed genes obtained from deseq analysis, enrichr  <cit>  was used to find enrichment of transcription factors and the corresponding targets for the differentially expressed transcription factors from the transfac  <cit>  and jasper  <cit>  databases.
enrichr applies a fisher’s exact test to calculate significance, which is further corrected for multiple testing and the resulting adjusted p-values are used for selecting significant transcription factors for the differentially expressed genes.
RESULTS
characterization of the postnatal brown to white adipose transformation
at birth  the perirenal adipose tissue macroscopically appeared dark brown.
the brown color fainted steadily during the time course, and the tissue ended up being white in appearance at postnatal days  <dig> and  <dig> .
accompanying the “whitening”, the volume of the tissue gradually increased .
he-stained sections were prepared from all lambs, and representative sections from days  <dig>   <dig>   <dig>   <dig> and  <dig> are presented in figure 1a.
in the first week of life, the tissue was an apparent mixture of brown adipocytes with multilocular lipid droplets and white adipocytes with large unilocular lipid droplets.
the perirenal adipose tissue contained by appearance mostly brown adipocytes at early ages , whereas white adipocytes were predominant from day  <dig> figure  <dig>
characterization of the postnatal brown to white adipose transformation.
hematoxylin-eosin  staining of perirenal adipose tissue at postnatal days  <dig>   <dig>   <dig>   <dig> and  <dig>  representative he-stained sections are shown for the indicated time points .
total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis.
relative expression was measured for uncoupling protein  <dig> , type ii iodothyronine deiodinase  and peroxisome proliferator-activated receptor γ  co-activator 1α .
the mrna expression levels were normalized to expression of β-actin .
data are mean + sem ; *, p <  <dig>  vs. day  <dig>   the level of uncoupling protein  <dig>  and atp synthase subunit β  was determined by immunoblotting on protein pools, one for each day during the time course.
transcription factor iib  was used as a loading control.
to approach the bat to wat transformation in molecular terms, we measured mrna and protein levels of selected marker genes by rt-qpcr and immunoblotting, respectively .
expression of ucp <dig>  the brown adipocyte-specific key thermogenic factor, was high and relatively stable until day  <dig>  after which it became nearly undetectable.
the bat-enriched factors type ii iodothyronine deiodinase  and peroxisome proliferator-activated receptor γ  co-activator 1α  were also highly expressed at days − <dig> and  <dig>  but displayed a faster and stepwise decrease in expression, being considerably reduced already at days  <dig>  and  <dig> and poorly expressed after day  <dig> .
in summary, at the level of macroscopic, microscopic and molecular analyses, we observed the expected postnatal transformation of bat to wat.
mitochondrial density declined during brown to white adipose transformation
the ultra-structure of the perirenal adipose tissue was investigated at selected days by tem .
tem confirmed the mixed presence of multilocular and unilocular adipocytes at days  <dig> to  <dig> and the predominant presence of the latter at day  <dig>  adipocyte mitochondrial density was very high at days  <dig> to  <dig> and appeared lower at day  <dig>  to estimate mitochondrial density quantitatively, we determined mtdna content by qpcr as the ratio of mtdna and ndna .
this ratio decreased approximately 7-fold between days  <dig> and  <dig>  indicating that the number of mitochondria per cell diminished during the bat to wat transformation.figure  <dig>
mitochondrial density declines during brown to white adipose transformation.
total dna was isolated from perirenal adipose tissues and analyzed by qpcr with primers specific for mtdna ) and ndna ).
the relative mtdna copy number was obtained as the ratio of mt-co <dig> to st <dig> levels.
total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis.
relative expression was measured for citrate synthase .
the mrna expression levels were normalized to expression of β-actin .
enzyme activity  of cs was determined spectrophotometrically in homogenates of perirenal adipose tissue and normalized to protein content.
relative expression of cytochrome c <dig>  was measured by rt-qpcr as described in panel b. data are mean + sem ; *, p <  <dig>  vs. day  <dig>
the ultra-structural observations and the mtdna/ndna ratios prompted us to investigate more carefully gene expression of relevance for mitochondrial abundance and function.
the mrna levels of the tricarboxylic acid  cycle enzyme cs decreased gradually during the time course .
cs activity, on the other hand, was high and stable until day  <dig>  after which it dropped .
two other mitochondrial genes were analyzed: the electron transporter cytochrome c <dig>  and atp5b.
levels of cyc <dig> mrna  and atp5b protein  displayed a time profile similar to that of cs activity.
a number of nuclear transcription factors and co-regulators regulate expression of genes encoding mitochondrial proteins.
these factors include pgc- <dig> family members, nuclear respiratory factor  <dig>  and a number of nuclear receptors.
in addition to ppargc1a , we measured the expression of ppargc1b, ppara , estrogen-related receptor α  and nrf <dig> by rt-qpcr .
the expression pattern of ppargc1b and erra was similar to cyc <dig>  with stable expression until day  <dig>  followed by lower expression at subsequent time points.
expression of ppara and nrf <dig> transiently decreased after birth .
the decrease in expression of ppargc1a, ppargc1b and erra is consistent with the decline in mitochondrial density.
accordingly, based on ultra-structure, relative mtdna measurements, expression and activity of key mitochondrial enzymes as well as expression of transcription factors and co-regulators controlling levels of mitochondrial factors, we concluded that mitochondrial density and function declined remarkably during the transition from bat to wat.
global gene expression analysis of postnatal brown to white adipose transformation
to obtain a global view of gene expression changes during the bat to wat transformation in the perirenal adipose depot, tag-based sequencing was performed on pools of mrna from days − <dig>   <dig>   <dig> ,  <dig>   <dig>   <dig> and  <dig>  the resulting reads were mapped to the october  <dig> release of the sheep genome   <cit> .
the library sizes obtained for the seven samples are approximately  <dig> , <dig>  out of these, approximately 94% of the reads mapped to the reference genome, with approximately 56% of reads mapping uniquely to the reference genome .
nearly 40% of the reads, those mapping to multiple locations, were discarded.
the uniquely mapped reads covered  <dig>  out of  <dig>  annotated genes in the sheep genome, with an average read depth of 16x.
the relatively low number of reads mapping to gene-annotated regions  accounts for the low annotation coverage available for the sheep genome.
the number of mapped reads per annotated gene was counted, and normalized read counts per gene for the  <dig> time points were calculated .
to facilitate downstream analyses, the human genes homologous to the sheep genes were mined, and the results from the gene expression analysis are discussed using the human protein symbols and names.
next, we compared the expression of the genes measured by rt-qpcr in figures  <dig>   <dig> and additional file 2: figure s1b to their expression in the tag-based sequencing data.
expression of ucp <dig>  dio <dig>  ppargc1a, ppargc1b, cs, cyc <dig> and erra decreased from day  <dig> to day  <dig> in both the sequencing data and when measured by rt-qpcr .
in general, there was a relatively high correlation in the expression data obtained by the two methods.
the gene expression profiles were made from whole perirenal adipose tissue samples containing adipose as well as non-adipose cells.
the amount and composition of non-adipose cells might have changed during the transformation of the tissue, thereby potentially influencing the differential gene expression between time points.
however, the most highly expressed gene at both day  <dig> and day  <dig> was fatty acid-binding protein  <dig> , a gene known to be strongly enriched in adipocytes .
among the  <dig> genes with the highest expression level at day  <dig> and day  <dig> were several genes encoding ribosomal proteins, fabp <dig>  the fatty acid transporter cluster of differentiation  <dig> , the glycolytic enzyme aldolase a  and regulator of cell cycle , a cell cycle regulator and kinase modulating protein.
genes highly expressed at day  <dig> included the pentose phosphate pathway enzyme transaldolase  and catalase .
when comparing the  <dig> most highly expressed genes, genes related to fatty acid oxidation, electron transport chain and atp synthase activity were more prevalent at day  <dig> compared to day  <dig>
identification of three phases in the brown to white adipose tissue transformation process
to analyze the distribution of gene expression, a pca was performed on the total gene expression data set .
the pca plot indicated that total gene expression at the different time points clustered into three groups: a group including days − <dig> and  <dig>  a second group including days  <dig> ,  <dig>   <dig> and  <dig>  and a third group comprising day  <dig>  hierarchical clustering of the total gene expression data set clustered the  <dig> time points into the same three groups .
we interpreted the three clusters as distinct phases in the bat to wat transition .
at days − <dig> and  <dig> the tissue is in the brown adipose phase.
during the phase composed of days  <dig> ,  <dig>   <dig> and  <dig> the tissue is expected to be in a thermogenic state in order to keep the lambs warm.
at the same time points expression of some bat-associated genes starts to drop, e.g.
illustrated by the decrease in ppargc1a expression from day  <dig> to day  <dig>  .
therefore, we designate this period the transition phase.
day  <dig> represents the white adipose phase, as was also suggested by tissue morphology, mitochondrial numbers and function as well as expression level of ucp <dig> .figure  <dig>
identification of the brown adipose phase, transition phase and white adipose phase.
principal component analysis  plot for the expression data from the seven time points showing the clustering of time points in the first two components.
heatmap showing the hierarchical clustering based on euclidean distances between the time points.
allocation of the different time points to the three phases and summary of numbers of induced and repressed genes between phases.
the expression of  <dig> genes changed significantly  between the brown adipose phase and the transition phase .
of these,  <dig> genes were up-regulated and  <dig> genes were down-regulated .
a heatmap with euclidian distances for the  <dig> genes is shown in figure 4a.
go enrichment analysis  of the  <dig> up-regulated genes revealed that they were enriched for genes related to “negative regulation of adaptive immune response” and “muscle cell migration”, whereas the  <dig> down-regulated genes were enriched for genes related to “organic acid metabolic processes” .figure  <dig>
gene expression changes in the two phase shifts.
heatmap of the  <dig> genes differentially expressed from the brown adipose phase to the transition phase.
heatmap of the  <dig> genes differentially expressed from the transition phase to the white adipose phase.
go enrichment analysis of genes differentially expressed from the brown adipose to the transition phase
go term
total number of genes in go term
number of differentially expressed genes
p
-value
enrichment from up-regulated genes
enrichment from down-regulated genes
between the transition phase and the white adipose phase, the expression of  <dig> genes changed significantly, of which  <dig> genes were up-regulated and  <dig> were down-regulated .
these differentially expressed genes are presented in a heatmap in figure 4b.
a go enrichment analysis  demonstrated that the  <dig> up-regulated genes were enriched for genes related to “cell death” and “negative regulation of cell death” .
the  <dig> down-regulated genes were enriched for genes related to “metabolic process”, including “fatty acid beta-oxidation” .table  <dig>
go enrichment analysis of genes differentially expressed from the transition to the white adipose phase
go term
total number of genes in go term
number of differentially expressed genes
p
-value
enrichment from up-regulated genes
enrichment from down-regulated genes
the changes in gene expression related to the go term “fatty acid beta-oxidation” were investigated in more detail by rt-qpcr .
of notice, the white adipose phase included samples from days  <dig>   <dig> and  <dig> for rt-qpcr measurements, whereas the white adipose phase for the global gene expression analysis included samples from day  <dig> only .
two key enzymes in β-oxidation are carnitine palmitoyltransferase 1b  and the hydroxyacyl-coa dehydrogenase complex .
the relative mrna expression levels of both cpt1b and the catalytic subunit α of hadh  decreased from the brown adipose phase to the transition phase and from the transition phase to the white adipose phase .
we also measured expression of two genes involved in fatty acid synthesis by rt-qpcr: acetyl-coa carboxylase  <dig>  and fatty acid synthase .
expression of both tended to increase during the postnatal adipose transformation , suggesting a higher rate of fatty acid synthesis in wat compared to bat.figure  <dig>
expression of selected metabolic enzymes related to fatty acid metabolism.
total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis.
relative expression was measured for:  carnitine palmitoyltransferase 1b  and hydroxyacyl-coa dehydrogenase subunit α ;  acetyl-coa carboxylase  and fatty acid synthase ;  diacylglycerol o-acyltransferase  <dig>  and dgat <dig>  the mrna expression levels were normalized to expression of β-actin .
data are mean + sem ; *, p <  <dig> .
the go term “metabolic process” included  <dig> differentially expressed genes, a number of which have been measured by rt-qpcr, including ucp <dig>  cyc <dig> and cs.
the two isoforms of the tag synthesis enzymes diacylglycerol o-acyltransferase  <dig>  and dgat <dig>  were also among the regulated metabolic genes.
rt-qpcr measurements confirmed a decreased expression of dgat <dig> and dgat <dig> in the white adipose phase compared to the transition phase .
of the  <dig> genes differentially expressed between the brown adipose and transition phase and  <dig> genes differentially expressed between the transition and the white adipose phase,  <dig> genes were in common.
a venn diagram of the  <dig> regulated genes is shown in additional file 7: figure s <dig>  fifteen of the  <dig> common genes were down-regulated at both phase shifts, whereas  <dig> genes were up-regulated at both phase shifts.
among the  <dig> consistently down-regulated genes were several mitochondrial genes, e.g.
the tca cycle enzyme isocitrate dehydrogenase 3α , and the two transcription factors myeloid leukemia factor  <dig>  and autoimmune regulator  .
among the consistently up-regulated genes were two receptors involved in cellular lipid uptake: low density lipoprotein receptor-related protein  <dig>  and macrophage scavenger receptor  <dig> .
the  <dig> genes also included  <dig> genes transiently up-regulated and  <dig> genes transiently down-regulated during the transition phase .
among the genes up-regulated during the transition phase were two enzymes involved in tag synthesis: the mitochondrial glycerol-3-phosphate acyltransferase  and 1-acylglycerol-3-phosphate o-acyltransferase  <dig> .
transcriptional components regulated between the three phases of adipose tissue transformation
expression of  <dig> transcription factors and co-regulators was significantly changing between the brown adipose and the transition phase, of which  <dig> were up-regulated .
between the transition and the white adipose phase,  <dig> transcriptional components were differently expressed, with  <dig> being up-regulated .
four transcription factors exhibited differential expression at both phase shifts.
using p <  <dig>  as the cut-off for adjusted p-value, two of the  <dig> transcriptional components, differently expressed between the brown adipose and the transition phase, had consensus putative response elements in an enriched set of the  <dig> genes displaying altered expression in the same phase shift.
the two transcription factors were nuclear receptor subfamily  <dig>  group h, member  <dig>  and v-myc avian myelocytomatosis viral oncogene homolog .
of the  <dig> differently expressed genes between the transition and the white adipose phase, an enriched set of genes  contained consensus putative response elements for six transcription factors that were themselves regulated in the same phase shift.
the six transcription factors are nr1h <dig>  myc, b-cell lymphoma  <dig> , estrogen receptor  <dig> , v-rel reticuloendotheliosis viral oncogene homolog a  and krüppel-like factor  <dig> .
the expression levels of these six transcription factors in the three phases were validated by rt-qpcr .
expression of nr1h <dig> and myc was significantly increased and decreased, respectively, in the transition phase compared to both the brown and the white adipose phase .
expression of the three transcription factors esr <dig>  rela and klf <dig> was up-regulated between the transition and white adipose phase, whereas bcl <dig> was significantly down-regulated from the transition to the white adipose phase .figure  <dig>
expression of regulated transcriptional components with consensus putative response elements in an enriched set of regulated genes.
total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis.
relative expression was measured for nuclear receptor subfamily  <dig>  group h, member  <dig> , v-myc avian myelocytomatosis viral oncogene homolog , b-cell lymphoma  <dig> , estrogen receptor  <dig> , v-rel reticuloendotheliosis viral oncogene homolog a  and krüppel-like factor  <dig> .
the mrna expression levels were normalized to expression of β-actin .
data are mean + sem ; *, p <  <dig> .
figure 7a lists genes with differential expression between the brown adipose and transition phase that have a consensus putative response element for either myc or nr1h <dig>  among the up-regulated genes that were potentially regulated by myc from the brown adipose to the transition phase were the adhesion protein thrombospondin  <dig>  and the rab <dig> gtpase activator tbc <dig> domain family member  <dig> .
among the down-regulated genes from the brown adipose phase to the transition phase potentially regulated by myc were a 9-cis-retinoic acid synthesizing enzyme, aldehyde dehydrogenase  <dig> family member a <dig>  and the transcription factor basic helix-loop-helix family member e <dig> .figure  <dig>
genes with altered expression that potentially are controlled by transcription factors regulated during the transformation.
list of genes regulated from the brown adipose phase to the transition phase, which have consensus putative response elements for nuclear receptor subfamily  <dig>  group h, member  <dig>  and v-myc avian myelocytomatosis viral oncogene homolog .
subcellular localization of differentially expressed genes from the transition phase to the white adipose phase that have consensus putative response elements for nr1h <dig>  myc, b-cell lymphoma  <dig> , estrogen receptor  <dig> , krüppel-like factor  <dig>  and v-rel reticuloendotheliosis viral oncogene homolog a .
green nodes indicate down-regulated genes in the white adipose phase as compared to the transition phase and red nodes indicate up-regulated genes in the white adipose phase as compared to the transition phase.
the corresponding gene names are listed in additional file 10: table s <dig>
figure 7b depicts the subcellular distribution of the genes potentially regulated by myc, esr <dig>  rela, bcl <dig>  klf <dig> or nr1h <dig> between the transition and the white adipose phase.
gene names corresponding to figure 7b are presented in additional file 10: table s <dig>  forty of the regulated genes were mitochondrial genes,  <dig> of which were down-regulated.
this is in accordance with the decreased activity and amount of mitochondria in the white adipose phase .
half of the down-regulated mitochondrial genes have been described to be regulated by rela.
nineteen genes encoded secreted proteins,  <dig> of which were up-regulated, including two pro-angiogenic factors: vascular endothelial growth factor b  and angiopoietin-related protein  <dig> , and one anti-angiogenic factor, serpin peptidase inhibitor f <dig>  .
expression of white, brite/beige and brown adipose markers in the three phases of adipose tissue transformation
to study the three phases in the transformation process in more detail, we measured a number of brown and white adipose marker genes by rt-qpcr.
as evident from figure  <dig>  we observed a down-regulation of ucp <dig> between the transition and white adipose phase, and a stepwise decrease in expression of dio <dig> and ppargc1a through the three phases of the transformation .
expression of two transcriptional co-regulators promoting white adipogenesis: nuclear receptor-interacting protein  <dig>  and retinoblastoma  <dig> , was increased between the transition and the white adipose phase .
a typical white adipose marker gene leptin  displayed decreased expression in the transition phase compared to both the brown and white adipose phase .
expression of the key transcriptional driver of brown adipogenesis, pr domain containing  <dig> , was not changed significantly between the phases .
overall, these measurements supported the brown to white adipose transformation occurring in the sheep perirenal adipose tissue within the first two weeks after birth.figure  <dig>
expression of genetic markers for white, brown and brite/beige adipose tissue during the three phases of the transformation.
gene expression was determined by rt-qpcr.
relative levels of genes associated with white adipocytes: nuclear receptor-interacting protein  <dig> , retinoblastoma  <dig>  and leptin .
relative levels of the brown adipose associated and marker genes: pr domain containing  <dig> , solute carrier family  <dig> member  <dig> , lim homeobox  <dig>  and myelin protein zero-like  <dig> .
zinc finger protein  <dig>  was not detectable in any of the adipose samples.
relative levels of the brite/beige adipose markers: homeobox c <dig> , hoxc <dig>  tumor necrosis factor receptor superfamily member  <dig> , transmembrane protein  <dig>  and t-box  <dig> .
the mrna expression levels were normalized to expression of β-actin .
data are mean + sem ; *, p <  <dig> .
to address if the sheep perirenal adipose tissue qualified as being brown, brite/beige or a mixture of brown and brite/beige at birth, and whether this status of the tissue changed over time, we measured a number of recently proposed marker genes selectively expressed in brown versus brite/beige adipose tissue and adipocytes  <cit> .
we determined the expression of the classical brown adipose marker genes solute carrier family  <dig> member  <dig> , lim homeobox  <dig> , myelin protein zero-like  <dig>  and zinc finger of the cerebellum  <dig> .
slc29a <dig> was expressed at birth and elicited a stepwise down-regulation through the two adipose phase shifts .
mpzl <dig> expression did not significantly change, whereas the lhx <dig> mrna increased steadily through the three phases .
zic <dig> was not detectable in any of the perirenal adipose samples, but was easily detectable in sheep brain .
we also measured the expression of the three brite/beige marker genes homeobox c <dig> , hoxc <dig> and tumor necrosis factor receptor superfamily member  <dig> .
hoxc <dig> and hoxc <dig> are marker genes for both wat and brite/beige adipose tissue  <cit> .
the expression of all three genes increased from the transition phase to the white adipose phase .
however, expression of the brite/beige marker genes transmembrane protein  <dig>  and t-box protein  <dig>  did not change .
in summary, three out of four markers of classical bat were detectable, and two out of these three changed expression over time.
all five measured brite/beige markers were detectable: the expression of three increased and two remained unchanged from the transition to the white adipose phase.
thus, markers of both brown and brite/beige adipose tissue were expressed in perirenal adipose tissue from sheep, and most of these markers displayed altered expression over time.
discussion
plasticity of adipose tissues is important for adaptation to changing physiological conditions  <cit> .
in response to prolonged cold exposure, subcutaneous wat depots of rodents undergo a transformation process during which numerous brite/beige adipocytes appear, thereby increasing overall thermogenic capacity of the animal  <cit> .
in large mammals, a substantial part of bat present in the newborn converts to wat after birth, which may reflect a diminished need for endogenous thermogenesis after the early postnatal period.
as little molecular insight into this conversion in large mammals is available, we have in the present study conducted a detailed analysis of the postnatal transformation of perirenal adipose tissue in sheep.
we chose this particular tissue, as it is the most frequently studied bat depot in large mammals.
moreover, we reasoned that the transformation of this depot was suitably modeling the postnatal brown to white adipose transformation in humans.
the perirenal adipose depot from adult humans has recently been characterized at the morphological and transcriptional level  <cit> .
this characterization revealed an enormous variation in expression levels of ucp <dig>
the postnatal transformation process from bat to wat in sheep perirenal adipose tissue occurred within the first two weeks after birth as determined by changes in tissue morphology, gene expression and mitochondrial density.
adipocyte morphology changed from being mainly multilocular to unilocular and the amount of mitochondria decreased.
the expression of brown adipocyte-selective genes, e.g.
ucp <dig>  dio <dig> and ppargc1a, declined, as did expression of additional genes encoding mitochondrial proteins.
to understand the adipose transformation in more detail, we performed a global gene expression analysis with  <dig> time points ranging from approximately two days before birth to two weeks after birth.
by two independent analyses of the gene expression data, we determined that the transformation clustered into three phases: a brown adipose phase, a transition phase and a white adipose phase.
regulated transcription factors and co-regulators
between the brown adipose and the transition phase were  <dig> genes differentially expressed, including  <dig> transcriptional regulators,  <dig> of which were down-regulated .
five of these have chromatin modifying activity: circadian locomotor output cycles kaput , nuclear receptor co-activator  <dig> , proviral insertion site in moloney murine leukemia virus lymphomagenesis , and the swi/snf-related matrix-associated actin-dependent regulator of chromatin subfamily members smarcc <dig> and smarcd <dig>  this leaves open the possibility that extensive remodeling of chromatin is occurring between the brown adipose and the transition phase.
in accordance with its down-regulation in the perirenal adipose transformation , ncoa <dig> has been reported to promote bat activity in mice  <cit> .
between the transition and the white adipose phase  <dig> genes were differentially expressed, of which  <dig> were transcription factors or co-regulators .
the list of regulated transcriptional regulators included a few factors known to be differently expressed in mouse wat and bat, e.g.
nrip <dig> and cell death-inducing dffa-like effector a .
among the down-regulated transcriptional regulators from the transition to the white adipose phase were some related to brown adipocyte function: early b-cell factor  <dig> , which have been described to induce expression of brown adipose-specific pparg target genes  <cit> , leucine-rich ppr motif-containing protein , a ppargc1a co-activator playing an important role in bat differentiation and function  <cit>  and y box binding protein  <dig> , an inducer of bone morphogenetic protein  <dig>  transcription and brown adipocyte differentiation  <cit> .
regulated transcription factors with a consensus putative response element in an enriched set of differentially expressed genes
expression of four transcription factors nr1h <dig>  myc, aire and mlf <dig> was regulated at both phase shifts.
the former two have a consensus putative response element in an enriched set of genes displaying altered expression at the two phase shifts.
expression of nr1h <dig> and myc was transiently increased and decreased, respectively, during the transition phase .
consistent with the opposite regulation during the transition phase, nr1h <dig> have been reported to suppress myc expression in colon cancer cells  <cit>  .
myc is known to inhibit adipogenesis  <cit> , which might explain why it is down-regulated in the transition phase, which includes tissue expansion.
nr1h <dig> has been described to regulate gene expression linked to several important aspects of both brown and white adipocyte biology, including adipogenesis, energy expenditure, lipolysis and glucose transport  <cit> .
nr1h <dig> was reported to be present at higher levels in mouse bat than wat  <cit>  and to suppress pparγ-induced ucp <dig> expression by binding to the ucp <dig> enhancer together with nrip <dig> in mouse adipocytes  <cit> .
accordingly, nr1h <dig> can regulate the expression of brown adipocyte-selective genes.
however, the increased nr1h <dig> expression in the transition phase did not correlate with decreased ucp <dig> expression in our study, which might be explained by the relative low expression of nrip <dig> in the transition phase .
beside nr1h <dig> and myc, four other transcription factors, rela, klf <dig>  esr <dig> and bcl <dig>  were regulated between the transition and the white adipose phase and found to have consensus putative response elements in an enriched number of genes regulated between these two phases.
the former three were up-regulated from the transition to the white adipose phase, whereas bcl <dig> was down-regulated .
both rela and esr <dig> have been described to stimulate myc expression  <cit> , which would be consistent with the increased expression of myc in the white adipose phase .
of interest, rela and esr <dig> have opposite effects on adipogenesis, as both knockdown of rela and activation of esr <dig> by estrogen supplementation attenuated adipogenesis  <cit> .
wat from mice with an adipocyte-specific knockout of rela have decreased lipid droplet size, increased glucose uptake and reduced expression of adipogenic marker genes such as lep, pparg and adiponectin .
furthermore, the mice are obesity resistant and less sensitive to cold  <cit>  .
esr <dig> knockout mice have increased fat mass caused by adipocyte hyperplasia  <cit> .
in addition, esr <dig> can inhibit the transcriptional activity of rela  <cit>  .
based on this, we speculate that rela and esr <dig> might contribute to the regulation of tag accretion during the adipose transformation.
rela might also contribute to the observed mitochondrial depletion, as rela negatively impacts mitochondrial content in c2c <dig> myocytes  <cit> .
rela has putative response elements in the promoter of  <dig> genes encoding mitochondrial proteins regulated between the transition and the white adipose phase, of these  <dig> were down-regulated.
this is in accordance with rela functioning both as a transcriptional activator and repressor  <cit> .
rela has been described to repress bcl <dig> expression through interferon regulatory factor  <dig>  <cit> .
this is in accordance with the increased expression of rela and the decreased expression of bcl <dig> in the white adipose phase .
bcl <dig> is a transcriptional repressor with the ability to reduce the expression of e.g.
myc  <cit>  .
the expression of bcl <dig> is strongly down-regulated by growth hormone in 3t3-f442a adipocytes  <cit> .
apart from this, bcl <dig> has not been linked to adipocyte or adipose tissue function.
klf <dig> and rela have been reported to be functionally intertwined, as they directly interact to induce expression of selected genes  <cit> , but also to compete for interaction with a co-activator, thereby inhibiting each other’s activity  <cit>  .
klf <dig> is important for induction of adipogenesis in vitro and its expression was reported to be induced in pre-adipocytes by camp.
klf <dig> stimulates ccaat/enhancer-binding protein β  expression, and c/ebpb in turn down-regulates klf <dig> expression, thereby forming a negative feedback loop  <cit> .
in summary, six transcription factors with differential expression during the adipose transformation have consensus putative response elements in an enriched set of the regulated genes, suggesting that they are involved in the control of the overall gene expression changes and thus potentially have an impact on remodeling of the tissue.
moreover, the six factors are mutually functionally linked, leaving open the possibility that they are part of a transcriptional network .
brown and brite/beige markers
a number of marker genes selectively expressed in white, brite/beige and brown adipose tissue and adipocytes have been reported  <cit> .
some of these marker genes are not identified consistently between studies.
moreover, some markers have been identified from gene expression profiling of whole tissue samples, whereby non-adipose cells cannot be excluded to be a significant source of expression  <cit> .
it is being discussed whether human bat is composed of brown or brite/beige adipocytes or a mixture of these.
moreover, it is not fully established to what extent expression of white, brite/beige and brown adipose marker genes changes in a particular adipose depot during development or remodeling.
to elucidate this in sheep, we analyzed the expression of selected marker genes in the brown adipose, transition and white adipose phase of the perirenal adipose depot.
although we only measured marker gene expression in the perirenal depot, and accordingly have not compared expression levels to those in other adipose depots, we could detect both brown  and brite/beige  adipocyte marker genes in the newborn sheep .
co-expression of marker genes selective for brown and brite/beige adipose tissues have also been observed in a recent study of the human supraclavicular bat depot  <cit> .
in this human study, expression of ucp <dig> in supraclavicular biopsies was positively associated with expression of both bat markers  and brite/beige adipose markers , whereas expression of the two wat and brite/beige adipose markers hoxc <dig> and hoxc <dig> correlated with low ucp <dig> expression  <cit> .
in our time course study, we did not observe a correlation between high ucp <dig> expression and high expression of zic <dig> , lhx <dig>  tbx <dig> or tmem <dig>  but we did detect higher expression of hoxc <dig> and hoxc <dig> in the white adipose state, where ucp <dig> expression was low .
a similar hoxc <dig> profile in sheep perirenal adipose tissue has been reported by others  <cit> .
bat, brite/beige adipose and wat marker gene expression have not previously been studied in detail during adipose tissue remodeling in large mammals.
based on the selective expression profile in mice, our observation that hoxc <dig> and hoxc <dig> are induced in the white adipose phase suggests that the perirenal adipose depot converts from brown  to white .
the brown adipose origin of the perirenal depot might be supported by the down-regulation of the bat marker gene slc29a <dig> during whitening.
however, the 5-fold increase in expression of lhx <dig> from the brown to the white adipose phase and the absence of zic <dig> expression were not consistent with this model.
in addition to hoxc <dig> and hoxc <dig>  a number of other brite/beige marker genes were expressed shortly after birth .
the expression of these was either unchanged or up-regulated during whitening.
in summary, markers of both brite/beige and brown adipose tissue are expressed in the sheep perirenal adipose tissue at birth and the expression of a number of these changes substantially over time.
the latter might be important to consider when analyzing adipose tissue type-selective gene expression.
model of the transformation process
the postnatal transformation from brown  to white in the perirenal adipose tissue can occur through at least three different mechanisms: 1) through transdifferentiation of brown  to white adipocytes; 2) through proliferation and differentiation of white adipogenic precursor cells and death of mature brown  adipocytes; 3) through a combination of the two.
in mice, brite/beige adipocytes can transdifferentiate into white adipocytes  <cit> , but whether this observation extends to large mammals is unclear.
if the white adipocytes arise exclusively from proliferation and differentiation of precursor cells, it would require an enormous cell turnover during the transition phase of the transformation, including extensive death of mature brown  adipocytes.
we would expect this to result in induction of cell cycle genes, but we did not observe this in the go term analysis.
moreover, the expression profile of key cyclins  was not consistent with the hypothesis of massive cell cycling during the transition phase .
however, we cannot rule out that a small population of proliferating white adipose precursor cells contributed to the whitening of the tissue.
in the shift from transition phase to white adipose phase, we did observe an up-regulation of genes related to cell death, but nearly half of the up-regulated cell death associated genes are negative regulators of cell death .
thus, it is not obvious from the gene expression data if cell death is increased or not.
furthermore, we did not observe evidence for significant cell death in any of the tissue sections analyzed.
consistently, lomax et al.
<cit>  failed to detect apoptotic cells during the transformation of the perirenal adipose tissue in sheep.
an alternative to the transdifferentiation model is that the white adipocytes present in the white adipose phase are in fact masked brite/beige adipocytes, possibly of a different lineage than white adipocytes.
these cells might have the potential to return to a thermogenic state, which would be compatible with existence of such masked adipocytes in mice  <cit> .
arguing against this scenario in sheep is that after postnatal day  <dig>  ucp <dig> and other thermogenic genes are no longer inducible in perirenal adipose tissue in response to repeated injections of a β-adrenergic agonist  <cit> .
in summary, we consider it plausible that transdifferentiation of brown  to white adipocytes is a significant component of the postnatal transformation of the perirenal adipose depot in sheep.
clearly, additional studies are required to validate to what extent transdifferentiation takes place, including a time course with more time points and a dedicated search for evidence of cell proliferation and death.
CONCLUSIONS
by global gene expression profiling, we provide novel information of the postnatal bat to wat transformation in sheep.
this transformation process is poorly understood in molecular terms, but is of significant interest, as a similar transformation occurs in human infants after birth.
an improved understanding of this tissue remodeling increases insight into adipose plasticity and might allow identification of targets suitable for interfering with the balance between energy-storing and energy-dissipating adipose tissue.
our results reveal novel transcription factors linked to the adipose transformation process in large mammals.
clearly, validation of their relevance in adipose function will require dedicated functional studies.
finally, we show that expression of adipose tissue-type selective marker genes change substantially over time, which might be an underappreciated variable in such analyses.
additional files
additional file 1: table s <dig>  primers used for qpcr.
additional file 2: figure s <dig>  transmission electron microscopy and expression of mitochondria-related genes during brown to white adipose transformation.
transmission electron microscopy and expression of mitochondria-related genes during brown to white adipose transformation.
transmission electron micrographs of perirenal adipose tissue at postnatal days  <dig>   <dig>   <dig> and  <dig>  representative images are shown for each of the indicated time points  in three different magnifications.
total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis.
relative expression was measured for peroxisome proliferator-activated receptor γ  co-activator 1β , ppara, estrogen-related receptor α  and nuclear respiratory factor  <dig> .
the mrna expression levels were normalized to expression of β-actin .
data are mean + sem ; *, p <  <dig>  vs. day  <dig>
additional file 3: table s <dig>  read statistics for the sequencing data when mapped and annotated to the sheep genome.
a table listing the counts of raw reads from the  <dig> time points, reads mapped to the sheep genome, uniquely mapped reads and reads mapped to  <dig>  annotated genes in the sheep genome.
the table also lists the percentage of total reads in the above mentioned classes.
additional file 4: table s <dig>  normalized counts of genes from perirenal adipose tissue at postnatal days − <dig>   <dig>   <dig> ,  <dig>   <dig>   <dig> and  <dig>
additional file 5: table s <dig>  list of the  <dig> differentially expressed genes between the brown adipose phase and the transition phase.
additional file 6: table s <dig>  list of the  <dig> differentially expressed genes between the transition phase and the white adipose phase.
additional file 7: figure s <dig>  venn diagram of differentially expressed genes between the three phases.
additional file 8: figure s <dig>  heatmap of the  <dig> genes regulated between the three phases.
heatmap of the  <dig> genes regulated between the brown adipose phase and the transition phase as well as between the transition phase and the white adipose phase.
transcription factors are highlighted in yellow.
additional file 9: table s <dig>  transcriptional regulators with changed expression levels between the three phases.
transcription factors and co-regulators with changed expression levels in the brown adipose to transition phase shift or in the transition to white adipose phase shift.
additional file 10: table s <dig>  list of differentially expressed genes from the transition phase to the white adipose phase that have consensus putative response elements for nr1h <dig>  myc, bcl <dig>  esr <dig>  klf <dig> or rela.
list of differentially expressed genes from the transition phase to the white adipose phase that have consensus putative response elements for nuclear receptor subfamily  <dig>  group h, member  <dig> , v-myc avian myelocytomatosis viral oncogene homolog , b-cell lymphoma  <dig> , estrogen receptor  <dig> , krüppel-like factor  <dig>  or v-rel reticuloendotheliosis viral oncogene homolog a .
genes are organized according to the subcellular localization of their encoded product.
additional file 11: figure s <dig>  relative levels of brown adipose-associated genes during the three phases of the postnatal adipose transformation.
relative levels of brown adipose-associated genes during the three phases of the postnatal adipose transformation.
these data are an alternative presentation of the data presented in figure 1b.
total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis.
relative expression was measured for uncoupling protein  <dig> , type ii iodothyronine deiodinase  and peroxisome proliferator-activated receptor γ  co-activator 1α .
the mrna expression levels were normalized to expression of β-actin .
data are mean + sem ; *, p <  <dig> .
additional file 12: figure s <dig>  mutual interactions between differentially expressed transcription factors with putative response elements in an enriched set of regulated genes.
mutual interactions between the six differentially expressed transcription factors with putative response elements in an enriched set of genes with altered expression during the adipose transformation.
the brown oval-shaped circles represent the differentially expressed transcription factors; white oval-shaped circles represent other transcriptional components functionally linked to the differentially expressed transcription factors; stop arrows indicate repression/inhibition; arrows indicate activation/stimulation.
abbreviations
acacaacetyl-coa carboxylase 1
agpat91-acylglycerol-3-phosphate o-acyltransferase 9
aireautoimmune regulator
aldh8a1aldehyde dehydrogenase  <dig> family member a1
aldoaglycolytic enzyme aldolase a
angptl2angiopoietin-related protein 2
atp5batp synthase β
batbrown adipose tissue
bcl6b-cell lymphoma 6
bhlhe40basic helix-loop-helix family member e40
bmp7bone morphogenetic protein 7
c/ebpbccaat/enhancer-binding protein β
catcatalase
ccncyclin
cd36cluster of differentiation 36
cideacell death-inducing dffa-like effector a
clockcircadian locomotor output cycles kaput
cpt1bcarnitine palmitoyltransferase 1b
cscitrate synthase
cyc1cytochrome c1
dgatdiacylglycerol o-acyltransferase
dio2type ii iodothyronine deiodinase
ebf2early b-cell factor 2
edtaethylenediaminetetraacetic acid
epas1endothelial pas domain-containing protein 1
erraestrogen-related receptor α
esr1estrogen receptor 1
fabp4fatty acid-binding protein 4
fasnfatty acid synthase
fdrfalse discovery rate
gpammitochondrial glycerol-3-phosphate acyltransferase
hadhhydroxyacyl-coa dehydrogenase complex
hadhahadh catalytic subunit α
hgnchugo gene nomenclature committee
hehematoxylin-eosin
hoxc8homeobox c8
hugohuman genome organization
idh3aisocitrate dehydrogenase 3α
klf4krüppel-like factor 4
lepleptin
lhx8lim homeobox 8
lrp1low density lipoprotein receptor-related protein 1
lrpprcleucine-rich ppr motif-containing protein
mlf1myeloid leukemia factor 1
mpzl2myelin protein zero-like 2
msr1macrophage scavenger receptor 1
mt-co1mitochondrially encoded cytochrome c oxidase i
mtdnamitochondrial dna
mycv-myc avian myelocytomatosis viral oncogene homolog
ncoa1nuclear receptor co-activator 1
ndnanuclear dna
nr1h3nuclear receptor subfamily  <dig>  group h, member 3
nrf1nuclear respiratory factor 1
nrip1nuclear receptor-interacting protein 1
pcaprincipal component analysis
pim1proviral insertion site in moloney murine leukemia virus lymphomagenesis
ppargperoxisome proliferator-activated receptor γ
ppargc1apparg co-activator 1α
prdm16pr domain containing 16
rb1retinoblastoma 1
relav-rel reticuloendotheliosis viral oncogene homolog a
rgccregulator of cell cycle
rt-qpcrreverse transcription-quantitative polymerase chain reaction
serpinf1serpin peptidase inhibitor f1
slc29a1solute carrier family  <dig> member 1
smarcc2swi/snf-related matrix-associated actin-dependent regulator of chromatin subfamily member c2
st7suppression of tumorigenicity 7
tagtriacylglycerol
taldo1transaldolase
tbx1t-box protein 1
tcatricarboxylic acid
temtransmission electron microscopy
tfiibtranscription factor iib
tmem26transmembrane protein 26
tnfrsf9tumor necrosis factor receptor superfamily member 9
ucp1uncoupling protein 1
vegfbvascular endothelial growth factor b
watwhite adipose tissue
ybx1y box binding protein 1
zic1zinc finger of the cerebellum 1
astrid l basse, karen dixen and rachita yadav contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
alb, kd, mpt, kk, bq, jw and jbh conceived and designed the experiments.
alb, kd, ry, mpt, kq, bq and jbh performed the experiments.
alb, kd, ry, rg and jbh analyzed and interpreted the data.
alb, kd, ry and kq prepared the figures.
alb, kd, ry and jbh wrote the paper.
all authors read and approved the final manuscript.

 two types of adipose tissue exist based on morphological appearance and biological function. white adipose tissue stores energy in the form of triacylglycerol for later release and use by other tissues, whereas brown adipose tissue metabolizes fatty acids and glucose for heat production. thermogenesis through uncoupled respiration in bat depends on a high mitochondrial density and expression of uncoupling protein. larger mammals such as primates and ruminants are born fully developed and able to thermoregulate minutes after birth due to the presence of relatively large amounts of functional bat, which gets activated at birth. the majority of this innate bat disappears after birth and is replaced by wat. besides brown adipocytes in bat, another type of thermogenic adipocytes exists: the brite or beige adipocytes, which are known to be recruited in subcutaneous wat of rodents in response to cold exposure or stimulation with β-adrenergic agonists. the developmental origin of brite/beige adipocytes and their contribution to human bat are still a matter of debate. rodents as opposed to larger mammals are born with immature bat which matures only postnatally and is largely retained throughout life. it is relevant to understand the brown to white adipose tissue remodeling in large mammals, as it is likely to mimic the transition occurring in human infants. the most frequently studied adipose tissue transition in a large mammal is the postnatal transformation of the perirenal adipose tissue in sheep. around the time of birth, all visceral adipose depots in lambs are brown of nature. lambs are normally born with approximately 30 g perirenal adipose tissue constituting 80% of all their adipose tissue. the brown characteristics of the perirenal adipose depot change dramatically to a white adipose phenotype within a few weeks after birth. although some gene expression details have been reported, relatively little is known about this transition at the molecular level. here we report a comprehensive time course analysis of the postnatal bat to wat transformation process of the perirenal adipose depot in lambs, including histological, biochemical and molecular examination as well as analyses of global gene expression profiles. we provide evidence for dramatic changes in mitochondrial function and fatty acid metabolism during the adipose remodeling and we identified a number of transcriptional components linked to this adipose tissue transformation process. methods animals and tissues experimental procedures were in compliance with guidelines laid down by the danish inspectorate of animal experimentation. lambs from cross-bred ewes in their second or third parturition sired by purebred texel ram, born and raised at a commercial farm in denmark, were used. during gestation ewes were fed hay ad libitum, 200 g barley and 200 g commercial concentrate per day. ewes were housed in groups of until lambing. after lambing they were housed individually for 2 days and subsequently housed in groups of until they were transferred to pasture approximately one week after lambing. the ewe-reared lambs were kept on pasture being a mixture of 70% ray grass and 30% white clover. lambs were killed by bolt pistol and bled by licensed staff. perirenal adipose tissue was carefully dissected and frozen in liquid nitrogen for biochemical or molecular analyses or fixed for histology as described below. lambs at the following ages were used: −,,,,,,, and. live weights of the lambs were kept similar within groups. the ratio of females to males was similar in the three phases of the transformation. hematoxylin-eosin staining samples were fixed in 4% neutral buffered formaldehyde at room temperature for 24 h and subsequently at 4°c until preparation. the tissue was processed to paraffin and sectioned in 4 μm sections. he staining was performed according to standard procedures. transmission electron microscopy samples were fixed in karnowsky’s fixative for 3– days at room temperature and subsequently stored in  m cacodylate buffer at 4°c until further processing. the samples were rinsed three times in  m sorensen’s phosphate buffer and subsequently post fixed in 1% oso in  m sodium cacodylate buffer for 2 h. the specimens were dehydrated in graded series of ethanol, transferred to propylene oxide and embedded in epon according to standard procedures. ultra-thin sections were cut with a reichert-jung ultracut e microtome and collected on single slot copper grids with formvar supporting membranes. sections were stained with uranyl acetate and lead citrate and examined with a philips cm- transmission electron microscope, operated at an accelerating voltage of 80 kv. digital images were recorded with a sis megaview camera and the analysis software package. reverse transcription-quantitative polymerase chain reaction tissues were homogenized in trizol using a dispomix and total rna was purified. reverse transcriptions were performed in 25 μl reactions containing 1st strand buffer, 2 μg random hexamers,  mm of each dntp, units of rnaseout, 1 μg of total rq dnase -treated rna and units of moloney murine leukemia virus reverse transcriptase. reactions were left for 10 min at room temperature, followed by incubation at 37°c for 1 h. after cdna synthesis, reactions were diluted with 50 μl of water and frozen at −80°c. the cdna was analyzed by rt-qpcr using the stratagene mx3000p qpcr system. each pcr mixture contained, in a final volume of 20 μl,  μl of 1st strand cdna, 10 μl of sensifast™ sybr lo-rox kit and pmol of each primer. all reactions were run using the following cycling conditions: 95°c for 10 min, then 40 cycles of 95°c for 15 s, 55°c for 30 s and 72°c for 15 s. pcr was carried out in 96-well plates and each sample was run in duplicate. target gene mrna expression was normalized to the stably expressed β-actin mrna. protein extracts and immunoblotting tissues were homogenized in a gg-buffer containing 25 mm glycyl-glycin, 150 mm kcl, 5 mm mgso and 5 mm ethylenediaminetetraacetic acid as well as freshly added dithiothreitol, bovine serum albumin and triton x-. homogenization was performed with a tissuelyser using 5 mm stainless steel beads, and homogenates were subsequently frozen in liquid nitrogen. protein concentrations were determined by the lowry method and equal amounts of protein from each animal were pooled according to age and diluted in a buffer containing % sds and 10% glycerol. proteins were separated on 4-12% bis-tris gradient gels, blotted onto immobilon pvdf membranes and stained with amido black 10b. membranes were blocked in tris-buffered saline or phosphate-buffered saline with 5% nonfat dry milk and % tween and subsequently probed with antibodies. primary antibodies used were against transcription factor iib, atp synthase β and ucp. secondary antibodies were horseradish peroxidase-conjugated. enhanced chemiluminescence was used for detection. quantification of relative mitochondrial dna copy numbers relative mtdna amount was measured as the ratio between mtdna and nuclear dna. tissues were homogenized using a tissuelyser in lysis buffer containing 100 mm tris-base, 5 mm edta, % sodium dodecyl sulphate, 200 mm nacl and 100 mg/ml proteinase k and incubated overnight at 55°c with rotation. dna was precipitated with two volumes of 99% ethanol and fished out with inoculation loops, washed in 70% ethanol and dissolved in tris-edta buffer containing 10 mg/ml rnase a at 55°c overnight. dna concentrations were determined on the eppendorf biophotometer at 260 nm and 50 ng dna was used for qpcr. pcr reactions and cycling conditions were as described above, and primers were against cytochrome c oxidase i and suppression of tumorigenicity. citrate synthase activity tissue homogenates were generated in gg-buffer as described above. homogenates were thawed on ice and centrifuged at 4°c at  g for 2 min. supernatants were used for activity measurements. cs activity was measured spectrophotometrically at 25°c and 412 nm in cs buffer containing 100 mm tris-base, 10 mm ′-dithiobis, 5 mm acetyl-coa and 50 mm oxaloacetic acid and activity was measured as described. each sample was measured in duplicate and the mean was used for subsequent calculations. activities were normalized to the amount of total protein determined by the lowry method. statistical analyses of qpcr data the time course study was analyzed for statistical significance using one-way anova and student’s t-test with bonferroni correction for multiple testing as post hoc test. a p-value <  was considered statistically significant. targeted rna-sequencing and data analysis isolation of mrna and synthesis of first strand cdna equal amounts of total rna from perirenal adipose tissue from lambs at the same age were pooled. mrna was isolated from 4 μg of total rna by magnetic oligo beads, which was used to synthesize bead-bound cdna, according to the instructions of the manufacturer. tag library construction the library for digital gene expression analysis was constructed according to the instructions of the manufacturer. bead-bound cdna was digested with nlaiii, followed by ligation of the gex adapter to the bead-bound nlaiii-digested cdna. this was then digested with mmei, releasing the gex adaptor linked to 17 bp cdna from the beads. the released fragment was ligated to gex adapter the 17 bp tags of cdna were pcr amplified using two primers that anneal to the two adapters. the resultant tag library was used for illumina sequencing. data analysis for rna-seq data quality control, trimming and adapter removal was performed using fastqc and fastx_clipper from the fastx-toolkit. the bases catg were added to the 5’ end of reads to increase the specificity of mapping. bwa was employed for the alignment and mapping of reads to the sheep genome. the october release of the sheep genome v was used as reference for the alignment. mapped reads were sorted and indexed with samtools. since the rna-seq data was used for expression quantification, duplicated reads were not removed. ht-seq was used for counting mapped reads per annotated gene, using the annotation file provided for the sheep genome v release. deseq and r were used for the post processing and statistical analysis of these read counts. principal component analysis and hierarchical clustering of time points a two-dimensional pca plot was employed to visualize the overall effect of experimental covariates. hierarchical clustering of the total gene expression was performed using a distance matrix to assess the relationship between the samples and identify clusters amongst the time points. grouping of time points based on the pca and hierarchical clustering of the total gene expression, days − and were used as replicates of the “brown adipose state”, days, and as replicates of the “transition state” and day as the “white adipose state”. using the deseq package of bioconductor, differentially expressed genes were found between the brown adipose and the transition state as well as between the transition and the white adipose state. for the differential gene expression between “transition state” and “white adipose state”, the partial replicate function of deseq for calculation of dispersion of the genes was used. this method ignores the sample from day when estimating dispersion for the genes and would use only days, for calculating the dispersion per gene, which is an indicator of variability in that gene, and is used to fit the negative binomial model on the count data. heatmaps representing clustering for the differentially expressed genes were created using the ggplots package in r. the sheep proteins were queried against the human non-redundant protein database using blast to find human homologous genes for further functional analysis. reciprocal blast, a computation method used to countercheck the blast results, was employed to filter the correct mapping between the sheep and human proteins. the biomart tool on ensemble version was used for gene identification and conversion, and for obtaining human genome organization gene nomenclature committee approved gene names for the human homologous proteins. uniprot was used to annotate the proteins for function, transcriptional activity and subcellular localization. go term enrichment was done using explain™ from biobase corporation. the explain tool uses false discovery rate correction for finding significantly enriched go classes. for the two sets of differentially expressed genes obtained from deseq analysis, enrichr was used to find enrichment of transcription factors and the corresponding targets for the differentially expressed transcription factors from the transfac and jasper databases. enrichr applies a fisher’s exact test to calculate significance, which is further corrected for multiple testing and the resulting adjusted p-values are used for selecting significant transcription factors for the differentially expressed genes.  characterization of the postnatal brown to white adipose transformation at birth the perirenal adipose tissue macroscopically appeared dark brown. the brown color fainted steadily during the time course, and the tissue ended up being white in appearance at postnatal days and. accompanying the “whitening”, the volume of the tissue gradually increased. he-stained sections were prepared from all lambs, and representative sections from days and are presented in figure 1a. in the first week of life, the tissue was an apparent mixture of brown adipocytes with multilocular lipid droplets and white adipocytes with large unilocular lipid droplets. the perirenal adipose tissue contained by appearance mostly brown adipocytes at early ages, whereas white adipocytes were predominant from day figure characterization of the postnatal brown to white adipose transformation. hematoxylin-eosin staining of perirenal adipose tissue at postnatal days and representative he-stained sections are shown for the indicated time points. total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis. relative expression was measured for uncoupling protein, type ii iodothyronine deiodinase and peroxisome proliferator-activated receptor γ co-activator 1α. the mrna expression levels were normalized to expression of β-actin. data are mean + sem; *, p <  vs. day the level of uncoupling protein and atp synthase subunit β was determined by immunoblotting on protein pools, one for each day during the time course. transcription factor iib was used as a loading control. to approach the bat to wat transformation in molecular terms, we measured mrna and protein levels of selected marker genes by rt-qpcr and immunoblotting, respectively. expression of ucp the brown adipocyte-specific key thermogenic factor, was high and relatively stable until day after which it became nearly undetectable. the bat-enriched factors type ii iodothyronine deiodinase and peroxisome proliferator-activated receptor γ co-activator 1α were also highly expressed at days − and but displayed a faster and stepwise decrease in expression, being considerably reduced already at days and and poorly expressed after day. in summary, at the level of macroscopic, microscopic and molecular analyses, we observed the expected postnatal transformation of bat to wat. mitochondrial density declined during brown to white adipose transformation the ultra-structure of the perirenal adipose tissue was investigated at selected days by tem. tem confirmed the mixed presence of multilocular and unilocular adipocytes at days to and the predominant presence of the latter at day adipocyte mitochondrial density was very high at days to and appeared lower at day to estimate mitochondrial density quantitatively, we determined mtdna content by qpcr as the ratio of mtdna and ndna. this ratio decreased approximately 7-fold between days and indicating that the number of mitochondria per cell diminished during the bat to wat transformation.figure mitochondrial density declines during brown to white adipose transformation. total dna was isolated from perirenal adipose tissues and analyzed by qpcr with primers specific for mtdna ) and ndna ). the relative mtdna copy number was obtained as the ratio of mt-co to st levels. total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis. relative expression was measured for citrate synthase. the mrna expression levels were normalized to expression of β-actin. enzyme activity of cs was determined spectrophotometrically in homogenates of perirenal adipose tissue and normalized to protein content. relative expression of cytochrome c was measured by rt-qpcr as described in panel b. data are mean + sem; *, p <  vs. day the ultra-structural observations and the mtdna/ndna ratios prompted us to investigate more carefully gene expression of relevance for mitochondrial abundance and function. the mrna levels of the tricarboxylic acid cycle enzyme cs decreased gradually during the time course. cs activity, on the other hand, was high and stable until day after which it dropped. two other mitochondrial genes were analyzed: the electron transporter cytochrome c and atp5b. levels of cyc mrna and atp5b protein displayed a time profile similar to that of cs activity. a number of nuclear transcription factors and co-regulators regulate expression of genes encoding mitochondrial proteins. these factors include pgc- family members, nuclear respiratory factor and a number of nuclear receptors. in addition to ppargc1a, we measured the expression of ppargc1b, ppara, estrogen-related receptor α and nrf by rt-qpcr. the expression pattern of ppargc1b and erra was similar to cyc with stable expression until day followed by lower expression at subsequent time points. expression of ppara and nrf transiently decreased after birth. the decrease in expression of ppargc1a, ppargc1b and erra is consistent with the decline in mitochondrial density. accordingly, based on ultra-structure, relative mtdna measurements, expression and activity of key mitochondrial enzymes as well as expression of transcription factors and co-regulators controlling levels of mitochondrial factors, we concluded that mitochondrial density and function declined remarkably during the transition from bat to wat. global gene expression analysis of postnatal brown to white adipose transformation to obtain a global view of gene expression changes during the bat to wat transformation in the perirenal adipose depot, tag-based sequencing was performed on pools of mrna from days −, and the resulting reads were mapped to the october release of the sheep genome. the library sizes obtained for the seven samples are approximately, out of these, approximately 94% of the reads mapped to the reference genome, with approximately 56% of reads mapping uniquely to the reference genome. nearly 40% of the reads, those mapping to multiple locations, were discarded. the uniquely mapped reads covered out of annotated genes in the sheep genome, with an average read depth of 16x. the relatively low number of reads mapping to gene-annotated regions accounts for the low annotation coverage available for the sheep genome. the number of mapped reads per annotated gene was counted, and normalized read counts per gene for the time points were calculated. to facilitate downstream analyses, the human genes homologous to the sheep genes were mined, and the results from the gene expression analysis are discussed using the human protein symbols and names. next, we compared the expression of the genes measured by rt-qpcr in figures  and additional file 2: figure s1b to their expression in the tag-based sequencing data. expression of ucp dio ppargc1a, ppargc1b, cs, cyc and erra decreased from day to day in both the sequencing data and when measured by rt-qpcr. in general, there was a relatively high correlation in the expression data obtained by the two methods. the gene expression profiles were made from whole perirenal adipose tissue samples containing adipose as well as non-adipose cells. the amount and composition of non-adipose cells might have changed during the transformation of the tissue, thereby potentially influencing the differential gene expression between time points. however, the most highly expressed gene at both day and day was fatty acid-binding protein, a gene known to be strongly enriched in adipocytes. among the genes with the highest expression level at day and day were several genes encoding ribosomal proteins, fabp the fatty acid transporter cluster of differentiation, the glycolytic enzyme aldolase a and regulator of cell cycle, a cell cycle regulator and kinase modulating protein. genes highly expressed at day included the pentose phosphate pathway enzyme transaldolase and catalase. when comparing the most highly expressed genes, genes related to fatty acid oxidation, electron transport chain and atp synthase activity were more prevalent at day compared to day identification of three phases in the brown to white adipose tissue transformation process to analyze the distribution of gene expression, a pca was performed on the total gene expression data set. the pca plot indicated that total gene expression at the different time points clustered into three groups: a group including days − and a second group including days, and and a third group comprising day hierarchical clustering of the total gene expression data set clustered the time points into the same three groups. we interpreted the three clusters as distinct phases in the bat to wat transition. at days − and the tissue is in the brown adipose phase. during the phase composed of days, and the tissue is expected to be in a thermogenic state in order to keep the lambs warm. at the same time points expression of some bat-associated genes starts to drop, e.g. illustrated by the decrease in ppargc1a expression from day to day. therefore, we designate this period the transition phase. day represents the white adipose phase, as was also suggested by tissue morphology, mitochondrial numbers and function as well as expression level of ucp.figure identification of the brown adipose phase, transition phase and white adipose phase. principal component analysis plot for the expression data from the seven time points showing the clustering of time points in the first two components. heatmap showing the hierarchical clustering based on euclidean distances between the time points. allocation of the different time points to the three phases and summary of numbers of induced and repressed genes between phases. the expression of genes changed significantly between the brown adipose phase and the transition phase. of these, genes were up-regulated and genes were down-regulated. a heatmap with euclidian distances for the genes is shown in figure 4a. go enrichment analysis of the up-regulated genes revealed that they were enriched for genes related to “negative regulation of adaptive immune response” and “muscle cell migration”, whereas the down-regulated genes were enriched for genes related to “organic acid metabolic processes”.figure gene expression changes in the two phase shifts. heatmap of the genes differentially expressed from the brown adipose phase to the transition phase. heatmap of the genes differentially expressed from the transition phase to the white adipose phase. go enrichment analysis of genes differentially expressed from the brown adipose to the transition phase go term total number of genes in go term number of differentially expressed genes p -value enrichment from up-regulated genes enrichment from down-regulated genes between the transition phase and the white adipose phase, the expression of genes changed significantly, of which genes were up-regulated and were down-regulated. these differentially expressed genes are presented in a heatmap in figure 4b. a go enrichment analysis demonstrated that the up-regulated genes were enriched for genes related to “cell death” and “negative regulation of cell death”. the down-regulated genes were enriched for genes related to “metabolic process”, including “fatty acid beta-oxidation”.table go enrichment analysis of genes differentially expressed from the transition to the white adipose phase go term total number of genes in go term number of differentially expressed genes p -value enrichment from up-regulated genes enrichment from down-regulated genes the changes in gene expression related to the go term “fatty acid beta-oxidation” were investigated in more detail by rt-qpcr. of notice, the white adipose phase included samples from days and for rt-qpcr measurements, whereas the white adipose phase for the global gene expression analysis included samples from day only. two key enzymes in β-oxidation are carnitine palmitoyltransferase 1b and the hydroxyacyl-coa dehydrogenase complex. the relative mrna expression levels of both cpt1b and the catalytic subunit α of hadh decreased from the brown adipose phase to the transition phase and from the transition phase to the white adipose phase. we also measured expression of two genes involved in fatty acid synthesis by rt-qpcr: acetyl-coa carboxylase and fatty acid synthase. expression of both tended to increase during the postnatal adipose transformation, suggesting a higher rate of fatty acid synthesis in wat compared to bat.figure expression of selected metabolic enzymes related to fatty acid metabolism. total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis. relative expression was measured for: carnitine palmitoyltransferase 1b and hydroxyacyl-coa dehydrogenase subunit α; acetyl-coa carboxylase and fatty acid synthase; diacylglycerol o-acyltransferase and dgat the mrna expression levels were normalized to expression of β-actin. data are mean + sem; *, p < . the go term “metabolic process” included differentially expressed genes, a number of which have been measured by rt-qpcr, including ucp cyc and cs. the two isoforms of the tag synthesis enzymes diacylglycerol o-acyltransferase and dgat were also among the regulated metabolic genes. rt-qpcr measurements confirmed a decreased expression of dgat and dgat in the white adipose phase compared to the transition phase. of the genes differentially expressed between the brown adipose and transition phase and genes differentially expressed between the transition and the white adipose phase, genes were in common. a venn diagram of the regulated genes is shown in additional file 7: figure s fifteen of the common genes were down-regulated at both phase shifts, whereas genes were up-regulated at both phase shifts. among the consistently down-regulated genes were several mitochondrial genes, e.g. the tca cycle enzyme isocitrate dehydrogenase 3α, and the two transcription factors myeloid leukemia factor and autoimmune regulator. among the consistently up-regulated genes were two receptors involved in cellular lipid uptake: low density lipoprotein receptor-related protein and macrophage scavenger receptor. the genes also included genes transiently up-regulated and genes transiently down-regulated during the transition phase. among the genes up-regulated during the transition phase were two enzymes involved in tag synthesis: the mitochondrial glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate o-acyltransferase. transcriptional components regulated between the three phases of adipose tissue transformation expression of transcription factors and co-regulators was significantly changing between the brown adipose and the transition phase, of which were up-regulated. between the transition and the white adipose phase, transcriptional components were differently expressed, with being up-regulated. four transcription factors exhibited differential expression at both phase shifts. using p <  as the cut-off for adjusted p-value, two of the transcriptional components, differently expressed between the brown adipose and the transition phase, had consensus putative response elements in an enriched set of the genes displaying altered expression in the same phase shift. the two transcription factors were nuclear receptor subfamily group h, member and v-myc avian myelocytomatosis viral oncogene homolog. of the differently expressed genes between the transition and the white adipose phase, an enriched set of genes contained consensus putative response elements for six transcription factors that were themselves regulated in the same phase shift. the six transcription factors are nr1h myc, b-cell lymphoma, estrogen receptor, v-rel reticuloendotheliosis viral oncogene homolog a and krüppel-like factor. the expression levels of these six transcription factors in the three phases were validated by rt-qpcr. expression of nr1h and myc was significantly increased and decreased, respectively, in the transition phase compared to both the brown and the white adipose phase. expression of the three transcription factors esr rela and klf was up-regulated between the transition and white adipose phase, whereas bcl was significantly down-regulated from the transition to the white adipose phase.figure expression of regulated transcriptional components with consensus putative response elements in an enriched set of regulated genes. total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis. relative expression was measured for nuclear receptor subfamily group h, member, v-myc avian myelocytomatosis viral oncogene homolog, b-cell lymphoma, estrogen receptor, v-rel reticuloendotheliosis viral oncogene homolog a and krüppel-like factor. the mrna expression levels were normalized to expression of β-actin. data are mean + sem; *, p < . figure 7a lists genes with differential expression between the brown adipose and transition phase that have a consensus putative response element for either myc or nr1h among the up-regulated genes that were potentially regulated by myc from the brown adipose to the transition phase were the adhesion protein thrombospondin and the rab gtpase activator tbc domain family member. among the down-regulated genes from the brown adipose phase to the transition phase potentially regulated by myc were a 9-cis-retinoic acid synthesizing enzyme, aldehyde dehydrogenase family member a and the transcription factor basic helix-loop-helix family member e.figure genes with altered expression that potentially are controlled by transcription factors regulated during the transformation. list of genes regulated from the brown adipose phase to the transition phase, which have consensus putative response elements for nuclear receptor subfamily group h, member and v-myc avian myelocytomatosis viral oncogene homolog. subcellular localization of differentially expressed genes from the transition phase to the white adipose phase that have consensus putative response elements for nr1h myc, b-cell lymphoma, estrogen receptor, krüppel-like factor and v-rel reticuloendotheliosis viral oncogene homolog a. green nodes indicate down-regulated genes in the white adipose phase as compared to the transition phase and red nodes indicate up-regulated genes in the white adipose phase as compared to the transition phase. the corresponding gene names are listed in additional file 10: table s figure 7b depicts the subcellular distribution of the genes potentially regulated by myc, esr rela, bcl klf or nr1h between the transition and the white adipose phase. gene names corresponding to figure 7b are presented in additional file 10: table s forty of the regulated genes were mitochondrial genes, of which were down-regulated. this is in accordance with the decreased activity and amount of mitochondria in the white adipose phase. half of the down-regulated mitochondrial genes have been described to be regulated by rela. nineteen genes encoded secreted proteins, of which were up-regulated, including two pro-angiogenic factors: vascular endothelial growth factor b and angiopoietin-related protein, and one anti-angiogenic factor, serpin peptidase inhibitor f. expression of white, brite/beige and brown adipose markers in the three phases of adipose tissue transformation to study the three phases in the transformation process in more detail, we measured a number of brown and white adipose marker genes by rt-qpcr. as evident from figure  we observed a down-regulation of ucp between the transition and white adipose phase, and a stepwise decrease in expression of dio and ppargc1a through the three phases of the transformation. expression of two transcriptional co-regulators promoting white adipogenesis: nuclear receptor-interacting protein and retinoblastoma, was increased between the transition and the white adipose phase. a typical white adipose marker gene leptin displayed decreased expression in the transition phase compared to both the brown and white adipose phase. expression of the key transcriptional driver of brown adipogenesis, pr domain containing, was not changed significantly between the phases. overall, these measurements supported the brown to white adipose transformation occurring in the sheep perirenal adipose tissue within the first two weeks after birth.figure expression of genetic markers for white, brown and brite/beige adipose tissue during the three phases of the transformation. gene expression was determined by rt-qpcr. relative levels of genes associated with white adipocytes: nuclear receptor-interacting protein, retinoblastoma and leptin. relative levels of the brown adipose associated and marker genes: pr domain containing, solute carrier family member, lim homeobox and myelin protein zero-like. zinc finger protein was not detectable in any of the adipose samples. relative levels of the brite/beige adipose markers: homeobox c, hoxc tumor necrosis factor receptor superfamily member, transmembrane protein and t-box. the mrna expression levels were normalized to expression of β-actin. data are mean + sem; *, p < . to address if the sheep perirenal adipose tissue qualified as being brown, brite/beige or a mixture of brown and brite/beige at birth, and whether this status of the tissue changed over time, we measured a number of recently proposed marker genes selectively expressed in brown versus brite/beige adipose tissue and adipocytes. we determined the expression of the classical brown adipose marker genes solute carrier family member, lim homeobox, myelin protein zero-like and zinc finger of the cerebellum. slc29a was expressed at birth and elicited a stepwise down-regulation through the two adipose phase shifts. mpzl expression did not significantly change, whereas the lhx mrna increased steadily through the three phases. zic was not detectable in any of the perirenal adipose samples, but was easily detectable in sheep brain. we also measured the expression of the three brite/beige marker genes homeobox c, hoxc and tumor necrosis factor receptor superfamily member. hoxc and hoxc are marker genes for both wat and brite/beige adipose tissue. the expression of all three genes increased from the transition phase to the white adipose phase. however, expression of the brite/beige marker genes transmembrane protein and t-box protein did not change. in summary, three out of four markers of classical bat were detectable, and two out of these three changed expression over time. all five measured brite/beige markers were detectable: the expression of three increased and two remained unchanged from the transition to the white adipose phase. thus, markers of both brown and brite/beige adipose tissue were expressed in perirenal adipose tissue from sheep, and most of these markers displayed altered expression over time. discussion plasticity of adipose tissues is important for adaptation to changing physiological conditions. in response to prolonged cold exposure, subcutaneous wat depots of rodents undergo a transformation process during which numerous brite/beige adipocytes appear, thereby increasing overall thermogenic capacity of the animal. in large mammals, a substantial part of bat present in the newborn converts to wat after birth, which may reflect a diminished need for endogenous thermogenesis after the early postnatal period. as little molecular insight into this conversion in large mammals is available, we have in the present study conducted a detailed analysis of the postnatal transformation of perirenal adipose tissue in sheep. we chose this particular tissue, as it is the most frequently studied bat depot in large mammals. moreover, we reasoned that the transformation of this depot was suitably modeling the postnatal brown to white adipose transformation in humans. the perirenal adipose depot from adult humans has recently been characterized at the morphological and transcriptional level. this characterization revealed an enormous variation in expression levels of ucp the postnatal transformation process from bat to wat in sheep perirenal adipose tissue occurred within the first two weeks after birth as determined by changes in tissue morphology, gene expression and mitochondrial density. adipocyte morphology changed from being mainly multilocular to unilocular and the amount of mitochondria decreased. the expression of brown adipocyte-selective genes, e.g. ucp dio and ppargc1a, declined, as did expression of additional genes encoding mitochondrial proteins. to understand the adipose transformation in more detail, we performed a global gene expression analysis with time points ranging from approximately two days before birth to two weeks after birth. by two independent analyses of the gene expression data, we determined that the transformation clustered into three phases: a brown adipose phase, a transition phase and a white adipose phase. regulated transcription factors and co-regulators between the brown adipose and the transition phase were genes differentially expressed, including transcriptional regulators, of which were down-regulated. five of these have chromatin modifying activity: circadian locomotor output cycles kaput, nuclear receptor co-activator, proviral insertion site in moloney murine leukemia virus lymphomagenesis, and the swi/snf-related matrix-associated actin-dependent regulator of chromatin subfamily members smarcc and smarcd this leaves open the possibility that extensive remodeling of chromatin is occurring between the brown adipose and the transition phase. in accordance with its down-regulation in the perirenal adipose transformation, ncoa has been reported to promote bat activity in mice. between the transition and the white adipose phase genes were differentially expressed, of which were transcription factors or co-regulators. the list of regulated transcriptional regulators included a few factors known to be differently expressed in mouse wat and bat, e.g. nrip and cell death-inducing dffa-like effector a. among the down-regulated transcriptional regulators from the transition to the white adipose phase were some related to brown adipocyte function: early b-cell factor, which have been described to induce expression of brown adipose-specific pparg target genes, leucine-rich ppr motif-containing protein, a ppargc1a co-activator playing an important role in bat differentiation and function and y box binding protein, an inducer of bone morphogenetic protein transcription and brown adipocyte differentiation. regulated transcription factors with a consensus putative response element in an enriched set of differentially expressed genes expression of four transcription factors nr1h myc, aire and mlf was regulated at both phase shifts. the former two have a consensus putative response element in an enriched set of genes displaying altered expression at the two phase shifts. expression of nr1h and myc was transiently increased and decreased, respectively, during the transition phase. consistent with the opposite regulation during the transition phase, nr1h have been reported to suppress myc expression in colon cancer cells. myc is known to inhibit adipogenesis, which might explain why it is down-regulated in the transition phase, which includes tissue expansion. nr1h has been described to regulate gene expression linked to several important aspects of both brown and white adipocyte biology, including adipogenesis, energy expenditure, lipolysis and glucose transport. nr1h was reported to be present at higher levels in mouse bat than wat and to suppress pparγ-induced ucp expression by binding to the ucp enhancer together with nrip in mouse adipocytes. accordingly, nr1h can regulate the expression of brown adipocyte-selective genes. however, the increased nr1h expression in the transition phase did not correlate with decreased ucp expression in our study, which might be explained by the relative low expression of nrip in the transition phase. beside nr1h and myc, four other transcription factors, rela, klf esr and bcl were regulated between the transition and the white adipose phase and found to have consensus putative response elements in an enriched number of genes regulated between these two phases. the former three were up-regulated from the transition to the white adipose phase, whereas bcl was down-regulated. both rela and esr have been described to stimulate myc expression, which would be consistent with the increased expression of myc in the white adipose phase. of interest, rela and esr have opposite effects on adipogenesis, as both knockdown of rela and activation of esr by estrogen supplementation attenuated adipogenesis. wat from mice with an adipocyte-specific knockout of rela have decreased lipid droplet size, increased glucose uptake and reduced expression of adipogenic marker genes such as lep, pparg and adiponectin. furthermore, the mice are obesity resistant and less sensitive to cold. esr knockout mice have increased fat mass caused by adipocyte hyperplasia. in addition, esr can inhibit the transcriptional activity of rela. based on this, we speculate that rela and esr might contribute to the regulation of tag accretion during the adipose transformation. rela might also contribute to the observed mitochondrial depletion, as rela negatively impacts mitochondrial content in c2c myocytes. rela has putative response elements in the promoter of genes encoding mitochondrial proteins regulated between the transition and the white adipose phase, of these were down-regulated. this is in accordance with rela functioning both as a transcriptional activator and repressor. rela has been described to repress bcl expression through interferon regulatory factor. this is in accordance with the increased expression of rela and the decreased expression of bcl in the white adipose phase. bcl is a transcriptional repressor with the ability to reduce the expression of e.g. myc. the expression of bcl is strongly down-regulated by growth hormone in 3t3-f442a adipocytes. apart from this, bcl has not been linked to adipocyte or adipose tissue function. klf and rela have been reported to be functionally intertwined, as they directly interact to induce expression of selected genes, but also to compete for interaction with a co-activator, thereby inhibiting each other’s activity. klf is important for induction of adipogenesis in vitro and its expression was reported to be induced in pre-adipocytes by camp. klf stimulates ccaat/enhancer-binding protein β expression, and c/ebpb in turn down-regulates klf expression, thereby forming a negative feedback loop. in summary, six transcription factors with differential expression during the adipose transformation have consensus putative response elements in an enriched set of the regulated genes, suggesting that they are involved in the control of the overall gene expression changes and thus potentially have an impact on remodeling of the tissue. moreover, the six factors are mutually functionally linked, leaving open the possibility that they are part of a transcriptional network. brown and brite/beige markers a number of marker genes selectively expressed in white, brite/beige and brown adipose tissue and adipocytes have been reported. some of these marker genes are not identified consistently between studies. moreover, some markers have been identified from gene expression profiling of whole tissue samples, whereby non-adipose cells cannot be excluded to be a significant source of expression. it is being discussed whether human bat is composed of brown or brite/beige adipocytes or a mixture of these. moreover, it is not fully established to what extent expression of white, brite/beige and brown adipose marker genes changes in a particular adipose depot during development or remodeling. to elucidate this in sheep, we analyzed the expression of selected marker genes in the brown adipose, transition and white adipose phase of the perirenal adipose depot. although we only measured marker gene expression in the perirenal depot, and accordingly have not compared expression levels to those in other adipose depots, we could detect both brown and brite/beige adipocyte marker genes in the newborn sheep. co-expression of marker genes selective for brown and brite/beige adipose tissues have also been observed in a recent study of the human supraclavicular bat depot. in this human study, expression of ucp in supraclavicular biopsies was positively associated with expression of both bat markers and brite/beige adipose markers, whereas expression of the two wat and brite/beige adipose markers hoxc and hoxc correlated with low ucp expression. in our time course study, we did not observe a correlation between high ucp expression and high expression of zic, lhx tbx or tmem but we did detect higher expression of hoxc and hoxc in the white adipose state, where ucp expression was low. a similar hoxc profile in sheep perirenal adipose tissue has been reported by others. bat, brite/beige adipose and wat marker gene expression have not previously been studied in detail during adipose tissue remodeling in large mammals. based on the selective expression profile in mice, our observation that hoxc and hoxc are induced in the white adipose phase suggests that the perirenal adipose depot converts from brown to white. the brown adipose origin of the perirenal depot might be supported by the down-regulation of the bat marker gene slc29a during whitening. however, the 5-fold increase in expression of lhx from the brown to the white adipose phase and the absence of zic expression were not consistent with this model. in addition to hoxc and hoxc a number of other brite/beige marker genes were expressed shortly after birth. the expression of these was either unchanged or up-regulated during whitening. in summary, markers of both brite/beige and brown adipose tissue are expressed in the sheep perirenal adipose tissue at birth and the expression of a number of these changes substantially over time. the latter might be important to consider when analyzing adipose tissue type-selective gene expression. model of the transformation process the postnatal transformation from brown to white in the perirenal adipose tissue can occur through at least three different mechanisms: 1) through transdifferentiation of brown to white adipocytes; 2) through proliferation and differentiation of white adipogenic precursor cells and death of mature brown adipocytes; 3) through a combination of the two. in mice, brite/beige adipocytes can transdifferentiate into white adipocytes, but whether this observation extends to large mammals is unclear. if the white adipocytes arise exclusively from proliferation and differentiation of precursor cells, it would require an enormous cell turnover during the transition phase of the transformation, including extensive death of mature brown adipocytes. we would expect this to result in induction of cell cycle genes, but we did not observe this in the go term analysis. moreover, the expression profile of key cyclins was not consistent with the hypothesis of massive cell cycling during the transition phase. however, we cannot rule out that a small population of proliferating white adipose precursor cells contributed to the whitening of the tissue. in the shift from transition phase to white adipose phase, we did observe an up-regulation of genes related to cell death, but nearly half of the up-regulated cell death associated genes are negative regulators of cell death. thus, it is not obvious from the gene expression data if cell death is increased or not. furthermore, we did not observe evidence for significant cell death in any of the tissue sections analyzed. consistently, lomax et al. failed to detect apoptotic cells during the transformation of the perirenal adipose tissue in sheep. an alternative to the transdifferentiation model is that the white adipocytes present in the white adipose phase are in fact masked brite/beige adipocytes, possibly of a different lineage than white adipocytes. these cells might have the potential to return to a thermogenic state, which would be compatible with existence of such masked adipocytes in mice. arguing against this scenario in sheep is that after postnatal day ucp and other thermogenic genes are no longer inducible in perirenal adipose tissue in response to repeated injections of a β-adrenergic agonist. in summary, we consider it plausible that transdifferentiation of brown to white adipocytes is a significant component of the postnatal transformation of the perirenal adipose depot in sheep. clearly, additional studies are required to validate to what extent transdifferentiation takes place, including a time course with more time points and a dedicated search for evidence of cell proliferation and death.  by global gene expression profiling, we provide novel information of the postnatal bat to wat transformation in sheep. this transformation process is poorly understood in molecular terms, but is of significant interest, as a similar transformation occurs in human infants after birth. an improved understanding of this tissue remodeling increases insight into adipose plasticity and might allow identification of targets suitable for interfering with the balance between energy-storing and energy-dissipating adipose tissue. our results reveal novel transcription factors linked to the adipose transformation process in large mammals. clearly, validation of their relevance in adipose function will require dedicated functional studies. finally, we show that expression of adipose tissue-type selective marker genes change substantially over time, which might be an underappreciated variable in such analyses. additional files additional file 1: table s primers used for qpcr. additional file 2: figure s transmission electron microscopy and expression of mitochondria-related genes during brown to white adipose transformation. transmission electron microscopy and expression of mitochondria-related genes during brown to white adipose transformation. transmission electron micrographs of perirenal adipose tissue at postnatal days and representative images are shown for each of the indicated time points in three different magnifications. total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis. relative expression was measured for peroxisome proliferator-activated receptor γ co-activator 1β, ppara, estrogen-related receptor α and nuclear respiratory factor. the mrna expression levels were normalized to expression of β-actin. data are mean + sem; *, p < vs. day additional file 3: table s read statistics for the sequencing data when mapped and annotated to the sheep genome. a table listing the counts of raw reads from the time points, reads mapped to the sheep genome, uniquely mapped reads and reads mapped to annotated genes in the sheep genome. the table also lists the percentage of total reads in the above mentioned classes. additional file 4: table s normalized counts of genes from perirenal adipose tissue at postnatal days −, and additional file 5: table s list of the differentially expressed genes between the brown adipose phase and the transition phase. additional file 6: table s list of the differentially expressed genes between the transition phase and the white adipose phase. additional file 7: figure s venn diagram of differentially expressed genes between the three phases. additional file 8: figure s heatmap of the genes regulated between the three phases. heatmap of the genes regulated between the brown adipose phase and the transition phase as well as between the transition phase and the white adipose phase. transcription factors are highlighted in yellow. additional file 9: table s transcriptional regulators with changed expression levels between the three phases. transcription factors and co-regulators with changed expression levels in the brown adipose to transition phase shift or in the transition to white adipose phase shift. additional file 10: table s list of differentially expressed genes from the transition phase to the white adipose phase that have consensus putative response elements for nr1h myc, bcl esr klf or rela. list of differentially expressed genes from the transition phase to the white adipose phase that have consensus putative response elements for nuclear receptor subfamily group h, member, v-myc avian myelocytomatosis viral oncogene homolog, b-cell lymphoma, estrogen receptor, krüppel-like factor or v-rel reticuloendotheliosis viral oncogene homolog a. genes are organized according to the subcellular localization of their encoded product. additional file 11: figure s relative levels of brown adipose-associated genes during the three phases of the postnatal adipose transformation. relative levels of brown adipose-associated genes during the three phases of the postnatal adipose transformation. these data are an alternative presentation of the data presented in figure 1b. total rna was isolated from perirenal adipose tissue and used for rt-qpcr analysis. relative expression was measured for uncoupling protein, type ii iodothyronine deiodinase and peroxisome proliferator-activated receptor γ co-activator 1α. the mrna expression levels were normalized to expression of β-actin. data are mean + sem; *, p <. additional file 12: figure s mutual interactions between differentially expressed transcription factors with putative response elements in an enriched set of regulated genes. mutual interactions between the six differentially expressed transcription factors with putative response elements in an enriched set of genes with altered expression during the adipose transformation. the brown oval-shaped circles represent the differentially expressed transcription factors; white oval-shaped circles represent other transcriptional components functionally linked to the differentially expressed transcription factors; stop arrows indicate repression/inhibition; arrows indicate activation/stimulation. abbreviations acacaacetyl-coa carboxylase 1 agpat91-acylglycerol-3-phosphate o-acyltransferase 9 aireautoimmune regulator aldh8a1aldehyde dehydrogenase family member a1 aldoaglycolytic enzyme aldolase a angptl2angiopoietin-related protein 2 atp5batp synthase β batbrown adipose tissue bcl6b-cell lymphoma 6 bhlhe40basic helix-loop-helix family member e40 bmp7bone morphogenetic protein 7 c/ebpbccaat/enhancer-binding protein β catcatalase ccncyclin cd36cluster of differentiation 36 cideacell death-inducing dffa-like effector a clockcircadian locomotor output cycles kaput cpt1bcarnitine palmitoyltransferase 1b cscitrate synthase cyc1cytochrome c1 dgatdiacylglycerol o-acyltransferase dio2type ii iodothyronine deiodinase ebf2early b-cell factor 2 edtaethylenediaminetetraacetic acid epas1endothelial pas domain-containing protein 1 erraestrogen-related receptor α esr1estrogen receptor 1 fabp4fatty acid-binding protein 4 fasnfatty acid synthase fdrfalse discovery rate gpammitochondrial glycerol-3-phosphate acyltransferase hadhhydroxyacyl-coa dehydrogenase complex hadhahadh catalytic subunit α hgnchugo gene nomenclature committee hehematoxylin-eosin hoxc8homeobox c8 hugohuman genome organization idh3aisocitrate dehydrogenase 3α klf4krüppel-like factor 4 lepleptin lhx8lim homeobox 8 lrp1low density lipoprotein receptor-related protein 1 lrpprcleucine-rich ppr motif-containing protein mlf1myeloid leukemia factor 1 mpzl2myelin protein zero-like 2 msr1macrophage scavenger receptor 1 mt-co1mitochondrially encoded cytochrome c oxidase i mtdnamitochondrial dna mycv-myc avian myelocytomatosis viral oncogene homolog ncoa1nuclear receptor co-activator 1 ndnanuclear dna nr1h3nuclear receptor subfamily group h, member 3 nrf1nuclear respiratory factor 1 nrip1nuclear receptor-interacting protein 1 pcaprincipal component analysis pim1proviral insertion site in moloney murine leukemia virus lymphomagenesis ppargperoxisome proliferator-activated receptor γ ppargc1apparg co-activator 1α prdm16pr domain containing 16 rb1retinoblastoma 1 relav-rel reticuloendotheliosis viral oncogene homolog a rgccregulator of cell cycle rt-qpcrreverse transcription-quantitative polymerase chain reaction serpinf1serpin peptidase inhibitor f1 slc29a1solute carrier family member 1 smarcc2swi/snf-related matrix-associated actin-dependent regulator of chromatin subfamily member c2 st7suppression of tumorigenicity 7 tagtriacylglycerol taldo1transaldolase tbx1t-box protein 1 tcatricarboxylic acid temtransmission electron microscopy tfiibtranscription factor iib tmem26transmembrane protein 26 tnfrsf9tumor necrosis factor receptor superfamily member 9 ucp1uncoupling protein 1 vegfbvascular endothelial growth factor b watwhite adipose tissue ybx1y box binding protein 1 zic1zinc finger of the cerebellum 1 astrid l basse, karen dixen and rachita yadav contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions alb, kd, mpt, kk, bq, jw and jbh conceived and designed the experiments. alb, kd, ry, mpt, kq, bq and jbh performed the experiments. alb, kd, ry, rg and jbh analyzed and interpreted the data. alb, kd, ry and kq prepared the figures. alb, kd, ry and jbh wrote the paper. all authors read and approved the final manuscript. 
global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. using global gene expression profiling of the postnatal bat to wat transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. batbrite/beige adipose tissueglobal gene expression profilingmitochondrial numbersheeptranscription factorsucp1watissue-copyright-statement© the author 2015 the transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. large mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue. between the brown adipose and the transition phase genes were differentially expressed, and genes were differentially expressed between the transition and the white adipose phase. finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time. we analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. we observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from bat to wat. we identified a number of regulated transcription factors, including nr1h myc, klf esr rela and bcl which were linked to the overall changes in gene expression during the adipose tissue remodeling. the majority of bat disappears after birth and is replaced by white adipose tissue. moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.
 large mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue. the majority of bat disappears after birth and is replaced by white adipose tissue.  we analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. we observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from bat to wat. the transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. between the brown adipose and the transition phase genes were differentially expressed, and genes were differentially expressed between the transition and the white adipose phase. thirty-eight genes were shared among the two sets of differentially expressed genes. we identified a number of regulated transcription factors, including nr1h myc, klf esr rela and bcl which were linked to the overall changes in gene expression during the adipose tissue remodeling. finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time.  using global gene expression profiling of the postnatal bat to wat transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. moreover, our data set provides a useful resource for further studies in adipose tissue plasticity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords batbrite/beige adipose tissueglobal gene expression profilingmitochondrial numbersheeptranscription factorsucp1watissue-copyright-statement© the author 2015 