Abstract

The Study of Adipose Tissue Differentiation and Development

by

Olga Gulyaeva

Doctor of Philosophy in Endocrinology

University of California, Berkeley

Professor Hei Sook Sul, Chair

Two functionally different adipose tissues exist in mammals: white (WAT) and brown

(BAT). WAT is the primary energy storage site and a critical organ for regulation of

energy metabolism through secretion of various adipokines. In contrast, BAT is a key

thermogenic organ, capable of burning chemical substrates for heat generation through

unique protein UCP1. Interestingly, UCP1 is only expressed in brown or beige

adipocytes found in WAT, however all the transcriptional activators of UCP1 promoter

identified by far are not tissue-specific and, therefore, cannot solely explain BAT-specific

expression of UCP1. BAT is believed to form early in embryogenesis from muscle-like

progenitors, while WAT depending on the anatomical locations, develops in late

embryogenesis or postnatally from non-muscle–like progenitors. Although embryogenic

BAT precursors have been well characterized, the molecular identity of WAT precursors

remains controversial. The aim of this dissertation work was to identify novel tissuespecific

transcriptional regulator of UCP1 in BAT as well as to identify and characterize

WAT precursors at various stages of adipogenic commitment and differentiation.

Chapter 1 reviews developmental origin of adipose tissue. Most of subcutaneous WAT

depots develop perinatally, while visceral WAT develops postnatally from adipose

precursors expressing markers such as Myf5, Pref-1, Wt1, and Prx1 depending on the

anatomical location. BAT develops in early embryogenesis form Myf5+, Pax7+ musclelike

precursors. Brown fat-like cells found in WAT upon cold exposure or b3-adrenergic

stimulation may arise from transdifferentiation of white adipocytes or by de-novo

differentiation of yet to be characterized precursors. Finally, transcriptional and

epigenetic control of brown and beige adipocyte differentiation is discussed.

Chapter 2 demonstrates my efforts to characterize the hierarchy of adipose progenitors

in WAT in vivo. By using Pref-1 promoter-mediated conditional ablation of Sox9, one of

Pref-1 targets in inhibiting adipogenesis, coupled with fluorescent labeling, I identified

and characterized a population of early adipose progenitors that express Pref-1, Sox9

and CD24. This early WAT precursor population is highly proliferative and does not yet

express adipogenic markers and rather resembles stem cell-like population. Sox9

appeared to be critical for maintaining these early precursors in highly proliferative and

undifferentiated state. Loss of Sox9 in Pref-1+ cells lead to a depletion of this precursor

population and an increase in more committed PDGFRa+ preadipocytes that do not

proliferate and express early adipogenic markers. This switch in precursor populations

from Pref-1+ to PDGFRa+ in the absence of Sox9 leads to enhanced adipogenesis and

increased adiposity impairing insulin sensitivity in vivo.

Chapter 3 describes a role of novel BAT-enriched transcription factor, Zfp516 in

regulating UCP1 transcription and BAT differentiation and development. I found that

Zfp516 is induced upon cold exposure and directly binds and activates UCP1 and

PGC1a promoters. I also showed that knockdown of Zfp516 in cultured cells impairs

BAT-gene program and induces muscle-like gene signature. Thus, total body Zfp516

KO mice exhibit severe defect in BAT development, while transgenic mice

overexpressing Zfp516 in all adipose tissues show increased browning of WAT. This

leads to improved cold tolerance and reduces body weight accumulation upon high fat

diet improving metabolic parameters.

Chapter 4 focuses on my efforts to identify Zfp516 interacting partners for activation

BAT-gene program resulting in discovery of lysine-specific demethylase 1, Lsd1, as a

direct binding partner of Zfp516. Lsd1 through interaction with Zfp516 is recruited to

UCP1 and PGC1a promoters to demethylate H3K9 and therefore promoter

transcription. Lsd1 is induced during BAT differentiation and its ablation using Myf5-Cre

results in impaired embryonic BAT development. UCP1-Cre mediated ablation of Lsd1

impairs BAT gene program and results in formation of BAT that resembles WAT,

therefore reducing thermogenic capacity and causing obesity in mice. Lastly, Zfp516-

Lsd1 interaction is required for Lsd1 function for BAT gene program in vitro.

Supplement summarizes my effort to identify a novel gene critical for BAT function and

characterizes one such gene, C11orf54. I found that C11orf54 is highly expressed in

murine BAT, kidney and liver and is highly induced during BAT cell line differentiation.

C11orf54 is localized to the nucleus and cytoplasm and appears to be important for BAT

differentiation as CRISPR-mediated KO lines demonstrate dramatically impaired

adipogenic differentiation. Overexpression of C11 in Cos7 cells results in increased

formation of TAG through yet-to be identified mechanism.

Finally, chapter 5 summarizes my work describing importance of novel genes identified

for BAT and WAT development, future directions and remaining questions.

Thus, these studies provide a better understanding of how BAT and WAT development

and differentiation are regulated in vivo, as well as explain the hierarchy of adipose

progenitors with various degree of commitment and differentiation in WAT.

Adipose tissue is the major energy storage depot in mammals, storing energy in the form triacylglycerol (TAG) during times of energy excess, and mobilizing TAG, via lipolysis, during times of energy need to generate fatty acids. Desnutrin (also known as ATGL) is a novel TAG hydrolase expressed highly only in white and brown adipose tissue (WAT and BAT). To investigate the role of desnutrin in vivo, transgenic mice that overexpress desnutrin predominantly in adipose tissue (aP2-desnutrin mice) were generated. aP2-desnutrin mice had increased lipolysis in adipose tissue and were resistant to diet-induced obesity. Surprisingly, this increased lipolysis in adipose tissue did not result in higher levels of serum non-esterified fatty acids (NEFA), but instead promoted fatty acid oxidation directly within adipocytes resulting in higher energy expenditure and a leaner phenotype. To further investigate the adipose-specific role of desnutrin, gene targeting by homologous recombination in embryonic stem cells was performed to generate adipose-specific conditional desnutrin knockout mice (desnutrin ASKO mice). In contrast to aP2-desnutrin mice, these mice had markedly decreased lipolysis in both WAT and BAT with massive accumulation of TAG in these tissues. These mice had impaired thermogenesis and decreased oxygen consumption. Notably, BAT in these mice was drastically enlarged, and resembled WAT, indicating that desnutrin may be important in the conversion of WAT to BAT.

While the classic model of the endocrine regulation of lipolysis by catecholamines and insulin has been extensively studied, the local regulation of lipolysis in adipose tissue by autocrine/paracrine factors is not well understood. Identification of adipose-specific phospholipase A2 (AdPLA) revealed a surprisingly important and dominant role for adipocyte-derived PGE2 in the autocrine/paracrine regulation of lipolysis. As a PLA2, AdPLA catalyzes the release of fatty acids from the sn-2 position of phospholipids that is typically enriched in arachidonic acid, providing substrate for the initial, rate-limiting step in the synthesis of eicosanoids. To investigate the role of AdPLA in vivo, AdPLA null mice were generated. Through the PGE2/EP3/cAMP pathway, AdPLA dominantly regulates lipolysis in adipose tissue in an autocrine/paracrine manner. The unrestrained adipocyte lipolysis in AdPLA null mice resulted in resistance to both diet-induced and genetic obesity. Consistent with results from aP2-desnutrin mice, despite elevated lipolysis in adipose tissue, serum NEFA levels were not elevated in AdPLA null mice and fatty acid oxidation was increased in WAT, resulting in higher energy expenditure and resistance to obesity. Taken together, findings from aP2-desnutrin mice, desnutrin ASKO mice, and AdPLA null mice have led to the discovery of novel players in the lipolytic cascade, and revealed an unexpected link between lipolysis and the ability of adipose tissue to oxidize fatty acids and release heat, rather than to provide useful energy for work.

Obesity, an excess accumulation of white adipose tissue, has become a global epidemic. White adipose tissue (WAT) plays a critical role by serving as the major energy storage site and by secreting adipokines that control various biological processes such as appetite, metabolism and insulin sensitivity. In contrast, brown adipose tissue (BAT) or BAT-like tissue dissipate energy via non-shivering thermogenesis to maintain body temperature and is known to inversely correlate with adiposity. BAT not only has high capacity of glucose and fatty acid (FA) uptake, but also secretes adipokines, all potentially contributing to insulin sensitivity. Understanding how adipocyte differentiation (adipogenesis) and thermogenesis is regulated and supported can further effort of developing therapeutic strategies against obesity/lipodystrophy. The aims of this dissertation work were to identify the receptor of Pref-1, an adipose tissue-specific secreted factor that inhibits adipogenesis and to characterize Aifm2 function during thermogenesis in BAT.

Chapter 1 reviews the developmental of various adipose depots. While subcutaneous WAT develops perinatally, visceral WAT develops after birth. They are derived from adipose progenitors expressing distinctive markers depending on the anatomical location. Adipogenesis is controlled by transcription factors which is tightly regulated by secreted factors such as Wnt, and Pref-1. In contrast, BAT forms in early embryogenesis from Myf5+, Pax7+ precursors. Brown fat-like cells can be found in WAT upon cold exposure or -adrenergic stimulation. In this chapter, transcriptional and epigenetic control of brown and beige adipocyte differentiation and thermogenic program is discussed. Finally, how metabolites can regulate this transcriptional machinery and support thermogenesis in BAT and BAT-like tissues is also highlighted.

Chapter 2 profiles my effort to identify the receptor of Pref-1 by employing a new improved and more sensitive method of crosslinking in live cells. I found a high level of plasma membrane ST2 in 3T3-L1 preadipocytes and in preadipocytes of SVF from WAT. Like Pref-1, ST2 is downregulated during adipocyte differentiation. In contrast, IL-33, the ST2 known ligands, is upregulated during adipocyte differentiation. I also found that Pref-1 binds tightly to ST2 at the plasma membrane of adipose precursor cells to inhibit adipogenesis. ST2 KO prevented Pref-1 activation of phospho-ERK and its inhibitory effect on adipogenesis. Moreover, our ST2 loss-of function approaches in mice showed in vivo evidence of ST2 requirement for Pref-1 to exert its effects on adipogenesis.

Chapter 3 profiles my discovery of Aifm2 as a lipid droplet (LD) associated NADH oxidase that is expressed at a high level only in BAT, but not in other tissues, and is cold inducible. I found Aifm2 to increase cytosolic NAD/NADH, correlating with higher glycolytic rate leading to higher oxygen consumption/uncoupled respiration and heat production in BAT cells. The Aifm2 BAT specific knockout mice were cold-sensitive due to impaired thermogenesis and, with decreased energy expenditure, they exhibited a higher adiposity. Conversely, our transgenic mice overexpressing Aifm2 in UCP1+ cells had a higher thermogenic capacity to better maintain body temperature upon cold exposure, increasing energy expenditure with decreasing adiposity.

Adipose tissue has a central role in controlling mammalian metabolism. Whilewhite adipose tissue (WAT) is to store excess in the form of triglycerides, brown adipose tissue (BAT) is specialized in burning calories and generating heat. Brown adipose tissue is a key thermogenic organ and functions as a mechanism to combat hypothermia in small mammals such as rodents as well as in humans. Classic brown adipocytes contain a high density of mitochondria that constitutively express a protein called Uncoupling protein 1 (UCP1), which dissipates chemical energy by combusting energy and generating heat instead. Mice housed in the cold environment undergo a marked remodeling of their white fat and form beige fat, a third class of inducible cells that appears within white adipose depot that expresses UCP1 in response to cold stimulus. Importantly, there is emerging evidence of thermogenic tissues in human adults after chronic cold, and it is inversely correlated with body mass index and visceral fat. The aim of this dissertation work was to identify and characterize critical thermogenic factors that activate the UCP1 promoter and increase thermogenesis which may serve as promising avenues to combat obesity and associated metabolic diseases. Chapter 1 reviews advances made in understanding the molecular mechanism underlying the thermogenic gene program. A number of key transcriptional regulators critical for the thermogenic gene program centering on activating the UCP1 promoter, have been discovered. Thermogenic gene expression in brown adipocytes relies on coordinated actions of a multitude of transcription factors, including EBF2, PPARγ, Zfp516 and Zc3h10. Moreover, these transcription factors recruit epigenetic factors, such as LSD1 and Dot1l, for specific histone signatures to establish the favorable chromatin landscape. Additionally, this chapter discusses environmental signals that affect gene expression via various signaling pathways and by changes in intracellular metabolites, hence altering epigenome. Chapter 2 demonstrates my effort to characterize a key thermogenic transcription factor, Zc3h10 and discusses its molecular significance in thermogenesis both in vitro and in vivo. As a member of CCCH zinc finger proteins, Zc3h10 directly binds to the distal UCP1 promoter and increases the UCP1 gene expression. Upon sympathetic 2 stimulation, Zc3h10 is phosphorylated at S126 by p38 mitogen-activated protein kinase to increase binding to the distal region of the UCP1 promoter. Zc3h10 overexpression in mice increases thermogenic gene expression and energy expenditure, resulting in a lean phenotype. Conversely, Zc3h10 ablation in mice impairs thermogenic capacity, leading to weight gain. Chapter 3 focuses on the function of the epigenetic factor, Dot1l, the only known H3K79 methyltransferase, in the Zc3h10-mediated activation of the thermogenic program. Through a direct interaction, Dot1l is recruited by Zc3h10 to the promoter regions of thermogenic genes to function as a coactivator by methylating H3K79, and Dot1l and its H3K79 methyltransferase activity are required for activating the thermogenic gene program. Dot1l ablation in Ucp1+ cells in mice prevents activation of Ucp1 and other target genes to reduce thermogenic capacity and energy expenditure, promoting adiposity. Chapter 4 concludes my work describing the importance of key factors identified for thermogenesis and presents future directions and remaining questions.

Lipolysis is the biochemical process during which triacylglycerol (TAG) stored in cellular lipid droplets undergoes stepwise hydrolysis to release fatty acids (FAs). FAs produced from lipolysis in adipose tissue are released into circulation, whereas, in other tissues, FAs are used intracellularly. There are 3 major lipases involved in intracellular

TAG hydrolysis; Desnutrin/adipose triglyceride lipase (ATGL) catalyzes hydrolysis of TAG to form diacylglycerol (DAG). DAG is then hydrolyzed by hormone-sensitive lipase (HSL) to monoacylglycerol, which is further hydrolyzed by monoglyceride lipase (MGL).

By employing adipose-specific conditional desnutrin knockout strategy (desnutrin ASKO mice), we show that desnutrin ASKO mice not only manifests in obesity, but converts BAT to a WAT-like tissue. Desnutrin ASKO mice exhibit impaired thermogenesis with decreased expression of UCP-1, with lower PPAR alpha binding to its promoter, revealing the

requirement of desnutrin-catalyzed lipolysis in the maintenance of a BAT phenotype. We also found that ablation of desnutrin in islet beta cells by using RIP-Cre or RIP-CreER impairs glucose-stimulated insulin secretion (GSIS) and induces TAG accumulation in islets, leading to pronounced hyperglycemia. Furthermore, we show that desnutrin-catalyzed lipolysis activates PPAR delta, rather than PPAR alpha, which in turn is critical for expression of genes involved in mitochondrial function, and thus ATP production, required for GSIS.

We next examined the mode of desnutrin regulation. We found that desnutrin is phosphorylated on Ser406 by AMP-activated protein kinase (AMPK) to increase its TAG hydrolase activity. To study the in vivo significance for AMPK phosphorylation, adipose-specific ablation of AMPK catalytic subunits alpha1 and alpha 2 (AMPK alpha 1/alpha 2 ASKO) were performed. We detected decreased phosphorylation of desnutrin on Ser406, accompanied with a lower TAG hydrolase activity. Furthermore, the inhibitory phosphorylation of hormone sensitive-lipase (HSL) on Ser565 by AMPK was reduced in AMPK alpha 1/alpha 2 ASKO WAT, resulting in an increased activating phosphorylation of HSL on Ser563 and Ser660 by PKA. Overall, AMPK alpha 1/alpha 2 ASKO mice manifest lean phenotype, with decreased fat mass, as well as reduced TAG and DAG content in WAT. We conclude that, when desnutrin activity is low, HSL dominates the lipolytic rate. These in vivo 2 transgenic mice studies reveal a novel function of lipolysis to provide specific ligands for the maintenance and function in adipose and non-adipose tissues. These studies may offer promising therapeutic approaches for obesity, diabetes, and related disorders.

While white adipose tissue (WAT) stores excess energy as triglycerides, brown adipose tissue (BAT) dissipates heat through uncoupling protein 1 (UCP1) by utilizing fuels such as fatty acids and glucose. A third type of adipocytes are beige adipocytes, which arise in WAT depots upon certain stimuli and are capable of thermogenesis, whose process is called browning or beiging. Since the recent discovery of active BAT or BAT-like tissue in human adults, targeting BAT has been proposed as an attractive strategy to fight against obesity and its associated diseases as there are currently no effective treatments other than lifestyle changes. Regardless, the mechanisms underlying BAT differentiation and thermogenesis are not fully understood. The aim of my dissertation work was to identify and characterize novel regulators of BAT differentiation and thermogenesis.Chapter 1 reviews the current knowledge on signaling pathways that regulate thermogenesis in adipocytes. Upon cold exposure, norepinephrine is released and activates β3-adrenergic signaling in BAT. This causes activation of UCP1, upregulation of thermogenic genes, and increase in substrate supply for thermogenesis. Additionally, insulin/IGF-1 signaling is also important for BAT development as well as browning of WAT. The involvement of other signaling pathways such as those for thyroid hormone and TGFβ superfamily are also discussed. Chapter 2 is to identify and characterize novel thermogenic genes in brown adipocytes. By utilizing CRISPR-Cas9 screening along with a temperature-sensitive dye, I identified Apold1, which is enriched in BAT and responsive to β3-adrenergic stimuli. Through in vitro gain-of-function and loss-of-function experiments, I confirmed that Apold1 indeed promotes thermogenesis. Unexpectedly, Apold1 global knockout mice showed lower body weight due to reduced food intake. Thus, the tissue-specific role of Apold1 was examined by viral injection of Apold1 into BAT, which increased body temperature and oxygen consumption in mice. Chapter 3 describes another screening effort to identify novel thermogenic regulators. Using a different dye as a readout for thermogenesis, I found Rasl12 to promote thermogenesis in brown adipocytes. The role of Rasl12 on thermogenesis was verified by in vitro experiments. Interestingly, co-immunoprecipitation combined with mass spectrometry revealed the possible interaction of Rasl12 with nuclear transporters and RNA processing factors, which is an unexplored aspect of thermogenesis regulation. Chapter 4 profiles yet another screening effort to identify and characterize novel regulators for brown adipocyte differentiation. I found Zc2hc1c, which suppressed brown adipocyte differentiation when overexpressed. RNA-sequencing and RT-qPCR revealed that Zc2hc1c KO cells had increased expression of PPARγ, a master regulator of adipocyte differentiation, suggesting the inhibitory effect of Zc2hc1c on differentiation. Lastly, chapter 5 concludes my work describing the importance of novel genes identified in BAT thermogenesis and differentiation, future directions and remaining questions. All together, these studies have identified novel regulators of thermogenesis and differentiation in brown adipocytes, which will provide a better understanding of BAT biology and advance the field to combat obesity.

Transcription of genes encoding enzymes involved in fatty acid and triacylglycerol synthesis, including fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase, is coordinately induced in lipogenic tissues by feeding and insulin treatment. Dysregulation of lipognesis often contributes to metabolic diseases such as obesity, diabetes, and cardiovascular diseases. Transcription factors and signaling molecules involved in transcriptional activation of lipogenesis represent attractive targets for the prevention and treatment of metabolic diseases.

In transcriptional activation of fatty acid synthase by feeding/insulin, USF constitutively bound to the -65 E-box is required. In this study, USF was shown to function as a molecular switch by recruiting various interacting proteins during the fasting/feeding transition. First, USF was detected to directly interact with SREBP-1 that is induced by feeding and binds nearby -150 SRE. Cotransfection of USF and SREBP-1c with an FAS promoter-luciferase reporter construct resulted in high synergistic activation of the FAS promoter. Chromatin immunoprecipitation analysis of mouse liver demonstrated that USF binds constitutively to the fatty acid synthase promoter during fasting/feeding, whereas the binding of SREBP-1 was observed only during feeding, in a manner identical to that of the FAS promoter. These data show that USF recruits SREBP-1c to the lipogenic gene promoters upon feeding/insulin. Similar cooperative action of USF and SREBP-1c in the activation of the mitochondrial glycerol-3-phosphate acyltransferase promoter was also observed.

During feeding/insulin, USF-1 recruits three distinct families of proteins to the lipogenic promoters: 1) P/CAF which acetylates USF and functions as a coactivator, 2) DNA break/repair machinery including Ku70, Ku80, PARP-1, TopoIIB and DNA-PK, which causes a DNA break in the lipogenic gene promoter prior to the transcriptional initiation in vivo, and 3) signaling molecules including PP1 and DNA-PK. PP1 dephosphorylates and activates DNA-PK upon feeding/insulin treatment. Thus in the fed state, activated DNA-PK phosphorylates USF-1 at S262, allowing the recruitment of and acetylation by P/CAF at K237, leading to promoter activation. P/CAF-mediated acetylation of USF is reversed by HDAC9 in the fasted state. Although total HDAC9 levels do not change during fasting/feeding, nuclear abundance of HDAC9 increases upon fasting, suggesting regulation of HDAC9 by nuclear translocation. DNA break/repair components associated with USF also bring about transient DNA breaks during feeding-induced FAS activation. In DNA-PK deficient SCID mice, feeding induced USF-1 phosphorylation/acetylation, DNA-breaks, and FAS activation leading to lipogenesis are impaired, resulting in decreased liver and circulating triglyceride levels. This study demonstrates that DNA-PK mediates the feeding/insulin-dependent lipogenic gene activation. BAF60c was also detected to be an USF interacting protein recruited to lipogenic gene promoters upon feeding. Among the three isoforms of BAF60s, BAF60c is the only BAF60 specific to lipogenesis and recruits other BAF subunits including BAF155 and BAF190 for the formation of lipoBAF. BAF60 proteins function as the bridge between transcription factors and the BAF complex. BAF60c was found to be phosphorylated at S247 by aPKC upon feeding/insulin. BAF60c was translocated from the cytosol to the nucleus in response to feeding/insulin and the nuclear localization was dependent on its S247 phosphorylation. Furthermore, this BAF60c phosphorylation together with USF acetylation were required for the interaction between the two proteins. Overexpression of BAF60c activated the lipogenic transcription program in mice even in the fasted state. This study provides a novel mechanism to fine-tune lipogenic transcription in response to feeding/insulin. Closely positioned E-boxes and sterol regulatory elements found in the promoters of several lipogenic genes suggest that USF functions as a master molecular switch as a common mechanism of induction by feeding/insulin. Taken together, identification of SREBP-1c, DNA-PK and BAF60c as USF interacting proteins has led to the discovery of novel players in insulin signaling cascade and has revealed an unexpected link between DNA break/repair and metabolism.

Obesity has become a worldwide epidemic, which has lead to a surge of interest in therapeutics to combat this condition. Characterized by an increase in adipose tissue mass, obesity is associated with increased risk of a variety of detrimental diseases such as cardiovascular diseases and diabetes. Despite major advances in uncovering the root of this problem, the underlying basic biological process of adipose tissue development, as well as its ability to expand, is not well understood. To this end, many transcription factors have been identified to play a role in promoting the differentiation of precursor cells, or preadipocytes, into fully mature and lipid-filled adipocytes. Attempts have been made to identify early precursors of adipocytes, however the origin of these adipocytes are not clear, and classic lineage tracing has not been performed extensively. The aim of this dissertation work was to identify adipose precursor cells using the preadipocyte marker, Pref-1, to investigate adipose precursors during embryogenesis and the expansion of adipose tissue in adults.

Chapter 1 reviews the adipose tissue organ and the role of Pref-1 in the regulation of adipocyte differentiation. In addition to being the main energy storage organ, adipose tissue is now well recognized as an endocrine organ of the body, and there are a variety of hormones and cytokines that are secreted by adipose tissue for many physiological functions including energy homeostasis. Pref-1 is expressed prior to differentiation into adipocytes, and becomes extinguished during differentiation. Pref-1 activates MEK/ERK to upregulate Sox9 which binds to the promoter and blocks expression of several important transcription factors that promote adipocyte differentiation.

Chapter 2 focuses on understanding white adipose tissue development and the identification and characterization of adipose precursors. To investigate adipose tissue development, I generated transgenic mouse models to label and ablate Pref-1 expressing cells in vivo, with the overall goal of uncovering details of adipose development during embryogenesis, and diet induced expansion of adipose tissue in adults. Pref-1 expressing cells represent adipose precursors that are mesenchymal in origin, and not endothelial cells or pericytes. These precursors are capable of division and are required for the expansion of the adipose tissue both during embryogenesis and during high fat diet induced obesity.

Chapter 3 focuses on another type of fat, brown adipose tissue (BAT), which, unlike white adipose tissue (WAT), burns fatty acids to perform non-shivering thermogenesis, and which was recently recognized to be present even in adult humans. Brown adipocytes have a different morphological and gene expression profile compared to white adipocytes, and can be identified by the unique expression of Uncoupling Protein-1 (UCP-1). UCP-1 acts to uncouple respiration from ATP production, resulting in heat production. I generated transgenic mice to label UCP-1 expressing cells in vivo, and to characterize these cells during cold induced or beta-adrenergic agonist induced expansion of BAT.

Chapter 4, concludes this body of work, and discusses future directions and remaining questions. Thus, these studies provide a better understanding of both white and brown adipose tissue development and characterization of the precursors responsible for adipose tissue development, and may offer future therapeutic targets for obesity.