Translational Profiling Reveals the Transcriptome of Leptin Receptor Neurons and Its Regulation by Leptin

TRANSLATIONAL PROFILING REVEALS THE TRANSCRIPTOME OF LEPTIN RECEPTOR NEURONS AND ITS REGULATION BY LEPTIN

Margaret B. Allison

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Integrative Physiology) In the University of Michigan 2015

Doctoral Committee: Professor Martin G. Myers Jr., Chair Associate Professor Carol F. Elias Professor Malcolm J. Low Professor Suzanne Moenter Professor Audrey Seasholtz

Before you leave these portals

To meet less fortunate mortals

There's just one final message

I would give to you:

You all have learned reliance

On the sacred teachings of science

So I hope, through life, you never will decline

In spite of philistine defiance

To do what all good scientists do:

Experiment!

-- Cole Porter

There is no cure for curiosity.

-- unknown

2015 ACKNOWLEDGEMENTS

If it takes a village to raise a child, it takes a research university to raise a graduate student. There are many people who have supported me over the past six years at Michigan, and it is hard to imagine pursuing my PhD without them. First and foremost among all the people I need to thank is my mentor, Martin. Nothing I might say here would ever suffice to cover the depth and breadth of my gratitude to him. Without his patience, his insight, and his at times insufferably positive outlook, I don’t know where I would be today. Martin supported my intellectual curiosity, honed my scientific inquiry, and allowed me to do some really fun research in his lab. It was a privilege and a pleasure to work for him and with him.

I also have to thank the many members of the Myers lab over the years.

Research is sometimes a solitary endeavor, but I was lucky to pursue it in very good company. Particular thanks go out to Megan Greenwald-Yarnell, Christa Patterson

Polidori, Amy Sutton, and Paula Goforth in this regard. In addition to being a challenging and thoughtful group with whom to pursue scientific investigation, they made coming into lab each day a pleasure.

A special acknowledgement has to be extended to Dave Olson. Scientifically, without his eGFP-L10a mice, none of this dissertation would have been possible. More

ii valuable to me by far was his mentorship and friendship. He was an irreplaceable source of insight for many of my projects, and his thoughtful approach to science is one

I hope to emulate in the future. He also deserves a thank you for putting up with me for so long. I know it was painful, and he may be scarred for life.

A host of people remained to be thanked, and I will try to get them all. Thank you: to my dissertation committee, for their thoughtful and valuable questions, advice, and support over the years; to the MSTP program, including Ron, Ellen, Hilkka, and Laurie, for welcoming me to the University, and for their ability to solve almost any problem that a graduate or medical student might face; to MIP, including Michele Boggs, Scott

Pletcher, Sue Moenter, and Ormond Macdougald, for their assistance in navigating the sometimes treacherous waters of graduate school; to Matthew Brady, for getting me hooked on research, and for introducing me to Martin; to my fellow MIP students for their good company at Pub nights; to AARC for giving me a reason to wake up in the morning; and to Alex, Steve, and Andrew, for sushi nights that gave me something to look forward to every week.

I should note that some of this work has been published previously. The introduction is derived from a review article that appeared in the Journal of

Endocrinology in October 2014. The second chapter of this dissertation is currently in press at Molecular Metabolism (2015). The material in chapters 3 and 4 has not been published. I have to thank Christa Patterson in particular for her help in generating this data. Without her expert hypothalamic and brainstem dissections, I would not have been able to perform many, if not all, of the TRAP-Seq experiments described.

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Additionally, the efforts of the University of Michigan DNA Sequencing and

Bioinformatics cores were instrumental in the success of this project.

Finally, to my family: I love you very much. Thank you for supporting me in all of my endeavors, for investing so much in my education, and for reminding me that there are few problems that a strong cocktail, a long run, or a good night’s sleep can’t solve.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………………………………..ii

LIST OF FIGURES…………………………………………………………………………………………………..vi

LIST OF TABLES…………………………………………………………………………………………………….vii

LIST OF APPENDICES…………………………………………………………………………………………….viii

ABSTRACT…………………………………………………………………………………………………………...ix

CHAPTER

1. INTRODUCTION…………………………………………………………………………………1

2. TRAP-SEQ DEFINES MARKERS FOR NOVEL POPULATIONS OF HYPOTHALAMIC AND BRAINSTEM LEPRB NEURONS……………………………………………………………25

3. TRANSCRIPTIONAL AND TRANSLATIONAL PROGRAMS INDUCED BY LEPTIN IN LEPRB NEURONS………………………………………………………………………………………….59

4. REGULATION OF THE LEPRB TRANSCRIPTOME BY LEPTIN AND LEPRB-STAT3 SIGNALING…………………………………………………………………………………………87

5. CONCLUSIONS AND FUTURE DIRECTIONS…………………………………………139

APPENDICES……………………………………………………………………………………………………….150

LIST OF FIGURES

Figure

1.1 Hypothalamic leptin action………………………………………………………………………….....18

1.2 Leptin signaling and biological functions………………………………………………………….19

2.1 LepRbeGFP-L10a mice allow for TRAP-Seq in LepRb neurons………………………………..44

2.2 Transcripts enriched in hypothalamic and brainstem LepRb neurons………………46

2.3 Secreted proteins enriched or de-enriched in hypothalamic and brainstem LepRb neurons……………………………………………………………………………………………………………………….47

2.4 Pdyn expression in hypothalamic LepRb neurons…………………………………………….48

2.5 Distribution of Tac1-positive LepRb neurons……………………………………………………50

2.6 Colocalization of LepRb and CRH in the lateral hypothalamus………………………...51

2.7 Colocalization of LepRb and VIP in the brainstem…………………………………………...52

2.8 Metabolic phenotype of LepRPdynKO mice…………………………………………………...... 53

3.1 ATF3 is induced in hypothalamic LepRb neurons……………………………………………..79

3.2 ATF3 induction by leptin in POMC and NPY neurons…………………..…………………..81

3.3 ATF3 is induced by fasting in AgRP neurons…………………………………………………….82

4.1 SERPINA3N colocalization with LepRb, POMC, and AgRP neurons………………...119

4.2 Fold change in diet induced obese vs ob/ob mice………………………………………….123

4.3 Conditional ablation of STAT3 from LepRb neurons……………………………………….125

4.4 Fold change in STAT3LepRKO vs ob/ob mice……………………………………….…………..127

LIST OF TABLES

Table

3.1 Acute leptin treatment………………………………………………………………………………...77

3.2 QPCR for TRAP-isolated vs whole hypothalamic RNA...... ….……………….79

4.1 Fold change in gene expression in ob/ob and leptin treated mice ……………….117

4.2 Fold change in neuropeptide expression in ob/ob and leptin treated mice….120

4.3 Fold change in gene expression in diet-induced obese mice..…………..………….121

4.4 Fold change in gene expression in STAT3LepRKO mice……….………………………….128

4.5 Fold change in gene expression in STAT3LepRKO mice (cont)…………………………130

4.6 QPCR analysis of gene expression in STAT3LepRKO and Leprs/s mice………………133

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LIST OF APPENDICES

Appendix

1. Transcripts enriched in hypothalamic LepRb neurons……………………………...... 150

2. Transcripts enriched in brainstem LepRb neurons...... ….……………………………...181

3. Fold change in gene expression in ob/ob, DIO, and leptin treated mice ……………..205

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ABSTRACT

Two thirds of American adults are overweight and at risk for complications such as Type 2 diabetes, heart disease, stroke, and fertility problems. The adipose hormone, leptin, signals via the long isoform of its receptor (LepRb) in the central nervous system to regulate diverse determinants of energy balance, including food intake, energy expenditure, and neuroendocrine output. Previous studies have demonstrated that the lack of leptin or its receptor promotes hyperphagic obesity among other phenotypes. Importantly, the identity of many LepRb subpopulations, as well as the transcriptional effects of leptin in these populations, remain almost entirely unknown. Recently, the optimization of Translating Ribosome Affinity Purification (TRAP) technology has allowed for the isolation of mRNA from specific neuronal populations via the cre-dependent induction of affinity-tagged ribosomes.

We first examined the transcriptome of LepRb neurons to identify markers for LepRb subpopulations. We isolated mRNA from mouse hypothalamic and brainstem LepRb cells by TRAP and analyzed it by RNA-seq (TRAP-Seq). TRAP-Seq defined the LepRb neuron transcriptome and revealed novel markers for previously unrecognized subpopulations of LepRb neurons. LepRb mRNA was enriched for markers of peptidergic neurons, including Pdyn. Pdyncre-mediated ablation of Leprflox in Pdyn neurons (LepRbPdynKO mice) blunted energy expenditure to promote obesity during high-fat feeding.

To determine the regulation of the LepRb transcriptome by leptin, we employed LepRb- specific TRAP-seq on mRNA isolated from the hypothalami of mice treated with exogenous leptin, genetically leptin-deficient (ob/ob) mice, mice exposed to diet induced obesity (DIO), and mice in which STAT3 had been specifically ablated from LepRb neurons. Exogenous leptin treatment induced a number of transcription factors and intracellular proteins but did not affect neuropeptide transcription/translation. In contrast, states of extreme leptin deprivation

ix or repletion, such as in untreated ob/ob mice or in DIO mice, induced changes in multiple neuropeptide species, many of which were also altered in LepRb-specific STAT3 ablated mice. This analysis revealed a small number of transcripts that were altered in multiple treatment conditions, including Socs3, Atf3, Asb4 and members of the Serpina3 family, and which may represent direct and essential targets of hypothalamic LepRb action in the control of energy balance.

CHAPTER 1

INTRODUCTION

Chapter Summary

Hypothalamic leptin action promotes negative energy balance and modulates glucose homeostasis, as well as serving as a permissive signal to the neuroendocrine axes that control growth and reproduction. Since the initial discovery of leptin 20 years ago, we have learned a great deal about the molecular mechanisms of leptin action. An important aspect of this research has been the dissection of the cellular mechanisms of leptin signaling, and how specific leptin signals influence physiology. Leptin acts via the long form of the leptin receptor, LepRb. LepRb activation and subsequent tyrosine phosphorylation recruits and activates multiple signaling pathways, including STAT transcription factors, SHP2 and ERK signaling, the IRS-protein/ PI3Kinase pathway, and SH2B1. Each of these pathways controls specific aspects of leptin action and physiology. Important inhibitory pathways mediated by suppressor of cytokine signaling

(SOCS) proteins also limit physiologic leptin action. This chapter summarizes the signaling pathways engaged by LepRb and their effects on energy balance, glucose homeostasis, and reproduction. Particular emphasis is given to the multiple mouse models which have been used to elucidate these functions in vivo.

Introduction

Obesity and its many comorbidities present a significant challenge to public health in the United States. In 2008, the health care costs associated with obesity totaled more than $147 billion annually. In addition to the economic burden, obesity results in premature death and disability from stroke, cardiovascular disease, and type 2 diabetes mellitus (T2DM) (http://www.cdc.gov/obesity/data/adult.html accessed

6/29/14). Furthermore, the obesity epidemic is no longer confined to the United States.

Worldwide, more than 1.4 billion adults were overweight or obese in 2008[1]. Clearly the need for anti-obesity therapies is large and growing larger, yet no pharmacotherapies have achieved more than minimal success in promoting long-term weight loss.

At its most basic level, body weight is determined by the amount of energy taken in relative to energy expenditure[2]. If energy intake exceeds energy expenditure, excess energy accumulates in the form of triglycerides stored in adipose tissue, resulting in weight gain and obesity. The brain integrates signals of long-term energy stores with other physiologic inputs to modulate energy intake relative to energy expenditure, however. When adipose energy (fat) stores fall, hunger increases and energy expenditure decreases to defend body energy stores; conversely, the brain responds to nutritional surfeit by permitting increased energy expenditure and decreased feeding to maintain a constant body weight.

One of the most important and widely studied players in the control of energy balance is the hormone leptin [3-4]. Leptin was discovered by Friedman and colleagues in 1994; defects in leptin production underlie the massive obesity observed in ob/ob mice[5]. Leptin is produced in adipose tissue in proportion to triglyceride stores, and

serves as a critical indicator of an organism’s long-term energy status[6-7]. Leptin acts primarily in the brain, especially the hypothalamus, where its action is integrated with that of other adipokines, gastrokines, and other signals to coordinate energy homeostasis [3, 8-10]. In addition to leptin-deficient ob/ob mice, rare human mutations resulting in leptin deficiency have also been identified; leptin-deficient mice and humans display hyperphagia, decreased energy expenditure, and early-onset obesity[11-12].

Leptin receptor deficient humans and db/db mice display a similar phenotype [13-14].

Numerous studies have elaborated the critical role for leptin in the modulation of energy balance: the lack of leptin, as in starvation or genetic leptin deficiency, increases hunger while promoting an energy-sparing program of neuroendocrine and autonomic changes, including decreased sympathetic nervous system tone, thyroid function, growth and reproduction[15]. Leptin treatment largely reverses these changes [12, 16].

Decreased leptin also promotes a variety of other behavioral and physiologic changes to respond appropriately to low energy stores[17-19].

Despite the initial heralding of leptin as a potential cure for human obesity, most obese humans exhibit high circulating leptin concentrations[7]. Serum leptin increases in proportion to body fat percentage; obese patients secrete leptin at levels appropriate for their increased adipose mass and display elevated leptin concentrations

(“hyperleptinemia”) relative to lean controls[20]. Clearly, these high circulating leptin levels do not suffice to restore body adiposity to lean levels however, as might be predicted based on the sensitivity of mice and humans to decreases in leptin signaling.

Whether this inability of leptin to suppress feeding in the face of obesity results from an intrinsic or acquired defect in leptin action, or rather reflects the inability of homeostatic

controls to overcome hedonic feeding drives remains a matter of debate. This controversy serves to underscore the importance of developing a more complete understanding of leptin signaling, its cellular effects, target neural pathways, and integration with other determinants of energy homeostasis.

Leptin and the leptin receptor

Leptin is a 146 amino acid protein produced in white adipose tissue in proportion to triglyceride stores[21]. Once secreted into the circulation, leptin travels to the brain, where it enters the CNS, presumably via the choroid plexus and circumventricular organs. In the brain, leptin acts by binding and activating the long form of leptin receptor

(LepRb), which is expressed primarily on specialized subsets of neurons in certain hypothalamic and brainstem nuclei[22-25]. Mutations that inactivate LepRb, as well as antagonists of LepRb activation, confirm that leptin binding to LepRb is required for its biological activity[26-27]. While the Lepr gene encodes multiple isoforms (LepRa-f in rats), only LepRb contains the full intracellular domain necessary for the activation of critical second messenger pathways and normal leptin action [14, 22, 28-29]. Many functions for the other (“short”) forms of the receptor have been hypothesized, including actions as a serum binding protein that functions in leptin stabilization or sequestration

[30-32] , or as a leptin transporter[33] [34], but LepRb alone suffices for the control of energy balance, glucose homeostasis, and other leptin effects, and LepRb thus constitutes the focus of this dissertation

Peripheral actions of leptin

Multiple studies have attempted to assess the role of leptin in the periphery. Mice with ablated hepatic leptin signaling had normal body weight and blood glucose levels, but were protected from high fat diet or age induced insulin intolerance. Mice in which

LepRb was deleted from the pancreas using a Pdxcre or RIPcre also demonstrated improvements in glucose tolerance [35-36]. Interpretation of these results however is confounded by reports that demonstrate hypothalamic cre expression in both the Pdx and RIP models[37-38]. LepRb expression has also been demonstrated in perivascular intestinal cells, however the direct actions of leptin in the modulation of intestinal function have not been determined [39]. Studies examining the role of LepRb in the heart have been difficult to perform based on the negative effects of cre expression on cardiac function [40]. One model revealed an additive role for cardiac specific LepRb deletion in inducing cardiac failure, however, suggesting that LepRb may regulate the cardiovascular system through both central and peripheral mechanisms [41].

Central actions of leptin

Within the brain, leptin acts on multiple populations of LepRb neurons- primarily in the hypothalamus and brainstem [24-25]. While leptin action in the nucleus of the solitary tract plays a role in modulating satiety, and ventral tegmental area LepRb contributes to the control of reward and aversion, hypothalamic LepRb appears to mediate the lion’s share of leptin action on energy balance[9, 42-43]. Within the hypothalamus, leptin acts on LepRb-expressing neurons in multiple hypothalamic nuclei including those in the lateral hypothalamic area and the ventromedial, dorsomedial, ventral premammilary, and arcuate (ARC) nuclei [24-25] (Figure 1.1). These sites each

contain multiple distinct subtypes of LepRb cells which contribute uniquely to leptin action. The most studied site of leptin action is the ARC, where leptin inhibits orexigenic agouti-related protein/neuropeptide Y-containing (AgRP/NPY) neurons, and stimulates anorexigenic proopiomelanocortin (POMC)-containing neurons. POMC neurons produce anorexigenic neuropeptides, while AgRP is a potent antagonist of the melanocortin system and NPY mediates additional orexigenic signals [2].

LepRb signaling

LepRb is an IL6-type class I cytokine receptor consisting of an extracellular leptin binding domain, a single-pass membrane spanning domain, and an intracellular tail that contains binding domains for multiple signaling proteins [13, 44]. LepRb is present on the cell membrane as a mixture of monomers and dimers [45]. Unlike many other cytokine receptors, ligand binding does not appear to activate LepRb by promoting receptor dimerization, but rather promotes a conformational change that results in the autophosphorylation and activation of Janus kinase 2 (JAK2), which is constitutively bound to Box1 and Box2 motifs in the membrane-proximal portion of LepRb [46-47].

Activated JAK2 phosphorylates LepRb on three tyrosine residues in mice: Tyr985,

Tyr1077, and Tyr1138 [46, 48]. Each of these phosphorylated tyrosine (pY) residues represents a Src homology 2 (SH2) binding motif that recruits specific SH2-containing effector proteins to the receptor to mediate subsequent signaling.

Leptin binding to LepRb results in the activation of several major signaling pathways (Figure 1.2). Importantly, phosphorylation of Tyr1138 results in the recruitment of signal tranducer and activator of transcription 3 (STAT3) to LepRb, to permit its

phosphorylation (pSTAT3) and activation by JAK2[46, 49]. Activated pSTAT3 translocates to the nucleus, where it mediates changes in the expression of target genes, including Socs3 (which encodes a feedback inhibitor of LepRb signaling)[50].

Phosphorylation of Tyr985 recruits protein tyrosine phosphatase 2 (SHP2; PTPN1) to

LepRb, contributing to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway [46, 51]. Tyr985 also serves as the binding site for SOCS3, and thus plays a prominent role in the feedback inhibition of LepRb [52]. Phosphorylated Tyr1077 promotes the recruitment and activation of STAT5; Tyr1138 may also contribute to STAT5 activation[48].

Another SH2 domain protein, SH2B1, also participates in LepRb signaling. In addition to increasing the amplitude of LepRb signaling via JAK2, SH2B1 may control specific downstream LepRb signals, including insulin receptor substrate (IRS)-proteins

[53-54]. IRS-proteins also participate in leptin action; they control the phosphatidylinositol 3-kinase (PI3K) pathway, and the subsequent regulation of

AktFoxO1 and mTORC1 signaling [55-57]. The mechanism(s) whereby LepRb modulates this pathway remains obscure; some data suggest a potential role for poorly- understood LepRb signaling that occurs independently of LepRb pY sites [58].

LepRb Signaling & Physiology

LepRbSTAT3 signaling

Multiple LepRb signaling pathways coordinate the regulation of energy homeostasis. Of these, the Tyr1138pSTAT3 pathway plays an especially prominent role[8]. Mice containing a substitution mutation of LepRb Tyr1138 (which renders LepRb

incapable of recruiting and activating STAT3; Leprs/s mice) display hyperphagia and obesity approaching that of db/db animals (although linear growth, fertility, and glucose homeostasis are relatively protected in Leprs/s relative to db/db mice)[59-61].

Furthermore, brain-specific STAT3 knockout mice (STAT3N-/-) exhibit severe obesity[62]. Mice in which STAT3 was deleted specifically in LepRb neurons

(STAT3LepRKO) similarly develop hyperphagic obesity with some preservation of glucose homeostasis [63]. These studies highlight the importance of LepRb

Tyr1138STAT3 signaling for the regulation of body weight, but suggest some regulation of growth, reproduction, and glucose homeostasis by leptin independently of this pathway.

The role of STAT3 signaling in energy balance in discrete neural populations has been best characterized in the ARC. As might be expected, specific deletion of STAT3 from AgRP neurons results in moderate obesity, increased Npy expression, and decreased sensitivity to leptin [64]. STAT3 deletion from POMC neurons also increases adiposity, but the effect is milder than for the AgRP-specific knockout, suggesting a greater role for STAT3 in leptin action in AgRP neurons than in POMC cells[65]. In contrast to STAT3 deletion studies, the interpretation of studies in which a mutant transcriptionally active form of STAT3 (STAT3-C) is expressed in ARC neurons is more complicated. While STAT3-C expression in AgRP neurons promotes leanness, expression of STAT3-C in POMC neurons results in obesity[66-67]. Agrp expression is not altered in STAT3-CAgRP mice, consistent with the notion that Agrp expression is more sensitive to modulation by PI3K than by STAT3 (see below) [66]. It is possible that the mild obesity resulting from STAT3-C action in POMC neurons results from

altered transcriptional activity of this isoform relative to native STAT3, but STAT3-C also promotes Socs3 expression, which could limit endogenous leptin action despite increased transcription mediated by STAT3-C. Interestingly, although the Pomc promoter contains known STAT3 binding sites[68] and Pomc expression is decreased in both Leprs/s mice and animals with neuronal STAT3 ablation[59, 62], Pomc expression is decreased in STAT3-CPOMC animals [67]. This suggests that while Socs3 represents a direct STAT3 target, the control of ARC Pomc expression may be regulated by additional and/or downstream LepRb signals, as well. Taken together, the data from

STAT3-C and STAT3-KO mice may suggest that LepRb-STAT3 signaling is necessary but not sufficient for the regulation of Agrp and Pomc expression by leptin. Importantly, none of the phenotypes resulting from the modulation of LepRbSTAT3 signaling in

POMC or AgRP neurons approach that of brain or hypothalamus-wide modulation, suggesting that LepRbSTAT3 signaling in other, non-ARC LepRb cells contributes importantly to the control of energy balance by LepRbSTAT3 signaling.

Tyr985-dependent signaling, SOCS3, and SHP2

In contrast to the obese phenotype that results from disruption of LepRbSTAT3 signaling, mice with a mutation in Tyr985 display a lean phenotype (which is especially pronounced in females). These mice also display decreased hypothalamic Agrp expression, increased pSTAT3, exaggerated sensitivity to exogenous leptin, and resistance to DIO [69]. These results are consistent with increased LepRb signaling due to decreased LepRb feedback inhibition via disruption of SOCS3 binding. Indeed, as for mice mutant for LepRb Tyr985, disruption of SOCS3 in the brain decreases adiposity

(more dramatically in female than in male mice) and increases the response to exogenous leptin [70].

In addition to its role in feedback inhibition, Tyr985 may also coordinate energy homeostasis via SHP2/ERK signaling [51-52]. As a tyrosine phosphatase, SHP2 was initially investigated as a potential negative regulator of leptin signaling. Deletion of

Shp2 from the forebrain disrupts ERK signaling and promotes early onset obesity, however [71]. Furthermore, deletion of Shp2 from POMC neurons results in mild obesity and increased susceptibility to DIO [72]. Similarly, female mice expressing a dominant active SHP2 mutant in the brain are resistant to DIO [73]. Thus, these data are consistent with the notion that LepRbSHP2 signaling is important for leptin action and the control of energy homeostasis, rather than SHP2 mediating feedback inhibition on

LepRb. While SHP2 plays an essential role in the control of energy homeostasis, however, the promiscuity of SHP2 (which plays roles in many signaling pathways), renders it difficult to assess the specificity of SHP2 effects for LepRb signaling.

Tyr1077 and STAT5

LepRbSTAT5 signaling appears to have little impact on energy balance. While brain-wide STAT5 knockout mice develop late-onset obesity, this phenotype is quite mild [74]. LepRb Tyr1077 mutants develop only mildly increased food intake and adiposity [75]. Furthermore, a recent study deleting STAT5 specifically in LepRb neurons revealed no body weight phenotype; deleting both STAT3 and STAT5 did not produce a more robust phenotype than deleting STAT3 alone [76]. Also, Tyr1077 mutants enter puberty normally, but have a prolonged inter-estrus interval, suggesting mild subfertility in these animals. STAT5LepRKO animals display normal oestrus cycling

and fertility, however. Altogether, these studies suggest that Tyr1077 plays a minor role in the control of feeding and reproductive function, but that STAT5 may not be the binding partner that mediates this effect.

Other LepRb signals

Although the tyrosine phosphorylation of LepRb is essential for the majority of leptin’s actions, mice in which Tyr985,Tyr1077, and Tyr1138 have all been replaced with phenylalanine (LepRb3F) are slightly less obese than db/db animals and display significant improvements in glucose homeostasis and fertility relative to db/db mice [58].

In contrast, mice expressing a LepRb truncation mutant (LepRb65) that retains JAK2 signaling and activity but lacks Tyr985,Tyr1077, and Tyr1138 phenocopy db/db animals, and do not appear to be significantly protected from the obesity, diabetes and infertility that are hallmarks of impaired leptin signaling [77]. Thus the improved phenotype seen in

LepRb3F mice relative to db/db animals does not result from JAK2 signaling alone, since the LepRb65 model reveals that JAK2 signaling is not sufficient to mediate these improvements. The differing phenotypes between mice expressing LepRb3F and

LepRb65 thus suggests the existence of non-canonical signaling pathway which may emanate from a distal site on LepRb, independently of LepRb pY sites. Further work will be required to identify this presumptive pathway.

While SH2B1 and IRS-protein/PI3K signaling contribute to leptin action, the mechanism(s) of their activation by LepRb remain somewhat unclear- no LepRb pY site has been definitively shown to mediate their recruitment. Thus, it is possible that one or both of these pathways constitute the presumptive LepRb pY-independent signaling pathway. Furthermore, these pathways may overlap, since SH2B1 recruits the IRS-

protein/PI3K pathway during leptin signaling in cultured cells[53] [78]. The SH2B1 and

IRS-protein/PI3K pathways contribute to energy balance in vivo, however. SH2B1 null mice display severe early-onset obesity and hyperphagia [54]. Furthermore, neuron- specific restoration of SH2B1 throughout the CNS rescues this phenotype, suggesting that CNS SH2B1 is crucial for the control of body weight [79]. Unfortunately, the critical role of SH2B1 in insulin signaling (which is also significantly impacted by this deletion) as well as in signaling by other receptor tyrosine kinases, renders it challenging to determine whether this phenotype results from only from the disruption of

LepRbSH2B1 signaling.

The roles for PI3K signaling in leptin action and the control of energy balance are also complicated. Leptin administration activates IRS-protein/PI3K signaling in the mediobasal hypothalamus, and ICV treatment with PI3K inhibitors inhibits leptin’s anorexigenic effects [55], along with the ability of exogenous leptin to suppress Agrp mRNA expression in fasted rats [80]. Furthermore, deletion of IRS2 specifically in

LepRb neurons results in obesity, (although it does not impact the ability of LepRb to stimulate pSTAT3)[81]. Both in vitro and in vivo studies have also implicated PI3K signaling in the acute actions of leptin. Leptin treatment induces the depolarization of

POMC neurons in slice recordings; these effects are abrogated by pretreatment with

PI3K inhibitors [82]. This effect is also perturbed in mice lacking the PI3K regulatory subunits p85 and p85 in POMC neurons [82]. While these mice do not display gross phenotypic abnormalities, leptin’s ability to promote acute decreases in food intake is also disrupted. Studies in which the PI3K catalytic subunits p110 and p110 were deleted in AgRP or POMC neurons confirm these findings – mice lacking p110 in

AgRP neurons are mildly lean, whereas mice lacking p110 in POMC neurons are more sensitive to DIO [83]. It is unclear however, whether these results emanate from disrupted LepRb-PI3K signaling, or from alternations in IR-PI3K signaling, especially in light of data that suggests that leptin and insulin activate non-overlapping populations of

POMC neurons [84]. Together, these data suggest that leptin induced PI3K signaling has a limited effect on energy balance. However the importance of the LepRb-PI3K pathway for the glucoregulatory or reproductive functions of leptin is yet to be determined.

Negative regulation of leptin signaling

SOCS3 inhibition of leptin signaling

Multiple pathways and proteins inhibit LepRb function. As one of the genes predicted to cause leptin resistance, SOCS3 and its mechanisms of action in the inhibition of LepRb signaling have been a point of considerable interest[50]. SOCS3 binds to LepRb Tyr985 and mediates negative feedback by directly inhibiting JAK2 activity and/or targeting the receptor-JAK2 complex for proteasomal degradation [50,

52, 85]. Neuron-wide deletion of SOCS3 using either nestin-cre (SOCS3N-/-) or synapsin-cre confers significant resistance to diet-induced obesity [70]. SOCS3N-/- mice also display increased leptin sensitivity as measured by both leptin-induced food intake and STAT3 phosphorylation, as well as by increased PI3K activity. A more recent study demonstrated the SOCS3N-/- mice are lean on both chow and high fat diets, but concluded that the Nestincre allele alone induces leanness in the absence of SOCS3 disruption[86]. In contrast to whole brain SOCS3 deletion (mediated by the suspect

Nestincre), LepRb neuron specific deletion of SOCS3 did not promote leanness in mice fed either a chow or high fat diet. SOCS3LepRKO mice did demonstrate improvements in glucose homeostasis, as well as more sensitivity to exogenous leptin in the suppression of food intake[86]. Overexpression of SOCS3 in LepRb neurons (SOCS3LepROE) yields an unexpected phenotype of slightly increased leanness [87]. This may result from a compensatory increase of STAT3 at baseline and a corresponding increase in pSTAT3 levels after leptin treatment, although the mechanism for this is unclear and would seem counter-intuitive. Clearly, however, the function of Socs3 may not be as uniform or straightforward as initially thought.

Because high fat diet induces Socs3 expression in the ARC, ARC populations have been posited to be a major site of leptin resistance. As a result, the role of Socs3 in arcuate POMC and AgRP neurons has been extensively studied. As with SOCS3N-/- mice, SOCS3POMCKO mice are resistant to DIO, but display normal body weight on chow diet [88]. Interestingly, SOCS3POMCKO mice also have improved glucose homeostasis on a chow diet, suggesting that POMC neurons may be a critical site of

LepRb/SOCS3 signaling in the control of peripheral blood glucose levels. Unlike

SOCS3LepROE mice, mice overexpressing SOCS3 in POMC neurons develop mild obesity on a chow diet, and acute leptin resistance (as assessed by leptin induced inhibition of feeding) prior to any divergence in body weight [87]. These animals also display a POMC neuron-restricted reduction in the pSTAT3 response to leptin, suggesting that potential compensatory mechanisms induced in the SOCS3LepROE model were not activated in this more restricted cell population. SOCS3AgRPOE mice also display early onset leptin resistance, and slightly abnormal glucose homeostasis,

but no alterations in body weight [89]. Thus while decreasing SOCS3 levels may prove protective against obesity, the modest body weight changes that occur with overexpression of SOCS3 suggest that increased SOCS3 levels may reflect hyperleptinemia and increased overall leptin signaling, rather than promoting obesity, per se.

Phosphatase inhibition of LepRb

Protein tyrosine phosphatases (PTPases) also modulate the amplitude and duration of LepRb signaling. Protein tyrosine phosphatase 1B (PTP1B) has been the most extensively studied of these, but other PTPs such as TCPTP and RPTPe also play critical roles in both leptin and insulin signaling (see review by Tsou & Bence, 2013).

PTP1B is a promiscuous phosphatase that attenuates insulin receptor and LepRb signaling as well as inhibiting other receptors. In vitro, PTP1B dose-dependently suppresses the leptin-stimulated phosphorylation of Jak2 and pSTAT3 [90]. In vivo, whole body PTP1B knockout (PTP1BTKO) results in a lean phenotype, resistance to DIO, and increased sensitivity to exogenous leptin, consistent with the interpretation that

PTP1B is a negative regulator of LepRb signaling [91]. Interpretation of the PTP1BTKO model is complicated by the promiscuity of PTP1B and its broad pattern of expression, however, provoking more focused studies of the sites and mechanisms of its action.

Pan-neuronal deletion of PTP1B also induces a lean phenotype, whereas liver or muscle specific deletion has no effect, and adipose specific deletion actually causes weight gain (perhaps due to enhanced adipose insulin signaling) [92]. LepRb neuron- specific PTP1B deletion (PTP1BLepRKO ) results in a leaner phenotype than that observed in the PTP1BTKO mice, suggesting that this model may have unmasked an

even more important role for PTP1B in LepRb neurons that may have been opposed by other tissue (e.g., adipose) effects in the PTP1BTKO model [93]. The specificity of PTP1B action on LepRb for the development of the lean phenotype is supported by the similar phenotypes of hypothalamic LepRb knockout and LepRb/PTP1B double-knockout mice, suggesting the LepRb-dependence of the lean phenotype of PTP1B null animals [94].

Interestingly, heterozygous PTP1BLepR +/- mice display as strong a phenotype as

PTP1BLepRKO, underscoring the importance of appropriate levels of phosphatase action in the control of LepRb signaling [93].

Future Directions: Leptin signaling and gene transcription

Despite the early identification of LepRbSTAT3 signaling as the primary mechanism for leptin’s control of energy balance, LepRbSTAT3 target genes remain poorly defined. Currently, the list of genes known to be regulated by leptin in vivo is short: Socs3, Pomc, Cart, Agrp, and Npy. LepRbSTAT3 signaling is required for appropriate Socs3, Pomc, and Agrp gene expression, although (as noted above) Pomc and Agrp may represent indirect targets of STAT3 and/or may be partly controlled by other pathways; PI3K appears to play a role in the control of Agrp and Npy expression.

Furthermore, of these five genes, only Socs3 is thought to be induced in multiple LepRb populations; Pomc, Agrp, Npy and Cart expression are restricted to circumscribed populations, and do not contribute to leptin action in the majority of LepRb neurons.

This dearth of information about LepRbSTAT3 target genes can largely be attributed to the difficulty of specifically isolating LepRb neurons from the hypothalamic milieu;

LepRb neurons comprise approximately <5% of all hypothalamic neurons, making it

challenging to identify cell-autonomous changes in gene transcription for any subset of neurons. Clearly more work will be necessary to identify the hypothalamic gene targets of LepRb and STAT3 signaling. These transcripts will be responsible for much of leptin action, and may represent potential targets for therapy, in addition to shedding light on the mechanisms of leptin action.

Figure 1.1: Hypothalamic leptin action. Leptin acts on a series of interconnected hypothalamic nuclei to regulate satiety, neuroendocrine function, and autonomic tone. In the arcuate nucleus, leptin controls the melanocortin system through its opposing actions on POMC and AgRP neurons. ARC, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus; LHA, lateral hypothalamic area; PVH, paraventricular hypothalamic nucleus; MC4R, melanocortin 4 receptor; POMC, pro-opiomelanocortin; AgRP, agouti-related peptide; NPY, Neuropeptide Y.

Figure 1.2: Leptin signaling and biological function. Leptin binds to LepRb, activating the associated JAK2 tyrosine kinase. Activated JAK2 phosphorylates the intracellular tail of LepRb on 3 tyrosine residues. Phosphorylated Tyr985 recruits SHP2, which participates in ERK signaling; Tyr985 also serves as a binding site for the negative feedback regulator, SOCS3. Phosphorylated Tyr1077 partially mediates leptin’s control of reproduction; while STAT5 binds this site, STAT5 does not appear to participate in this effect of leptin. Phosphorylated Tyr1138 engages the STAT3 transcription factor. LepRbSTAT3 signaling represents the primary mechanism by which leptin regulates energy balance, although the target genes of STAT3 in LepRb neurons remain undiscovered. Leptin also recruits the IRS2PI3K and SH2B1 pathways, although the mechanism of their recruitment to LepRb remains unclear. Some glucoregulatory and reproductive actions of LepRb appear to be mediated by unknown signals that function independently of LepRb tyrosine phosphorylation sites.

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CHAPTER 2

TRAP-SEQ DEFINES MARKERS FOR NOVEL POPULATIONS OF HYPOTHALAMIC AND BRAINSTEM LEPRB NEURONS

Chapter Summary

Leptin acts via its receptor (LepRb) on multiple subpopulations of LepRb neurons in the brain, each of which controls specific aspects of energy balance. Despite the importance of LepRb-containing neurons, the transcriptome and molecular identity of many LepRb subpopulations remain undefined due to the difficulty of studying the small fraction of total cells represented by LepRb neurons. Here we sought to examine the transcriptome of LepRb neurons directly and identify markers for functionally relevant

LepRb subsets. We isolated mRNA from mouse hypothalamic and brainstem LepRb cells by Translating Ribosome Affinity Purification (TRAP) and analyzed it by RNA-seq

(TRAP-seq). TRAP mRNA from LepRb cells was enriched for markers of peptidergic neurons, while TRAP-depleted mRNA from non-LepRb cells was enriched for markers of glial and immune cells. Genes encoding secreted proteins that were enriched in hypothalamic and brainstem TRAP mRNA revealed subpopulations of LepRb neurons that contained neuropeptide-encoding genes (including prodynorphin, Pdyn) not previously used as functional markers for LepRb neurons. Furthermore, Pdyncre- mediated ablation of Leprflox in Pdyn-expressing neurons (LepRbPdynKO mice) blunted energy expenditure to promote obesity during high-fat feeding. Thus, TRAP-Seq of CNS

LepRb neurons defines the LepRb neuron transcriptome and reveals novel markers for previously unrecognized subpopulations of LepRb neurons.

Introduction

The hormone, leptin, which is produced by adipocytes to signal the repletion of fat stores, acts via the leptin receptor (LepRb) to modulate food intake and energy expenditure [1-3]. Leptin action is also crucial for the control of glucose homeostasis and other metabolic parameters [4-5]. Leptin controls energy balance and metabolism by acting on multiple subtypes of LepRb-expressing neurons in the central nervous system [6-9]. Given the centrality of leptin action to the control of body weight and metabolism, it is crucial to understand mechanisms of leptin action, including the roles of individual populations of LepRb neurons, since these represent points of potential therapeutic intervention.

Anatomically and molecularly distinct subpopulations of LepRb neurons each play specific roles in leptin action [10]. Although hypothalamic LepRb populations have historically been the best characterized, the brainstem also contains several substantial populations of LepRb neurons, including populations in the ventral tegmental area

(VTA), dorsal and linear raphe nuclei (DR, LR), Edinger-Westphal nucleus (EW), periaqueductal gray matter (PAG), parabrachial nucleus (PBN), and nucleus of the solitary tract (NTS)[11-14]. Only a few neuropeptides expressed in brainstem LepRb neurons have been identified, however, and (with few exceptions) brainstem LepRb neurons remain largely uncharacterized [15-17].

Hypothalamic LepRb neurons, including those that contain Nos1 or Vgat, play major roles in the control of energy balance and metabolism, but represent large,

26 heterogeneous and dispersed sets of cells that are not well-suited to circuit-level analysis [8, 18-19]. Smaller, circumscribed sets of LepRb neurons that reside in the hypothalamic arcuate nucleus (ARC) and express either Pomc and Cartpt or Agrp and

Npy each contribute to the control of energy balance and glucose homeostasis [20].

The modest effects observed upon manipulation of LepRb in these neurons suggest important roles for other hypothalamic LepRb neurons in leptin action, however [21-24].

Similarly, SF1/PACAP-containing ventromedial hypothalamic nucleus (VMH) neurons and the Prlh-expressing subset of dorsomedial hypothalamic (DMH) LepRb neurons each participate in the control of energy expenditure, but only modestly contribute to the overall regulation of body weight by leptin [25-27]. A subgroup of lateral hypothalamic

(LHA) LepRb neurons that express neurotensin (Nts) modulates the mesolimbic dopamine system, but also contributes only a small amount to the control of energy balance and metabolism by leptin [28-30]. Even taken together, these known subpopulations of hypothalamic LepRb neurons constitute only a fraction of total hypothalamic LepRb neurons, and fail to explain the totality (or even the majority) of leptin action on feeding, metabolic control, and body weight regulation. Thus, additional, uncharacterized, groups of hypothalamic and brainstem LepRb neurons must contribute importantly to overall leptin action.

To identify, manipulate, and understand the function of potentially important but currently unrecognized subpopulations of LepRb neurons, it is necessary to identify other genes, including neurotransmitters, expressed in these cells. Since LepRb- expressing neurons comprise only a fraction of the cells within the nuclei in which they reside, it has not been possible to disentangle the transcriptome of LepRb cells from

27 that of other cells within these areas [11-12, 31-32]. Fluorescent cell sorting can isolate labeled cells from complex populations, but this approach is suboptimal for LepRb neurons, since hypothalamic and brainstem neurons survive isolation procedures poorly at ages when fluorescent markers for LepRb neurons are robustly expressed. We thus set out to examine the transcriptome of LepRb neurons by expressing an enhanced green fluorescent protein-tagged ribosomal subunit (eGFP-L10a) selectively in LepRb neurons to enable immunopurification of ribosomes and their associated mRNA.

Results

Validation of TRAP-Seq in LepRb neurons

We generated Leprcre;Rosa26eGFP-L10a (LepRbeGFP-L10a) animals that express eGFP-L10a in LepRb neurons (Figure 2.1 A). To confirm the expression of eGFP-L10a in LepRb neurons in the hypothalamus and brainstem of these animals, we examined eGFP-L10a and its colocalization with phosphorylated STAT3 (pSTAT3; a marker of

LepRb signaling) in brain sections from leptin-treated animals. As for other LepRb reporter strains [11-12, 33], eGFP-immunoreactivity (-IR) and pSTAT3-IR colocalized in hypothalamic and brainstem regions known to contain LepRb (Figure 2.1 B-G).

We performed anti-eGFP TRAP on hypothalamic extracts from LepRbeGFP-L10a mice and utilized the resultant mRNA (as well as TRAP-depleted supernatant RNA) to generate multiplexed libraries for sequencing on the Illumina HiSeq2000 platform. We sequenced five independent samples, each containing material from the pooled hypothalami of 4-6 LepRbeGFP-L10a mice. To validate the derivation of TRAP mRNA from

LepRb neurons, we examined the enrichment of genes known to be expressed (or not

28 expressed) in LepRb neurons in TRAP-derived relative to TRAP-depleted sequences

(Figure 2.1 H). TRAP-depleted sequences, rather than input mRNA, were chosen for normalization in order to increase the power of the enrichment analysis. This analysis confirmed the enrichment of all examined markers of neurons known to express LepRb

(including Lepr, Pomc, and Agrp) [25-26, 29-30, 34-38] in TRAP mRNA relative to depleted supernatant. Furthermore, transcripts known to be expressed exclusively in non-LepRb neurons (e.g., Pmch, Hcrt, Oxt, Gnrh1) [33, 39-40] were enriched in the

TRAP-depleted samples relative to TRAP mRNA. Thus, TRAP-Seq identified genes preferentially expressed in LepRb cells by comparison to TRAP-depleted

(predominantly non-LepRb) samples.

The quantification of gene expression by next generation RNA sequencing not only permits the comparison of relative expression between two conditions, but also defines the level of expression for each mRNA species, since the frequency with which each sequence is detected reflects its abundance within the overall sample. In addition to revealing expression levels, these frequency data also enhance the statistical power of relative expression analysis compared to the single observation derived from each sample subjected to microarray analysis, permitting us to identify >1100 genes significantly enriched (>1.5-fold) in hypothalamic LepRb (TRAP) mRNA relative to non-

LepRb (TRAP-depleted) mRNA (Appendix 1).

Although brainstem LepRb neurons constitute a significant percentage of all

LepRb neurons, even less is known about these cells than for hypothalamic LepRb neurons. We thus also dissected brainstem tissue from LepRbeGFP-L10a mice and performed TRAP-seq on two independent samples, each derived from the brainstems of

6-8 animals. This analysis revealed ~900 genes that were significantly enriched in brainstem LepRb RNA relative to non-LepRb RNA (Appendix 2). Our analysis of the non-LepRb (TRAP-depleted) and LepRb (TRAP) mRNA also defined genes that were enriched in non-LepRb cells of the hypothalamus (~1800 genes) and brainstem (~900 genes) relative to LepRb cells.

To understand the common properties of brain cells that express LepRb (as well as of those that do not contain LepRb), we identified genes enriched in both hypothalamic and brainstem TRAP (and TRAP-depleted) mRNA. To characterize the types of cells contributing to TRAP- and TRAP-depleted mRNA, we plotted fold enrichment for hypothalamic and brainstem-derived mRNA to reveal the genes most highly enriched in common between the two sites (Figure 2.2). The genes most highly enriched in hypothalamic and brainstem TRAP mRNA included a number of neuropeptides (Npw, Ucn, Prok2, Ghrh, Cartpt, Tac1) and markers of dopaminergic

(DA) neurons (Slc6a3, Th), as well as some markers for subsets of vasculature- associated cells (Flt1, Abcb1a). Some cell surface receptors and intracellular signaling proteins known to be expressed in neurons important for the control of metabolism

(Mc3r, Gucy2c, Foxa2, Atg7) were also enriched in hypothalamic and brainstem LepRb cells[41-44]. These findings are consistent with the predominant expression of LepRb in peptidergic and DA neurons, as well as in a subset of vasculature-associated cells, in the hypothalamus and brainstem.

In contrast, the transcripts most highly enriched in TRAP-depleted (non-LepRb) mRNA relative to TRAP mRNA from both hypothalamus and brainstem included markers for glial cell types (Igf2, Fabp7, Cryab, Ermn), immune cells (Litaf) and neural

30 progenitor cells (Efhd1, Cdr1, Ppp1r14a). Thus, while some studies have suggested that a variety of non-neuronal cells express LepRb and respond directly to leptin [45-

46], TRAP-derived mRNAs from LepRbeGFP-L10a animals are enriched for transcripts from differentiated neurons (especially peptidergic and DA neurons) and vasculature- associated cells relative to these other cell types.

TRAP-Seq identifies novel subpopulations of LepRb neurons

The mRNA species that are both highly expressed and highly enriched in LepRb cells presumably represent the most functionally relevant genes within a specific class of proteins. Thus, to identify the neuropeptides most likely to be functionally relevant in

LepRb neurons, we examined expression level and fold enrichment for TRAP-enriched transcripts that encode secreted proteins as defined by gene-ontology (GO) analysis on the UniPROT platform [47] (Figure 2.3).

The secreted protein-encoding transcripts that were enriched in hypothalamic

LepRb neurons included peptides that define known subpopulations of hypothalamic

LepRb neurons (e.g., Pomc, Agrp, Prlh, Nts, Gal) (Figure 2.3 A). In addition to revealing the enrichment of Resp18 (a marker of peptide-secreting cells), this analysis also identified a number of highly-expressed and –enriched neuropeptide-encoding genes not previously examined as potential markers for subpopulations of LepRb neurons, including tachykinin-1 (Tac1), prodynorphin (Pdyn), corticotrophin releasing hormone (Crh), and growth hormone releasing hormone (Ghrh).

The genes that encode secreted proteins that were highly expressed and enriched in brainstem LepRb neurons included two neuropeptide-encoding transcripts

31 previously shown to be expressed in brainstem LepRb cells- Ucn and Cck [16-17, 48]

(Figure 2.3 B). Our analysis also revealed the expression of a number of additional neuropeptides (including Tac1 and vasoactive intestinal peptide (Vip)) that were highly expressed and -enriched in brainstem LepRb neurons.

Analysis of the transcripts encoding secreted proteins from TRAP-depleted (non-

LepRb) mRNA in the hypothalamus revealed genes that encode neuropeptides found exclusively in non-LepRb neurons (e.g., Pmch, Oxt, Avp, Hcrt, Ghrh1) and in pituitary gonadotrophs and lactotrophs (Lh, Cga, Prl), presumably derived from pituitary material that contaminated the hypothalamic tissue). In both hypothalamus and brainstem,

TRAP-depleted transcripts encoding secreted proteins were also enriched for markers for glial (Apoe, Apod, Apoc1, Ttr, Igf2, Ptgds, Scrg1, Metrn, Sparc) and immune (Il33,

Ly86) cells (Figures 2.3 C, D). These findings are consistent with the data above

(Figure 2.2 B), which suggest the failure to recover mRNA from many non-neuronal cell types by LepRb-specific TRAP.

To confirm the expression of Pdyn and Tac1 in LepRb neurons, we crossed

PdynIRES-Cre and Tac1IRES-Cre mice onto the cre-inducible Rosa26eYFP background, generating animals that express eYFP in Pdyn and Tac1 cells (PdyneYFP and Tac1eYFP mice, respectively), to examine their potential expression of LepRb. This analysis revealed the colocalization of leptin-stimulated pSTAT3-IR with Pdyn and Tac1 in largely distinct sets of hypothalamic neurons: LepRbPdyn cells lie primarily in the ARC,

VMH, and DMH; few LepRbPdyn cells were found in the LHA or ventral premammillary nucleus (PMv) (Figure 2.4 A-G). In the arcuate, approximately 40% of LepRbPdyn cells also contain POMC (data not shown). In contrast, hypothalamic LepRbTac1 cells were

32 detected primarily in the LHA and PMv; fewer LepRbTac1 cells were found in the DMH, and LepRbTac1 neurons were absent from the ARC and VMH (Figure 2.5). We also found that leptin-stimulated pSTAT3-IR and eYFP in Tac1eYFP mice colocalized in two brainstem areas: the NTS and the ventral lateral PAG.

Additionally, immunostaining for CRH or VIP peptide and eYFP in brain sections from colchicine-treated Leprcre;Rosa26eYFP (LepRbeYFP) reporter mice revealed that LHA

LepRb neurons contain CRH-IR (Figure 2.6) and that VIP-IR colocalized with LepRb in the brainstem DR and PAG nuclei of colchicine-treated LepRbeYFP mice (Figure 2.7).

Functional analysis of LepRbPdyn neurons

We hypothesized that TRAP-seq would identify physiologically relevant markers of LepRb subpopulations. To examine the role for LepRbPdyn neurons in leptin action, we generated Pdyncre/+;Leprflox/flox (LepRbPdynKO) animals (Figure 2.4 A), along with

Pdyncre;Lepr+/+ and Leprflox/flox (control) littermates. Leptin-stimulated pSTAT3-IR was largely ablated from Pdyn-expressing neurons in the VMH and DMH (along with the small populations of LepRbPdyn cells in the LHA and PMv) of LepRbPdynKO mice, and was reduced approximately 50% in ARC LepRbPdyn neurons (Figure 2.4 H). The reason underlying the incomplete penetrance of Pdyncre-mediated excision of Leprflox in ARC

LepRbPdyn cells is unclear, but similar idiosyncratic deletion patterns have been observed with other combinations of cre/flox alleles, including for Leprflox [48]. Thus,

LepRbPdynKO mice display LepRb ablation from most hypothalamic LepRbPdyn cells; roughly 7200 total neurons display LepRb disruption in these mice, ~50% of which lie in

33 the VMH; most of the remainder are distributed between the ARC and DMH (~20% of the total disrupted LepRb neurons each).

We detected no alterations in body weight or body composition for chow-fed

LepRbPdynKO male mice compared to controls (Figure 2.8 A, B). Weekly chow intake was also unchanged (data not shown). Body weight and chow intake were also unchanged in female LepRbPdymKO mice compared to controls (data not shown), however LepRbPdymKO females had increased adiposity on a chow diet (8.1% vs 6.7% body fat/weight, p=0.02). 24-hour fasting induced weight loss was not different between

LepRbPdynKO or controls of either sex. Consistent with an underlying defect in energy homeostasis in LepRbPdynKO mice, however, male LepRbPdynKO mice gained significantly more weight and adiposity than controls when challenged with 9 weeks of high fat diet (HFD) (Figure 2.8 A, B). As expected based upon their increased fat mass, circulating leptin concentrations were higher in LepRbPdynKO mice (Figure 2.8 C). While insulin concentrations trended up in ad libitum-fed LepRbPdynKO mice, this increase was not significant; fasted glucose levels were not different (Figure 2.8 D, E). While food intake assessed over three days in CLAMS was similar between high fat-fed

LepRbPdynKO mice and controls (Figure 2.8 F, 24hr data shown), calorimetry revealed

Pdyn decreased energy expenditure (VO2) in the HFD-fed LepRb KO mice compared to controls (Figure 2.8 G, H). Thus, leptin action via the Pdyncre-sensitive LepRbPdyn neurons promotes energy expenditure, rather than controlling food intake, to modulate overall energy balance. Furthermore, these data confirm the identification of a functionally relevant population of LepRb neurons by TRAP-Seq.

Discussion

We examined gene expression in brain LepRb neurons, revealing over 1100 mRNAs that are enriched in hypothalamic LepRb cells and approximately 900 genes whose expression is enriched in brainstem LepRb cells. These LepRb-enriched transcripts encode a variety of neuropeptides and other classes of proteins found primarily in peptidergic neurons (along with genes characteristic of DA neurons and an uncharacterized set of cells that express vascular markers). Most of these genes were not previously known to be expressed or enriched in LepRb neurons. Translational profiling of these cells thus revealed markers for previously unrecognized subpopulations of LepRb neurons, as well as genes of many classes that are likely to be important for the function of LepRb cells (and thus, for the control of body weight and metabolism). We have demonstrated that one of these populations (LepRbPdyn neurons, contained primarily in the VMH, DMH and ARC) play a crucial role in leptin-regulated energy balance through the control of metabolic rate/energy expenditure.

The TRAP enrichment of genes encoding known LepRb-expressed mRNAs

(e.g., Pomc, Agrp, Prlh, Nts, Gal, etc.) [25-26, 29-30, 34-38], together with our verification that many TRAP-enriched transcripts not previously known to be expressed in LepRb neurons colocalized with LepRb neurons, demonstrates the accuracy/specificity of the TRAP-seq method for cell type-specific transcriptome analysis. Although prodynorphin expression has previously been shown to be regulated by leptin (Janovic et al 2010), and prodynorphin-neurons have been shown to be activated following leptin treatment (Elias et al, 2000), this regulation has never been shown to be cell-autonomous. Furthermore, while some ARC LepRbPdyn neurons

35 contain POMC,LepRbTac1 and LepRbCRH neurons were absent from the ARC and thus distinct from both POMC and AgRP cells. Hence, TRAP-seq not only revealed previously unknown neuropeptide transmitters employed by discrete populations of

LepRb neurons, but also defined markers for previously unrecognized subsets of LepRb cells.

Markers for some previously-defined sets of hypothalamic LepRb neurons [18-

19], such as Nos1 (~1.5-fold enriched) and Slc32a1 (vGat; ~1.2-fold enriched) were poorly enriched. This presumably reflects the wide expression of Nos1 and Slc32a1 throughout the hypothalamus in both LepRb and non-LepRb neurons. Thus, TRAP-seq more robustly reveals the enrichment of genes with restricted expression (e.g., neuropeptides) than it does widely-expressed genes, since the fold enrichment of narrowly-expressed genes in TRAP mRNA is enhanced by comparison to the relative dilution of these genes within the tissue as a whole. Hence, the mRNAs that can be unambiguously assigned to the LepRb neuron transcriptome are biased toward genes more highly expressed in LepRb cells than other cells in the tissue, and this method is less sensitive for widely-expressed transcripts that are also found in some LepRb neurons. The genes that are highly enriched in TRAP mRNA, however, often represent the best markers for circumscribed, functionally-related, populations of cells that are tractable for circuit analysis.

The majority of LepRb-enriched mRNA species represent genes likely to be specific for neurons. While we did see enrichment of some vascular markers, including receptors Tie1, Tek and Eltd1, this is consistent with reports of LepRb expression in cells associated with the blood-brain barrier [49]). Conversely, genes enriched in non-

LepRb relative to LepRb mRNA not only contain markers for known non-LepRb neurons

(such as Pmch and Hcrt), but also for a variety of non-neuronal cell types, including glial and immune cells. This finding contrasts with reports of LepRb expression in subsets of these non-neuronal cell types [45-46]. It remains possible that some cell types

(including other neurons) might express low levels of LepRb, however, if cre expression from Leprcre and/or reporter expression from Rosa26eGFP-L10a is too low to detect in such cells. Thus our analysis is necessarily biased towards those cell populations which have active expression at both the Lepr and Rosa26 locus. In either case, our TRAP-Seq analysis appears to be specific, if not perfectly sensitive, for neurons (and some vasculature-associated cells) that contain LepRb.

Interestingly, while some genes were enriched in both hypothalamic and brainstem LepRb neurons, many highly-enriched neuropeptide-encoding mRNAs were found in LepRb neurons from only one region. Indeed, while a few neuropeptide- encoding genes enriched in brainstem LepRb neurons (including Tac1 and Cartpt) were also found in populations of hypothalamic LepRb cells, brainstem and hypothalamic

LepRb neurons contain relatively distinct sets of highly-expressed neuropeptide transmitters, as most neuropeptide-encoding genes that are enriched in hypothalamic

LepRb neurons are absent from brainstem LepRb cells. Similarly, Cck [16, 48] and Vip are enriched and expressed in brainstem, but not hypothalamic, LepRb neurons. These findings support the concept of discrete functions for individual groups of LepRb neurons and their uniquely expressed gene products [10].

In addition to providing markers for LepRb subpopulations, the products of transcripts enriched in LepRb neurons are likely to play important functional roles in

LepRb neurons. While the contribution of POMC and AgRP peptides to leptin action have been well described, the metabolic functions of the majority of the neuropeptides identified by TRAP-seq (including dynorphin) are as yet undetermined. Furthermore, not only do LepRb-expressed neuropeptides presumably contribute to the effects of leptin on neurotransmission, but the GPCRs, transcriptional regulators, signaling proteins, and other classes of proteins enriched in LepRb neurons also likely contribute to the function of these cells and thus to the control of energy balance and metabolism.

Some of these LepRb-enriched gene products may constitute potential targets for therapeutic intervention in obesity and diabetes.

Of the novel subsets of LepRb neurons that we identify here, we investigated the role for leptin action on LepRbPdyn neurons. LepRb ablation from LepRbPdyn neurons in

LepRbPdynKO mice decreased energy expenditure to increase adiposity during exposure to HFD. Thus, the Pdyncre-sensitive LepRb neurons promote energy expenditure in response to leptin. Approximately half of the LepRbPdyn cells from which LepRb was ablated in LepRbPdynKO mice lie in the VMH, and almost all VMH LepRb neurons contain Pdyn. It is technically challenging to assess the degree of colocalization between the LepRbPdyn and LepRbSF-1 populations; both appear to comprise ~80% of all

VMH LepRb neurons, suggesting a minimum of 60% of VMH LepRb neurons are affected in both models (25, 26). The consistent phenotype observed between these two models of VMH LepRb ablation however serves to highlight the known role for the

VMH in the control of energy expenditure, including in response to leptin [25-26]. Non-

VMH LepRbPdyn cells (primarily in the ARC and DMH) may also contribute to the

38 modulation of energy expenditure, but they do not appear to play major additional roles in the control of energy balance.

Overall, by elucidating the transcriptome of brainstem and hypothalamic LepRb neurons, our TRAP-seq analysis reveals markers for numerous subpopulations of

LepRb neurons, along with genes likely to contribute importantly to central leptin action.

In the future it will be important to define the roles in leptin action and metabolic control for new subpopulations of LepRb neurons and for a variety of gene products that are highly enriched in LepRb neurons. The LepRb-expressed genes defined by our TRAP- seq analysis thus provide an important resource for these and other future investigations.

Materials and Methods

Animals: Rosa26eGFP-L10a mice were generated as previously described [50]. The generation of Leprcre mice has also been previously described [51]. Leprcre mice were crossed to RosaeGFP-L10a mice to generate Leprcre/+;Rosa26eGFP-L10a/+ mice which were subsequently intercrossed to generate double homozygous Leprcre/cre;RosaeGFP-L10a/eGFP-

L10a (LepRbeGFP-L10a) study animals. Pdyncre mice [50] or Tac1cre mice (Tac1tm1.1(cre)Hze/J,

Jackson Laboratory Stock # 021877) were crossed to Rosa26eYFP mice

(Gt(ROSA)26Sortm1(EYFP)Cos/J, Jackson Laboratory, Stock # 006148) to generate

PdyneYFP or Tac1eYFP mice for study. Leprflox mice were as described previously [52].

Pdyncre/+ mice were bred to Leprflox/flox animals, producing Pdyncre/+;Leprflox/+ mice, which were bred to Leprflox/+ mice to generate Pdyncre/+;Leprflox/flox (LeprPdynKO) mice and littermate control (Pdyncre/+;Lepr+/+, Lepr+/+, and Leprflox/flox) mice for study. All procedures were approved by the University of Michigan University Committee on the

Use and Care of Animals in accordance with AAALAC and NIH guidelines. Animals were bred at the University of Michigan and maintained in a 12 hr light/12 hr dark cycle with ad libitum access to food and water.

Immunoprecipitation of ribosomes (TRAP): Adult homozygous LepRbeGFP-L10a mice were anesthetized and their brains removed to a mouse coronal brain matrix (1mm sections) to isolate the hypothalamus or brainstem; material from multiple animals was pooled to produce each sample. For hypothalamic dissections, a 3x3x3mm block was dissected from the ventral diencephalon immediately caudal to the optic chiasm. For brainstem dissections, serial 1mm sections were removed and individual LepRb-containing nuclei

(including the EW, DR, LR, PAG, PBN and NTS) were dissected by hand and pooled.

Messenger RNA was isolated from eGFP-tagged ribosomes, as well as from the eGFP- depleted fraction, as previously described [53-54]. RNA was assessed for quality using the TapeStation (Agilent, Santa Clara, CA). Samples with RINs (RNA Integrity

Numbers) of 8 or greater were prepped using the Illumina TruSeq mRNA Sample Prep v2 kit (Catalog #s RS-122-2001, RS-122-2002) (Illumina, San Diego, CA), where 0.1-

3ug of total RNA was converted to mRNA using a polyA purification. The mRNA was fragmented via chemical fragmentation and copied into first strand cDNA using reverse transcriptase and random primers. The 3’ ends of the cDNA were adenylated, and 6- nucleotide-barcoded adapters ligated. The products were purified and enriched by PCR to create the final cDNA library. Final libraries were checked for quality and quantity by

TapeStation (Agilent) and qPCR using Kapa’s library quantification kit for Illumina

Sequencing platforms (catalog # KK4835) (Kapa Biosystems,Wilmington MA). They

40 were clustered on the cBot (Illumina) and sequenced 4 samples per lane on a 50 cycle single end run on a HiSeq 2000 (Illumina) using version 2 reagents according to manufacturer’s protocols.

RNA-seq analysis: 50bp single-end reads underwent QC analysis prior to alignment to mouse genome build mm9 using TopHat and Bowtie alignment software [55].

Differential expression was determined using Cufflinks Cuffdiff analysis, with thresholds for differential expression set to fold change >1.5 or <0.66 and a false discovery rate of

Uniprot Database for gene ontology and protein class analysis [47].

Leptin treatment, colchicine treatment, and immunohistochemistry: LepRbeGFP-L10a mice had food removed at the onset of the light cycle. Animals were treated four hours later with metreleptin (5 mg/kg, i.p) (a generous gift from AstraZenica, Inc.) or vehicle and were subjected to perfusion 1.5 hours after leptin treatment. Treatment with ICV colchicine (10 µg) to concentrate neuropeptides in the soma for some experiments was for 2 days prior to perfusion.

For perfusion, mice were anesthetized with a lethal dose of intraperitoneal pentobarbital and transcardially perfused with phosphate buffered saline followed by

10% neutral buffered formalin. Brains were removed, post-fixed overnight, and dehydrated in 30% sucrose before coronal sectioning (30 µm) using a freezing microtome (Leica). Immunostaining was performed as previously described [33] using primary antibodies for pSTAT3 (Cell Signaling #9145, rabbit, 1:250), GFP (Aves Labs

#GFP1020, chicken, 1:1000), VIP (Phoenix #H06416, rabbit, 1:1000), CRF (Phoenix

#H01906, rabbit, 1:500). All antibodies were reacted with species-specific Alexa Fluor-

488 or -568 conjugated (Invitrogen, 1:200) secondary antibodies or processed via avidin-biotin/diaminobenzidine (DAB) method (ABC kit, Vector Labs; DAB reagents,

Sigma), and imaged as previously described [57]. DAB images were pseudocolored using Photoshop software.

Phenotyping of LepRbPdynKO and control mice: LepRbPdynKO and control littermates were weaned into individual housing at 21d and fed either chow (Purina Lab Diet 5001) or high fat diet (Research Diets D12492, 40% kcal from fat). Body weight was monitored weekly. A fasted (24hr) blood glucose sample was taken at 12-14 weeks of age. Analysis of body fat and lean mass was performed between 12–14 weeks of age using an NMR-based analyzer (Minispec LF90II, Bruker Optics). We also analyzed a subset of mice (13–16 weeks old) for oxygen consumption (VO2), food intake, and locomotor activity using the Comprehensive Laboratory Animal Monitoring System

(CLAMS, Columbus Instruments). Insulin was assessed using a double-antibody radioimmunoassay using an 125I-Human insulin tracer (EMD Millipore), a rat insulin standard (Novo), a guinea pig anti-rat insulin first antibody (EMD Millipore), and a sheep anti-guinea pig gamma globulin-PEG second antibody (MDRTC). Leptin was assayed by commercial ELISA (EMD Millipore). No significant differences were detected between the control (Pdyncre/+Lepr+/+, and Leprflox/flox) groups at the conclusion of the study and thus the data from these groups was combined for subsequent analysis.

Statistics: Physiological data are reported as mean +/- SEM. Statistical analysis of physiological data was performed using Prism (version 6.0) software. Unpaired t-tests were used to compare results between groups of two. Body weight gain between genotypes were analyzed by two-way ANOVA. p < 0.05 was considered statistically significant.

Figure 2.1: LepRbeGFP-L10a mice for TRAP-Seq of LepRb neurons. (A) Leprcre mediates the excision of the transcription blocking cassette from Rosa26eGFP-L10a in LepRb cells, promoting the expression of eGFP-L10a in these cells. (B,E)

Representative images of GFP-IR (green) in the hypothalamus (B) and midbrain (E) of LepRbeGFP-L10a mice. (C,D,F,G) Representative images of pSTAT3- (purple) and GFP- IR (green) in the indicated hypothalamic and brainstem nuclei of LepRbeGFP-L10a mice following leptin treatment (5mg/kg, i.p., 90 minutes). Shown are the ARC (C), DMH (D), DR (F), and PAG (G). Scale bar = 100 m. (H) Fold enrichment in TRAP relative to TRAP-depleted RNA subjected to RNA-Seq for transcripts known to be expressed in LepRb neurons (left of the dotted line) and to be excluded from LepRb neurons (right of the dotted line). Data represent 5 replicates, each from pooled hypothalami from 4-6 mice. Enrichment values for all genes shown were significant following correction for false discovery rate (p<0.05). IRES, internal ribosomal entry site; black arrow heads, LoxP sites; pA, polyadenylation signal; CAG, cytomegalovirus enhancer, chicken beta- actin promotor, rabbit beta-globin splice acceptor; NEO, neomycin selection cassette; DMH, dorsomedial hypothalamic nucleus; Arc, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; cDMH, compact zone of the DMH; vmDMH, ventromedial DMH; PAG, periaqueductal gray; DR, dorsal raphe nucleus.

Figure 2.2: A subset of proteins are enriched or de-enriched in both hypothalamic and brainstem LepRb neurons. (A) Transcripts enriched (expression in TRAP/Depleted >1.5) in both the hypothalamic and the brainstem TRAP-derived

46 fraction were plotted against each other to reveal the transcripts mostly highly enriched in both brain areas. (B) Transcripts de-enriched in the TRAP-derived fraction relative to background (TRAP/Depleted < 0.66) for both areas were also identified and plotted. Poorly-expressed transcripts (<1 fragments per kilobase of exon per millions reads mapped (FPKM)) in the TRAP-derived fraction or TRAP-depleted fraction of either brain area were excluded from the graph. All genes shown were differentially expressed between the TRAP and depleted fractions (p<0.05) as determined by CuffDiff analysis.

Figure 2.3: Secreted protein-encoding transcripts enriched or de-enriched in hypothalamic and brainstem LepRb neurons. Transcripts enriched >1.5-fold in the TRAP-derived fraction of the hypothalamus (A) or brainstem (B) were queried against the UniprotKB database to identify secreted proteins expressed in LepRb neurons of these areas. De-enriched (TRAP/depleted <0.66) transcripts from the hypothalamus (C) or brainstem (D) were similarly assayed. Following gene ontology analysis, genes were individually verified in the literature to confirm appropriate classification and sorted by expression level, as expressed in fragments per kilobase of exon per million reads mapped (FPKM), in the LepRb fraction. Transcripts expressed <1 FPKM in the LepRb fraction were excluded from the graph. All genes shown were differentially expressed between the TRAP and depleted fractions (p<0.05) as determined by CuffDiff analysis.

Figure 2.4: Pdyn expression defines a distinct subpopulation of LepRb neurons. (A) Pdyncre mediates the excision of the transcription blocking cassette from Rosa26eYFP in LepRb cells, promoting the expression of eYFP in these cells. Pdyncre can also mediate the excision of exon 17 from LepRflox mice resulting in the ablations of LepRb from Pdyn cells (LepRbPdynKO mice). (B-F) Representative images showing colocalization of pSTAT3- (purple) and GFP-IR (green; detects eYFP) in the ARC (B), VMH (C), DMH (D), LHA (E), and PMv (F), of PdyneYFP mice following leptin treatment (5mg/kg, i.p., 90 minutes). (G) Cells containing both Pdyn (eYFP-IR) and LepRb (pSTAT3-IR) were quantified in the hypothalamic regions shown (plotted as mean +/-

SEM). (H) Colocalization of pSTAT3 and Pdyn was reduced in LepRbPdynKO mice on the reporter background relative to control (C) PdyneYFP mice (mean +/-SEM is shown; *p<0.05 by t-test). Arrows indicate double labeled cells. Scale bar = 100 m. Cell counts were performed on serial sections (1:4) from n=3 control and n=2 LepRbPdynKO mice treated with leptin (5mg/kg, i.p., 90 minutes). For (G), cell counts were multiplied by 4 to approximate total hypothalamic cell numbers. NEO, neomycin selection cassette; black arrow heads, LoxP sites; pA, polyadenylation site.

Figure 2.5: LepRbTac1 neurons. Representative images showing colocalization of pSTAT3- (purple) and GFP-IR (green; detects eYFP) in the ARC, VMH, LHA, DMH, PMv, PAG, DR, and NTS of Tac1eYFP mice following leptin treatment (5mg/kg, i.p., 90 minutes). Arrows indicate double labeled cells. Scale bar = 100 m.

Figure 2.6: Colocalization of CRH and LepRb in the LHA. Representative images of GFP-IR (green; left; indicates eYFP/LepRb), CRH-IR (purple, middle) and merged channels (right) from the LHA of colchicine-treated LepRbeYFP mice. White arrowheads indicate colocalized neurons. Scale bar = 100 m.

Figure 2.7: Colocalization of VIP and LepRb in the brainstem. Representative merged images of GFP-IR (green; indicates eYFP/LepRb), VIP-IR (purple) from the DR and PAG of LepRbeYFP mice. White arrowheads indicate colocalized neurons. Scale bar = 100 m.

Figure 2.8: Leptin regulation of Pdyn neurons is required for normal energy homeostasis in DIO mice. Male LepRbPdynKO (KO) and littermate controls (C) were placed on chow or high fat diets, and body weight measured weekly (A) (mean +/- SEM is shown; *p<0.05 by ANOVA). At 12-14 weeks of age, animals underwent body composition analysis by NMR spectroscopy (B). Serum from HFD-fed control and LepRbPdynKO mice was assayed for leptin (C) or insulin (D). Blood glucose was measured following a 24 hour fast (E) in the HFD cohort. (B-E) mean +/-SEM is shown; *p<0.05 by t-test. (F-H) A subset of HFD-fed control and LepRbPdynKO mice were assessed in CLAMS metabolic cages for 3 days following body composition analysis.

Food intake (F) was measured on the final day. VO2 was also measured and is presented normalized to total body mass (G) and lean body mass (H) (Mean +/- SEM is shown; ***p<.0001 by ANOVA). HFD, high fat diet (Research diets, 60% kcal from fat). Black triangles = HFD fed LepRbPdynKO; white triangles = HFD fed controls; black circles = chow fed LepRbPdynKO; white circles = chow fed controls. Black bars = LepRbPdynKO; white bars = controls. N=8-14 for chow and HFD cohorts. N=6-7 animals per genotype for VO2 measurements.

References:

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CHAPTER 3

TRANSCRIPTIONAL AND TRANSLATIONAL PROGRAMS INDUCED BY LEPTIN IN LEPRB NEURONS

Chapter Summary

Leptin action in the hypothalamus is critical for the appropriate control of energy balance. Significant effort has led to the identification of the intracellular signaling pathways engaged by the activation of the leptin receptor (LepRb), and has determined that LepRb-STAT3 signaling is essential for the regulation of energy homeostasis by leptin. Despite the importance of LepRb neurons and their regulation, their transcriptome and its control have remained undefined because these cells only contribute a fraction to the aggregate transcriptome of the brain regions in which they reside. We thus treated mice with an acute bolus of leptin and isolated mRNA specifically from hypothalamic LepRb neurons by Translating Ribosome Affinity

Purification and subjected it to RNA sequencing (TRAP-Seq). This analysis revealed the transcriptome of LepRb neurons and its regulation by leptin. Acute (3-hour) leptin treatment promoted the expression of a limited set of genes in hypothalamic LepRb neurons, most of which represent known STAT3 targets. The preponderance of these genes encode transcription factors, including the most highly leptin-regulated gene,

Atf3- a previously unknown leptin target gene restricted to LepRb neurons in the

59 hypothalamus. Thus TRAP-seq analysis identifies specific targets of the STAT3- dominated transcriptional response to leptin.

Introduction

The hormone leptin, which is produced by adipocytes to signal the sufficiency of energy reserves, acts via the leptin receptor (LepRb) to modulate food intake and energy expenditure[1-2]. Leptin controls energy balance and metabolism by acting on

LepRb-expressing neurons in the hypothalamus and brainstem.[3-5]. Within the hypothalamus, direct leptin action on each of several circumscribed sets of LepRb neurons that reside in the arcuate nucleus (ARC), ventromedial hypothalamic nucleus

(VMH) and lateral hypothalamic area (LHA) (which express Pomc, Agrp, Nr5a1 (a.k.a. steroidogenic factor-1; SF-1), and Nts, respectively), contributes modestly to the control of body weight [6-8]. Other hypothalamic LepRb neurons that contain Nos1 or Vgat play substantial roles in the control of energy balance and metabolism [9-10].

Considerable effort has focused on understanding the individual roles of these LepRb populations in the control of energy homeostasis. In addition to understanding specific neuronal populations that mediate leptin action, however, it is essential to understand the cellular and molecular mechanisms by which leptin regulates these neurons.

The most critical LepRb signaling pathway for the control of energy balance is engaged when leptin-activated LepRb stimulates the tyrosine phosphorylation of

STAT3, promoting its nuclear translocation and subsequent modulation of gene transcription[11-12]. While a variety of animal models of perturbed LepRb signaling have revealed the indispensable role of the LepRbSTAT3 pathway for the control of energy balance, glucose homeostasis, and endocrine function by leptin,[2, 13-17] few direct transcriptional targets of leptin have been identified[18-21]. Furthermore, with the exception of Socs3 which appears to be widely regulated in LepRb neurons, the

61 majority of known leptin regulated transcripts (including Pomc, Agrp,and Npy) are not expressed outside of the arcuate nucleus, despite the arcuate LepRb population representing less than 25% of all hypothalamic LepRb neurons[22]. Since LepRb- expressing neurons comprise only a fraction of the cells within the complex nuclei in which they reside, however, it has not been possible to isolate the transcriptional effects of leptin on LepRb neurons from other cells within the hypothalamus.[23-24] Therefore, the regulation of the LepRb transcriptome has not been fully defined. We thus set out to examine the acute regulation of the LepRb neuron transcriptome by expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit selectively in LepRb neurons to enable anti-eGFP immunopurification of ribosomes and their associated mRNA for analysis.

Results

Hypothalamic transcriptional programs induced by acute leptin treatment

The difficulty of distinguishing the LepRb neuron-specific transcriptome from that of the surrounding cellular milieu has prevented the analysis of the cell-specific transcriptional programs activated by leptin. To define the cell-autonomous program of acute leptin-stimulated gene expression, we treated LepRbeGFP-L10a mice with pharmacological doses of leptin (5 mg/kg, i.p.) or vehicle for 3 hours followed by TRAP isolation. The resultant mRNA (as well as TRAP-depleted supernatant mRNA) was used to generate multiplexed libraries for sequencing on the Illumina HiSeq 2000 platform. We sequenced four independent vehicle samples and three independent leptin-treated samples, each comprised of TRAP extracts from pooled groups of 4-6

LepRbeGFP-L10a mice. We chose this acute treatment time to permit the full development

62 of the initial transcriptional response to leptin, while minimizing second order transcriptional changes and/or changes due to any alterations in physiology following leptin treatment. Approximately 1100 genes were enriched in the TRAP-derived versus

TRAP-depleted vehicle samples, and 98 genes were differentially expressed between the leptin and vehicle treated TRAP-derived samples.

Since it is possible for leptin to stimulate ubiquitously-expressed genes (which may not be highly enriched in LepRb neurons at baseline) in LepRb neurons, we specifically examined the regulation of genes that became enriched in LepRb neurons following leptin treatment, as well as the >1100 genes enriched at baseline, in TRAP mRNA. We also chose to focus our analysis on those genes that were upregulated by leptin treatment, as the short time point chosen seemed unlikely to accurately capture decreases in gene transcription. Overall, leptin increased the recovery of 25 genes that were enriched in hypothalamic LepRb neurons at baseline and/or following leptin treatment (Table 3.1). Although certain arcuate neuropeptide-encoding transcripts including Pomc, Agrp, and Npy are among the best studied leptin-response genes, we did not observe any changes in these transcript levels following acute leptin treatment.

Validation of RNA-Seq

In order to validate our RNA-seq analysis, TRAP-derived mRNA from vehicle and leptin treated samples was separately converted to cDNA by a reverse transcriptase reaction. cDNA samples were then subjected to RT-qPCR to examine the expression of genes shown to be differentially expressed by TRAP-seq. Unsurprisingly, those genes most highly regulated in the RNA-seq analysis (including Socs3, Atf3, Arid5a, Etv6, and

JunB) were also significantly different when analyzed by RT-qPCR (Table 3.2).

Although the magnitude of the detected fold change value varied between the two analyses, this likely reflects the low sample number (n=4 for vehicle, n=3 for leptin treatment) for use with qPCR, and the limitation of normalizing to a single housekeeping gene in qPCR, vs. the more robust methods available to RNA-seq analysis.

Transcriptional programs induced by leptin

The vast majority (17/25; 68%) of genes directly stimulated by leptin encoded two classes of proteins: cellular signaling proteins (8/25; 32%) and transcriptional regulators

(9/25; 36%) (Table 3.1). Interestingly, most of the genes that were stimulated by leptin were not enriched in LepRb neurons in untreated animals, but only became enriched following leptin treatment; neither did these transcripts represent the most highly- enriched mRNA species of their classes (data not shown), even following leptin treatment. This finding is consistent with the notion that these genes represent broadly- expressed transcripts that are induced specifically in LepRb neurons by acute leptin treatment.

Among the leptin-stimulated signaling proteins, most represent inhibitors of cytokine signaling that mediate feedback inhibition. Unlike the other members of this class of gene products, Socs3 was enriched in LepRb neurons at baseline and became the most highly enriched of the transcripts that encode signaling proteins following leptin treatment, consistent with the LepRb-specificity of SOCS3 function in the hypothalamus.

As for the genes encoding signaling proteins, most of the leptin-regulated transcription factors were not enriched at baseline but became enriched following leptin treatment, suggesting that they represent broadly-expressed genes that are induced by

64 acute leptin treatment in LepRb neurons. Junb, En1, and Atf3 were enriched in LepRb neurons at baseline, as well as being induced by leptin, however, suggesting some selectivity of these transcripts for expression in LepRb neurons in the hypothalamus. Of these, Atf3 (Activating Transcription Factor 3) was the most highly enriched transcription factor-encoding gene in LepRb neurons following leptin treatment, suggesting a possible leptin-specific role for ATF3 in the hypothalamus.

To assess our ability to detect changes in these broadly-expressed transcripts via non-TRAP-seq methods, we also replicated our TRAP-seq conditions in a cohort of

Leprcre mice. Mice were treated for 3 hours with leptin (5mg/kg i.p) or vehicle (n=10 per treatment), and RNA was isolated from the whole hypothalamus by standard methods.

Hypothalamic RNA was converted to cDNA by reverse transcriptase reaction and analyzed by RT-qPCR. Many of the changes in gene expression observed by TRAP-

Seq or TRAP-qPCR were not detectable in whole hypothalamic mRNA extracts (Table

3.2), highlighting the strength of cell-specific TRAP-seq for detecting changes in broadly expressed transcripts which would otherwise be lost by the ‘diluting’ effect of ubiquitous expression. Indeed, of the genes analyzed, only the highly enriched Socs3 and Atf3 were detectably different, further supporting the interpretation that their expression is restricted to LepRb neurons.

Leptin induces the expression of ATF3 specifically in LepR neurons

To examine the potential specificity of hypothalamic ATF3 for leptin signaling, we employed IHC to examine its induction by leptin and potential expression in LepRb neurons. We treated LepRbeGFP-L10a mice with leptin (5mg/kg, i.p.) or vehicle for 6 hours

(to permit sufficient time for the protein to be synthesized) and performed IHC for ATF3

(Figure 3.1 A, B). Vehicle-treated animals displayed only relatively light ATF3-IR in the

ARC and DMH (which is reminiscent of pSTAT3-IR in similarly-treated animals). In contrast, leptin treatment induced robust ATF3-IR in the ARC, DMH, and LHA in a pattern reminiscent of LepRb expression. Indeed, dual IHC for ATF3 and eGFP confirmed that most ATF3-IR colocalized with LepRb neurons in control and leptin- treated animals (Figure 3.1 C-F), demonstrating the specificity of ATF3 for LepRb neurons in the hypothalamus.

Multiple subpopulations of LepRb neurons play significant roles in the control of energy balance. The neurons of the melanocortin system, including those that express

Pomc or Agrp, are the most well studied; leptin activates anorexigenic POMC neurons and inactivates orexigenic AgRP neurons, while fasting activates AgRP neurons alone[25-28]. The role of ATF3 in these neurons, however, has not been assessed. We therefore determined the effect of leptin treatment on ATF3 expression in POMC and

AgRP neurons. POMC-dsRed transgenic mice, which express the dsRed fluorescent protein under the control of the Pomc promoter, or AgRPeGFP-L10a mice were treated for six hours with leptin (5mg/kg i.p) or vehicle, and ATF3 expression in fluorescently labeled POMC or AgRP neurons was examined by IHC (Figure 3.2 A,B). Leptin robustly induced ATF3-IR in a significant subset of POMC neurons as well as in a significant minority of fluorescently labeled AgRP neurons (Figure 3.2 C,D). This suggests that leptin may regulate ATF3 in multiple metabolically relevant neuronal populations.

ATF3 is a member of the AP1 transcription factor complex, which has been shown to be activated by multiple pathways, including ERK signaling and neuronal activation [29-31]. Therefore, it is possible that ATF3 serves as a specific marker of

66 activation of LepRb populations in addition to as a marker of leptin-LepRb signaling. To test this hypothesis, we also determined the effect of fasting on ATF3 expression in

AgRP neurons. AgRPeGFP-L10a mice were fasted for 24 hours or given ad libitum access to food prior to perfusion. ATF3 induction in eGFP-labeled AgRP neurons was assessed by IHC. Fasting robustly induced ATF3-IR in a subset of AgRP neurons

(Figure 3.3), suggesting that ATF3 is induced by neuronal activation in these neurons.

Discussion

We examined the acute regulation of gene expression by leptin in LepRb neurons, revealing over 25 transcripts enriched in LepRb neurons which were acutely induced by leptin. These leptin-induced transcripts encode primarily transcriptional regulators and intracellular signaling proteins, most of which were not previously known to be regulated by leptin. Translational profiling of LepRb neurons thus revealed the acute transcriptional program induced by leptin, and highlights the complex nature of leptin’s regulation of gene transcription. While the majority of these leptin responsive genes are widely expressed in the hypothalamus, several (including Socs3 and Atf3) are largely restricted to LepRb neurons.

When analyzing mRNA expression from a subpopulation of cells, only changes in transcripts known to be expressed in the cells of interest can be definitively assigned as cell-autonomous. Although TRAP isolation is in many ways superior to other methods of mRNA isolation from complex tissue, particularly in adult neuronal cells which do not tolerate cellular dispersion or FACS sorting techniques well, it still suffers from issues of contamination from the surrounding tissue. Thus, when examining the leptin-stimulated

67 induction of gene expression in LepRb neurons, we focused our analysis on transcripts that were enriched in LepRb neurons at baseline, or became enriched following leptin treatment.

We also choose to focus our analysis on those genes upregulated in response to leptin, since the short time course was unlikely to capture decreases in gene transcription, although decreases in translation might be detectable. In a similar vein, since TRAP is specific for mRNA actively bound to ribosomes, it is possible that increased translation of specific mRNAs, rather than increased transcription, contributes to the increased recovery of some transcripts following leptin treatment. This preferential isolation of ribosome bound mRNA species also means that our transcript pool is biased towards those populations that actively increase translation in response to leptin treatment, and biased against those populations (such as AgRP neurons) which may inhibit translation in response to leptin[32]. In the future, performing TRAP-

Seq on more circumscribed subsets of LepRb neurons may be necessary in order to evaluate this confounding effect.

Another limitation of LepRb specific TRAP-seq is that it may fail to identify certain transcripts which are present in LepRb neurons, but which are not enriched due to their broad expression. By examining the enrichment of leptin regulated genes both before and after treatment, however, we were able to identify 17 transcripts (including Arid5a and Etv6) which are expressed in LepRb neurons at baseline, but which only became enriched following leptin treatment. These genes, like Fos, presumably represent ubiquitously expressed transcripts that are nonetheless specifically induced by leptin action on LepRb neurons. This interpretation is supported by our inability to detect

68 changes in these transcripts when we examined their expression in RNA isolated from whole hypothalamus; any LepRb specific changes in expression are buffered by unchanging expression in neighboring cells. This result also highlights the value of cell- specific TRAP-seq in discovering cell-autonomous changes in gene transcription that would not be detectable by standard methods.

The vast majority (17/25; 68%) of genes directly stimulated by leptin encoded two classes of proteins: cellular signaling proteins (8/25; 32%) and transcriptional regulators

(9/25; 36%). Of the cellular signaling proteins, six are proteins that have an established role in the inhibition of cytokine receptor signaling- including four SOCS proteins. Thus, most members of this class presumably mediate feedback inhibition of LepRb to limit the duration and amplitude of the leptin signal. Indeed, Socs3, whose expression pattern and induction is consistent with a LepRb neuron-specific transcript, mediates feedback inhibition on LepRb and thus modulates leptin action and adiposity in mammals[12, 33-37]. The other leptin-stimulated Socs genes (Socs1, Socs2, Cish) are presumably expressed more widely, and may mediate feedback on other cytokine receptors or other tyrosine kinase systems in LepRb neurons. The mechanisms by which the products of Gadd45a and Gadd45g limit STAT3 signaling and mediate other cellular functions remain unclear, as are their roles in LepRb signaling and leptin action.

The bias toward transcriptional regulators among genes induced during the early response to leptin suggests that the transcriptional regulators (Atf3, JunB, etc.) induced during this initial wave of leptin-stimulated transcription might produce a distinct second wave of gene transcription events, including the other known leptin-regulated transcripts. Indeed, the expression of neuropeptides known to be regulated by leptin

(including Pomc, Cartpt, Agrp, and Npy) was not significantly changed by 3-hour leptin treatment[19, 38-39]. Although the failure to detect changes in leptin-repressed genes

(such as Agrp and Npy) could be due to the longer time generally required to detect decreases (relative to increases) in gene transcription, the failure to change Pomc and

Cartpt suggests that at least some leptin-dependent changes in neuropeptide gene expression may be mediated by processes downstream of the initial transcriptional response to leptin, rather than directly by STAT3.

The most highly regulated transcripts in our dataset, Atf3, was previously unknown as a target of leptin action. Deletion of Atf3 from an ill-defined population of

Pdxcre-expressing hypothalamic cells however, has been shown to alter energy balance and metabolism[40]. Given the expression of Pdxcre in a subpopulation of hypothalamic

LepRb neurons, the enrichment of Atf3 in LepRb neurons, and the dramatic induction of

ATF3 in LepRb neurons in response to leptin, we hypothesized that ATF3 plays an important role in the control of metabolism by Atf3-expressing hypothalamic LepRb neurons.

While its high degree of enrichment suggested a level of specificity for LepRb neurons, it was nonetheless possible that ATF3 was also expressed in non-LepRb hypothalamic neuronal populations. In order to examine the anatomical distribution of

ATF3 expression, we treated LepRbeGFP-L10a reporter mice with vehicle or leptin for 6 hours in order to allow for completion of translation, and examined ATF3 expression by immunohistochemistry. Our findings confirmed that ATF3 is largely absent from the hypothalamus in vehicle treated animals but is strongly induced by leptin treatment specifically in LepRb neurons. While ATF3 expression was specific for LepRb neurons

70 both before and after leptin treatment, not all hypothalamic LepRb neurons express

ATF3. Indeed, LepRb/ATF3 co-expression seems limited to the Arc, DMH, and LHA; whether the LepRb populations which express ATF3 have other commonalities (such as specific neurotransmitter or neuropeptide signatures) is a point for further study.

Due to its significant impacts on both food intake and energy expenditure, the melanocortin system represents a point of significant interest for the development of anti-obesity therapeutics. Thus we sought to determine the potential role for ATF3 in the control of the melanocortin system by treating POMC or AgRP reporter mice with leptin and examining ATF3 expression. Leptin treatment was found to markedly increase

ATF3 expression in a distinct subset of POMC neurons; a finding consistent with data that suggests that a significant proportion of POMC neurons do not express the leptin receptor in adulthood [41-42]. Indeed this more limited response in POMC neurons further supports the cell-autonomous nature of the leptin-LepRb-ATF3 response. Leptin treatment in AgRP reporter mice was also found to increase ATF3 expression in AgRP neurons, suggesting that LepRb-ATF3 signaling is important for multiple metabolicalkyl relevant cell types.

The finding that leptin induces ATF3 expression in antagonistic cell types is not straightforward to interpret. It may be best explained by the fact that ATF3 acts as part of the AP-1 transcription factor complex and that the individual transcription factor components of the AP-1 complex vary widely among cell types [43-46]. Indeed, we showed that ATF3 is merely one of a number of transcriptional regulators induced by leptin, many of which (such as Jun, JunB, Fosl2 and Fos) are AP-1 complex members; these regulators may be distributed or segregated across multiple LepRb

71 subpopulations which may or may not express ATF3. Thus it is possible that while

LepRb-induced ATF3 expression is ubiquitous across heterogenous (even antagonist)

LepRb populations, the transcriptional targets of ATF3 in these cell types are directed by the availability of different AP1 complex partners. This interpretation is supported by the known ability of ATF3 to activate or repress transcription in different cell types[47]; leptin treatment represses Agrp transcription, but stimulates Pomc transcription. Thus it is highly likely that certain transcriptional responses to leptin diverge at a point distal to the leptin-LepRb-ATF3 signaling pathway in these cells. Further work will be necessary to determine the potential binding partners for ATF3 in different LepRb populations, as well as the regulation of these factors by leptin.

The mechanism by which Atf3 transcription is activated by leptin is not known.

Although ATF3 and STAT3 are co-regulatory, the AP1 family of transcription factors has also been shown to respond to ERK signaling and neuronal firing [29-31, 48-50]. In order to test whether ATF3 can also be induced by neuronal activation, we fasted

AgRPeGFP-L10a mice for 24 hours, and examined ATF3 expression by IHC. Fasting is known to activate AgRP neurons, while leptin treatment is known to inhibit these cells[28]. Thus, the finding that ATF3 was induced both by fasting in AgRP neurons, and by leptin treatment, suggests that multiple mechanisms may engage ATF3 signaling in

LepRb neurons. Again it is likely that both fasting and leptin treatment induce different cohorts of transcriptional regulators in these neurons, and that as part of a generalized transcription factor complex, ATF3 represents a point of commonality between these groups. This result however, further highlights the necessity of future work to identify the

72 transcriptional targets of ATF3, and how they differ in response to metabolic perturbation.

Overall, by elucidating the acute regulation of gene expression in hypothalamic

LepRb neurons, our TRAP-seq analysis defined genes likely to contribute importantly to central leptin action, and revealed the initial steps of leptin action in vivo. To be sure, these findings raise as many new questions as they answer. In the future, it will be crucial to understand the roles for leptin-stimulated transcription factors in leptin action, especially as they relate to ATF3 and its associated transcripts. Furthermore, while we used an acute leptin treatment in order to gain insight into the ‘first order’ transcriptional responses to leptin, physiologically leptin serves as a chronic, rather than acute, signal of energy repletion. Thus, it will be necessary to elucidate the mechanisms by which leptin controls gene expression over the long term, commensurate with its physiological role in the control of energy balance.

Materials and Methods

Animals: Rosa26eGFP-L10a mice were generated as previously described [51]. The generation of Leprcre mice has also been previously described [52]. Leprcre mice were crossed to RosaeGFP-L10a mice to generate Leprcre/+;Rosa26eGFP-L10a/+ mice which were subsequently intercrossed to generate double homozygous Leprcre/cre;RosaeGFP-L10a/eGFP-

L10a (LepRbeGFP-L10a) study animals. Agrpcre mice (Jackson Laboratories stock #012899) and were crossed with Rosa26eGFP-L10a mice to generate Agrpcre/+;Rosa26eGFP-L10a/+

(AgRPeGFP-L10a) for use in fasting and leptin experiments. Pomc-dsRed transgenic mice were a gift from Dr. Malcolm J Low, and were generated as described [53]. All

73 experiments used mixed groups of male and female mice except where explicitly noted.

All procedures were approved by the University of Michigan University Committee on the Use and Care of Animals in accordance with AAALAC and NIH guidelines. Animals were bred at the University of Michigan and maintained in a 12 hr light/12 hr dark cycle with ad libitum access to food and water except as noted in experimental protocols.

Immunoprecipitation of ribosomes (TRAP): Messenger RNA was isolated from eGFP- tagged ribosomes, as well as from the eGFP-depleted fraction, as previously described

[54]. One tenth of each mRNA sample was set aside for conversion to cDNA and subsequent RT-PCR validation of RNA-seq results. The remainder of the RNA was assessed for quality using the TapeStation (Agilent, Santa Clara, CA). Samples with

RINs (RNA Integrity Numbers) of 8 or greater were prepped using the Illumina TruSeq mRNA Sample Prep v2 kit (Catalog #s RS-122-2001, RS-122-2002) (Illumina, San

Diego, CA), where 0.1-3ug of total RNA was converted to mRNA using a polyA purification. The mRNA was fragmented via chemical fragmentation and copied into first strand cDNA using reverse transcriptase and random primers. The 3’ ends of the cDNA were adenylated, and 6-nucleotide-barcoded adapters ligated. The products were purified and enriched by PCR to create the final cDNA library. Final libraries were checked for quality and quantity by TapeStation (Agilent) and qPCR using Kapa’s library quantification kit for Illumina Sequencing platforms (catalog # KK4835) (Kapa

Biosystems,Wilmington MA). They were clustered on the cBot (Illumina) and sequenced 4 samples per lane on a 50 cycle single end run on a HiSeq 2000 (Illumina) using version 2 reagents according to manufacturer’s protocols.

RNA-seq analysis: 50bp single-end reads underwent QC analysis prior to alignment to mouse genome build mm9 using TopHat and Bowtie alignment software [55].

Differential expression was determined using Cufflinks Cuffdiff analysis, with thresholds for differential expression set to fold change >1.5 or <0.66 and a false discovery rate of

Uniprot Database for gene ontology and protein class analysis [57].

RNA extraction of whole hypothalami and analysis by RT-qPCR: RNA was extracted from microdissected hypothalami using Trizol (Invitrogen) according to manufacturer’s protocol, or obtained from TRAP isolation, and subsequently converted to cDNA using iScript cDNA synthesis kit (Biorad # 170-8891) for use in reverse transcriptase PCR. cDNA was analyzed in duplicate by quantitative real time-PCR on an Applied

Biosystems StepOnePlus Real-Time PCR System for TBP (endogenous control) and each of the following: Socs3, Atf3, JunB, Arid5a, Etv6, Fos, Slco1a4, Ghrh, Agrp, Pomc,

Serina3h, Tbx19, Pltp, and Gadd45g. All Taqman assays were acquired from Applied

Biosystems (Foster City, CA).

Leptin treatment and fasting: For TRAP-seq and qPCR experiments, LepRbeGFP-L10a mice had food removed at the onset of the light cycle. Animals were treated four hours later with metreleptin (5 mg/kg, i.p) (a generous gift from AstraZenica, Inc.) or vehicle

(0.9% saline) and hypothalami were dissected 3 hours after treatment. For immunohistochemistry, LepRbeGFP-L10a-, Pomc-dsRed, or AgRPeGFP=L10a mice had food

75 removed at the onset of the light cycle. Animals were treated four hours later with metreleptin (5mg/kg i.p.) and subjected to perfusion 6 hours after leptin treatment. A separate cohort of AgRPeGFP-L10a mice was fasted for 24 hours prior to perfusion at the midpoint of the light cycle.

Perfusion and immunohistochemistry: For perfusion, mice were anesthetized with a lethal dose of intraperitoneal pentobarbital and transcardially perfused with phosphate buffered saline followed by 10% neutral buffered formalin. Brains were removed, post- fixed overnight, and dehydrated in 30% sucrose before coronal sectioning (30 µm) using a freezing microtome (Leica). Immunostaining was performed as previously described

[58] using primary antibodies for GFP (Aves Labs #GFP1020, chicken, 1:1000), ATF3

(Sigma #HPA001562, 1:1000), and DsRed (Clontech, # 632496 1:1000). All antibodies were reacted with species-specific Alexa Fluor-488 or -568 conjugated (Invitrogen,

1:200) secondary antibodies or processed via avidin-biotin/diaminobenzidine (DAB) method (ABC kit, Vector Labs; DAB reagents, Sigma), and imaged as previously described [59]. DAB images were pseudocolored using Photoshop software.

Statistics: RT-qPCR data are reported as mean fold change vs normalized vehicle.

Statistical analysis of RT-qPCR data was performed using Prism (version 6.0) software.

Unpaired t-tests were used to compare results between groups of two. p < 0.05 was considered statistically significant.

Enrichment Expression Enrichment Expression Fold Gene (Vehicle) (Vehicle) (Leptin) (Leptin) Change Protein Function Atf3 4.45 3.34 13.92 16.01 4.97 TR; AP1 Socs3 3.14 3.74 8.06 17.25 4.78 CS; CSI Rpl3 1.75 7.50 1.72 20.52 2.74 Ribosomal protein Arid5a 0.54 1.52 2.19 6.64 4.52 TR Etv6 0.73 2.61 1.59 6.67 2.65 TR Gadd45g 1.14 41.71 2.77 93.75 2.33 CS; CSI Serpina3h 6.07 4.88 13.62 10.31 2.19 Serine peptidase inhibitor Junb 2.04 28.05 3.78 54.35 2.01 TR; AP1 Jun 1.37 28.94 2.26 54.09 1.94 TR; AP1 Gadd45a 1.41 19.37 2.44 35.93 1.92 CS; CSI Socs2 0.99 3.56 1.56 6.58 1.92 CS; CSI Socs1 1.43 3.97 2.36 7.29 1.90 CS; CSI En1 4.99 9.18 5.98 16.77 1.89 TR Drd1a 1.01 2.11 1.94 3.67 1.80 GPCR Cish 1.14 5.41 1.87 9.36 1.79 CS; CSI Prokr2 1.42 1.23 2.41 2.13 1.79 GPCR Fosl2 1.22 3.30 1.98 5.60 1.76 TR; AP1 Sbno2 1.07 4.08 1.73 6.68 1.70 TR Fos 1.24 12.29 2.06 20.10 1.69 TR; AP1 Trim62 0.96 11.86 1.58 19.24 1.68 TR; AP1 regulator Rasl11a 0.97 13.02 1.56 21.11 1.68 Small GTPase Ier3 1.06 15.38 1.84 24.17 1.63 CS Aldh1a1 1.80 79.49 2.13 117.05 1.53 Retinal metabolism Ndufs5 1.45 105.76 1.71 155.56 1.52 NADH oxidoreductase Ddc 3.87 134.95 4.65 196.67 1.51 Dopamine synthesis Pomc 21.07 975.66 5.58 932.42 0.96 Neuropeptide Agrp 41.89 260.93 40.52 181.28 0.69 Neuropeptide Npy 21.55 1326.68 21.13 1056.25 0.80 Neuropeptide Cartpt 9.38 976.02 10.29 910.62 0.93 Neuropeptide Gal 3.46 159.47 3.79 130.97 0.82 Neuropeptide Nts 6.30 124.73 7.23 109.32 0.88 Neuropeptide

Table 3.1: Acute leptin treatment activates transcriptional programs in LepRb neurons. LepReGFP-L10 mice were treated for 3 hours with leptin (5mg/kg i.p.) or vehicle prior to hypothalamic TRAP-seq isolation. Transcripts levels were compared between TRAP-enriched and depleted fractions, as well as between treatments, to determine changes induced by leptin, and transcript enrichment in LepRb cells in both treatment groups. n=3-4 cohorts of pooled hypothalami from 4-6 mice per treatment. p<0.05 for all data shown except for fold change in neuropeptides. TR, transcriptional regulator; AP1,

77 activator protein-1 component; CS, cell signaling protein; CSI, cytokine signaling inhibitor.

Fold Change TRAP Fold Change Whole Hypo Gene Vehicle Leptin Vehicle Leptin Socs3 1.01 12.51 1.20 6.02 Atf3 1.14 18.90 1.10 2.40 JunB 1.02 4.49 1.08 1.29 Arid5a 1.13 10.67 1.03 1.10 Etv6 1.02 3.48 1.02 1.18 GHRH 1.09 0.72 1.02 0.91 Slco1a4 1.33 0.64 1.01 1.03 AgRP 1.06 0.74 1.06 1.17 POMC 1.00 0.97 NPY 1.01 0.92 Fos 1.18 2.45

Table 3.2: Leptin-induced genes are preferentially identified by TRAP isolation. LepReGFP-L10 or LeprCre mice were treated for 3 hours with leptin (5mg/kg i.p.) or vehicle prior to hypothalamic TRAP-seq isolation or RNA isolation by standard methods. RNA was converted to cDNA by reverse transcriptase reaction, and expression of target genes was measured by Taqman assay. Samples were normalized to Tbp (endogenous control), and vehicle was normalized to 1 for fold change analysis. n=4 per treatment for TRAP isolations, n=10 per treatment for whole RNA isolation. P<0.05 vs. vehicle for values in bold & italics.

Figure 3.1: ATF3 is induced specifically in LepRb neurons following acute leptin stimulation. LepRcreRos26eGFP-L10a mice were treated i.p with vehicle or leptin (5mg/kg i.p.) for 6 hours. Brains were harvested, sectioned, and stained for ATF3 and/or GFP. Images are representative of 3-4 mice per treatment group. (Upper panel) Immunohistochemical detection of ATF3-IR in the hypothalamus of control (A) and leptin treated (B) LepRcreRos26eGFP-L10a mice. (Lower panels) Dual IHC/IF for ATF3 and

80 eGFP in the Arc and DMH of control (C,E) and leptin treated (D,F) LepRcreRos26eGFP- L10a mice. White arrowheads indicate colocalized neurons. Scale bar = 100M.

Figure 3.2: ATF3 is induced by leptin treatment in both POMC and AgRP neurons. Representative images from POMC-dsRed (A-F) and AgRPeGFP-L10a (G-L) mice wreated with leptin (5mg/kg i.p.) or vehicle 60 minutes prior to perfusion. Brains were harvested, sectioned, and stained for ATF3 (red) and dsRed ((A-F), pseudocolored green, indicates POMC neurons) or GFP ((G-L), green indicates AgRP neurons). Images are representative of 3-4 mice per treatment group. White arrows indicate colocalized neurons.

Figure 3.3: ATF3 is induced by fasting in AgRP neurons. Representative images from AgRPeGFP-L10a mice given ad libitum access to food (Top panels) or fasted for 24 hours (Bottom panels) prior to perfusion. Brains were harvested, sectioned, and stained for ATF3 (red, Left) and GFP (green, Center; indicates AgRP neurons). Right; Merge. White arrows indicate colocalized neurons. Images are representative of 3-4 mice per treatment group.

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CHAPTER 4

REGULATION OF THE LEPRB TRANSCRIPTOME BY

LEPTIN AND LEPRB-STAT3 SIGNALING

Chapter Summary

Leptin activates the latent transcription factor, STAT3, in leptin receptor (LepRb)- expressing neurons in the brain, thereby controlling energy balance and metabolism.

Importantly, the effect of these signaling pathways on gene transcription in LepRb neurons has never been investigated. Here, we examined the regulation of gene transcription in LepRb neurons by leptin and by LepRb-STAT3 signaling. We employed

LepRb-specific TRAP to isolate mRNA from the hypothalami of mice treated with exogenous leptin, genetically leptin-deficient (ob/ob) mice, mice exposed to diet induced obesity (DIO), and mice in which STAT3 had been specifically ablated from LepRb neurons. TRAP-isolated mRNAs were subjected to RNA-Seq analysis, and changes in the LepRb transcriptome were identified. Exogenous leptin treatment again induced a number of transcription factors and intracellular proteins but did not affect neuropeptide transcription/translation in wild-type or ob/ob mice. In contrast, states of extreme leptin deprivation or repletion, such as in untreated ob/ob mice or in mice fed a high fat diet, induced changes in multiple neuropeptide species, many of which were also altered in

LepRb-specific STAT3 ablated mice. This analysis also revealed a small number of transcripts that were altered in multiple treatment conditions, including Socs3, Atf3,

Asb4 and members of the Serpina3 family, and which may represent direct and essential targets of hypothalamic LepRb action in the control of energy balance.

Introduction

The adipose hormone leptin is a critical central regulator of energy balance [1-2].

Produced in white adipocytes in proportion to their triglyceride content, leptin is released into the systemic circulation but acts primarily in the central nervous system to regulate diverse determinants of energy balance [3-4]. In the CNS, leptin acts on leptin receptor

(LepRb) expressing neurons in the hypothalamus and brainstem to control and coordinate a variety of metabolic parameters including food intake, energy expenditure, and glucose homeostasis[1-2, 5-11]. Importantly, the cellular and molecular mechanisms by which leptin controls these functions are poorly understood.

The leptin receptor is an IL6-type class I cytokine receptor that engages multiple intracellular signaling pathways upon its activation by leptin [12-13]. Binding of leptin to

LepRb results in the autophosphorylation and activation of Janus-activated Kinase 2

(JAK2) and subsequent JAK2-mediated phosphorylation of three critical tyrosine residues on the intracellular tail of LepRb [14-15]. Phosphorylation of Tyr985 results in the activation of SHP2/MAPK/ERK signaling, phosphorylation of Tyr1077 allows for the recruitment and activation of STAT5, and phosphorylation of Tyr1138 leads to the recruitment, phosphorylation, and activation of STAT3 [14, 16]. PI3K signaling is also activated by LepRb, but the mechanisms for this activation are unclear[17-19]. Negative feedback on the receptor is mediated by pSTAT3-induced increases in SOCS3, which also binds to Tyr985 and limits JAK2 signaling [14, 20].

The Tyr1138-STAT3 pathway is thought to be most important signaling pathway engaged by leptin for the control of energy balance [9]. LepRb-neuron specific STAT3 knockout mice are obese, hyperphagic, and hyperleptinemic, and mice in which Tyr1138

89 of LepRb was mutated to a serine, preventing its phosphorylation and subsequent recruitment of STAT3 (Leprs/s mice), are also highly obese, with body weights approaching that seen in leptin-receptor deficient db/db mice[21-24]. Although LepRb-

STAT3 signaling is necessary for leptin’s control of energy balance, the mechanisms by which STAT3 controls energy homeostasis are not clear. Activated STAT3 acts in the nucleus either in homo- or hetero-dimers to repress or activate target gene transcription, but the identity of these target genes in LepRb neurons is unknown [25-26].

STAT3 target genes have been identified in other tissues, especially in the context of wound healing and cancer, but understanding of STAT3 action in the hypothalamus is limited. This is likely due to the fact that STAT3 targets are highly cell- type dependent, and can also vary based on cofactor availability within a given cell type

[27-29]. Thus, while the regulation of a few specific transcripts in the hypothalamus

(most importantly, Socs3, but also the melanocortins Pomc and Agrp) have been ascribed to LepRb-STAT3 signaling, the full complement of LepRb-STAT3 responsive genes has never been rigorously assayed[30-32]. This is problematic, since disruption of leptin-melanocortin signaling fails to recapitulate the body weight phenotype observed in leptin deficient ob/ob mice or in Leprs/s mice, suggesting that other non- melanocortin genes must be essential for the regulation of energy balance by leptin [33-

34].

While LepRb-STAT3 signaling is essential for the control of energy balance, it is important to note that all of the LepRb-pY signaling pathways engaged by leptin terminate in the nucleus, either directly (as in the case of STAT3 and STAT5 phosphorylation and translocation) or indirectly (via SHP2/MAPK/ERK), where these

90 pathways also alter the expression of target genes. The PI3K pathway also activates the mTOR complex, leading to increases in gene translation, if not gene transcription per se [35-36]. Thus changes in gene expression in response to LepRb signaling may depend on signaling through multiple parallel pathways. Once again, the effects of these pathways on target gene expression are poorly defined. In short, there remains a significant gap in our knowledge of the gene targets of leptin, including those specific for

LepRb-STAT3 signaling, and thus in our understanding of cellular and molecular leptin action.

Since LepRb neurons comprise only a fraction of all neurons in the hypothalamus, it has been difficult to isolate LepRb neurons and their specific transcripts from the surrounding cellular milieu. Therefore we used cell specific translating ribosome affinity purification (TRAP) to isolate translating mRNA from LepRb neurons in mice treated with exogenous leptin, mice with diet-induced obesity, leptin deficient ob/ob mice, and mice in which STAT3 was specifically ablated only in LepRb neurons. We subjected the resulting transcripts to RNA-seq analysis (TRAP-Seq), and performed differential expression analysis. This data set allowed us to examine the regulation of the LepRb transcriptome by exogenous and endogenous leptin, as well as specifically identify LepRb-STAT3 regulated transcripts in LepRb neurons.

Results:

Regulation of the LepRb neuron transcriptome by leptin treatment n.b. We have included data from the acute 3 hour leptin treatment experiment (See

Chapter 3) throughout this chapter as it provides a context for interpreting the transcriptional changes observed with longer term leptin treatments.

Previous experiments examining the transcriptional effects of an acute (3 hour) leptin treatment demonstrated that in the short term, leptin increases the expression of a cohort of transcriptional factors specifically in LepRb neurons. We hypothesized that these transcriptional regulators modify the expression of a number of second or third order genes responsible for the majority of leptin’s effects in the control of energy balance, and that the expression of these genes might be regulated on a longer timescale. We therefore performed LepRb specific TRAP isolations in LepRbeGFP-L10a mice treated for 10 hours with leptin (5mg/kg i.p.) or vehicle. The resultant mRNA was used to generate multiplexed sequencing libraries for analysis by RNA-seq. We sequenced 3 independent samples per genotype and treatment group, each comprised of hypothalamic extracts from pooled groups of 4-6 adult LepRbeGFP-L10a mice. The 10 hour time point was chosen to allow the transcription factor transcripts seen to be increased at 3 hours (such as Junb, Atf3, En1, Arid5a and others) to be fully expressed and actively regulating gene transcription prior to dissection.

One limitation of exogenous leptin treatment in ad libitum fed mice is that the presence of endogenous leptin signaling may prevent the detection of transcripts that are already being actively regulated by leptin. We therefore also performed TRAP-Seq in leptin deficient LepRbeGFP-L10a;ob/ob mice subjected to the same 10 hour leptin or vehicle treatment. An added benefit of this model is that it allows us to examine the effects of chronic leptin deficiency on the LepRb transcriptome by comparing transcripts from vehicle treated LepRbeGFP-L10a mice to transcripts from vehicle treated LepRbeGFP-

L10a;ob/ob mice.

More than 700 genes were differentially regulated in response to leptin deficiency

(LepRbeGFP-L10a;ob/ob) or leptin treatment in these models. Due to the imperfect ability of

TRAP-seq to isolate mRNA from select cell populations without contamination from surrounding populations however, it is unlikely that this list represents only those genes that changed specifically in LepRb neurons. In order to generate a more focused list of genes cell-autonomously regulated by leptin, we chose to constrain our analysis to those genes that were significantly enriched in LepRb neurons at baseline, or that became enriched in the response to leptin treatment (or deficiency). This analysis reduced the number of regulated genes to approximately 200 (Appendix 3). Perhaps surprisingly, of the two hundred genes identified, only 51 were regulated in more than one treatment group. Of these, 33 genes displayed expression patterns that fall into easily distinguishable categories (Table 4.1).

Transcripts upregulated by leptin action

The genes regulated by sub-acute leptin treatment or leptin deficiency fell into a number of categories based on their expression patterns. Both Serpina3h and Socs3 were regulated in response to every treatment paradigm, suggesting that these genes are sensitive and specific targets of leptin action. These two genes were also upregulated by acute (3 hour) leptin treatment, and thus likely represent ‘first order’ genes which may be directly regulated by LepRb-STAT3 signaling. Other first order genes include those previously identified as upregulated in response to 3 hour leptin treatment; increases in many of these transcripts were also detectable 10 hours after leptin treatment in the LepRbeGFP-L10a or LepRbeGFP-L10a;ob/ob model. Whether this prolonged increase represents continual transcription of these genes or ongoing

93 translation of synthesized transcripts is not clear. Regardless, this group of acutely regulated transcripts might best be interpreted to be “stimulus response” genes in

LepRb neurons, as they were not regulated in the LepRbeGFP-L10a;ob/ob group, and most

(with the exception of Junb) had relatively low expression levels in vehicle treated samples. This is designation is consistent with the classification of many of these genes, including Junb and Atf3, as stress-inducible transcription factors [37-39].

As hypothesized, a separate group of transcripts did not respond to acute leptin treatment, but responded to longer term changes in leptin levels represented by the

LepRbeGFP-L10a;ob/ob model or by 10 hour leptin treatment of LepRbeGFP-L10a mice. These genes likely represent second or third order leptin target genes, which may be regulated either by other first order transcription factors such as Atf3 or Junb, or by leptin via

SHP2/ERK or PI3K/mTOR pathways.

The Serpina3 family

Multiple transcripts encoding the SerpinA3 family of genes were upregulated in response to leptin treatment. Indeed, Serpina3h, Serpina3i, and Serpina3n were among the most highly regulated genes across all groups; they also represent genes which were highly enriched in LepRb neurons at baseline, suggesting that they are not only highly regulated by leptin, but may also be specifically expressed in LepRb neurons.

The Serpina3 transcripts encode for the alpha-1-anti-chymotrypsin family of serine protease inhibitors [40]. Since protease inhibition may be a critical regulatory control mechanism in neuropeptidergic neurons (many of which express LepRb), we examined

94 the expression of these species in LepRb neurons as well as in two well-studied neuropeptidergic LepRb subpopulations: POMC and AgRP neurons[41-43].

We treated POMC-dsRed mice with leptin (5mg/kg i.p. for 60 minutes) and examined colocalization of pSTAT3-IR/Serpina3n-IR or POMC-dsRed/Serpina3n-IR.

Transgenic NPY-GFP mice were also used to examine Serpina3n-IR in AgRP neurons.

Unfortunately, whereas the human locus contains a single gene copy (SERPINA3), the murine Serpina3 locus has experienced multiple gene reduplication events [40]. As a result, the polyclonal antibody used to detect SERPINA3N was raised against an immunogen that shares >75% homology with SERPINA3C, -A3F, -A3G, -A3H, -A3I, and

-A3M, (although not with other SERPINS), making it difficult to determine the specificity of this antibody for a given murine isoform. Regardless, SERPINA3-IR was observed in almost all arcuate LepRb neurons as detected by leptin induced pSTAT3-IR (Figure 4.1

A-C). SERPINA3-IR was also detected in a majority of POMC neurons (Figure 4.1 D-F), although it was limited to the pSTAT-IR positive POMC population (data not shown), suggesting that SERPINA3 expression in POMC neurons may be regulated by leptin. In contrast, SERPINA3-IR was detected in all AgRP neurons; since not all AgRP neurons express LepRb, this suggests that Serpina3 expression may be less leptin-dependent in these neurons. It is also possible that the SERPINA3N antibody is detecting different

SERPINA3 isoforms in these cell types, however, and that the colocalization observed in AgRP neurons may represent a non-leptin-regulated SERPINA3 isoform.

Downregulation of gene transcription or translation by acute leptin

A final group of transcripts are those that were downregulated in response to both sub-acute leptin treatment, including transcripts such as Flt1, Fn1, and Abcb1a.

Many of these transcripts were also observed to be downregulated by 3 hour leptin treatment, although whether this observed decrease is due to changes in transcription or translation is unclear. Interestingly, all of these transcripts are highly preferentially expressed in AgRP neurons versus all arcuate LepRb neurons (data not shown). AgRP neurons are inhibited by acute leptin treatment, which may result in decreased phosphorylation of ribosomal protein S6, and subsequent inhibition of cap-dependent translation [44-45]. Thus the changes in these transcripts may represent preferential changes in translation of certain mRNA species rather than repression of gene transcription per se.

Regulation of neuropeptide expression by leptin

Many populations of LepRb neurons can be defined by the neuropeptides that they express, yet the role of leptin in regulating the expression of these neuropeptides has not been elucidated. Thus we also focused our analysis on the regulation of neuropeptides known to be expressed in LepRb neurons (Table 4.2). The majority of the neuropeptides did not respond to leptin treatment in LepRbeGFP-L10a;ob/ob or

LepRbeGFP-L10a mice, but were significantly altered at baseline in LepRbeGFP-L10a;ob/ob animals, suggesting these transcripts may be regulated by leptin over a longer timescale. This interpretation is supported by our understanding of leptin as a chronic, rather than acute, signal of energy repletion. It may also indicate the leptin is necessary, but not sufficient, for the expression of many of these neuropeptides.

Regulation of the LepRb transcriptome in diet-induced obese mice

A number of transcripts, particularly those encoding neuropeptides, were regulated in LepRbeGFP-L10a;ob/ob animals, but not in response to exogenous leptin treatment, suggesting that they may be regulated by chronic leptin signaling rather than acute changes in leptin levels. The developmental defects in the hypothalamus of

LepRbeGFP-L10a;ob/ob animals however, may confound the interpretation of these results

[46]. Furthermore, administration of bolus leptin does not appropriately recapitulate physiological leptin secretion, and thus may induce non-physiological changes in LepRb neurons. Therefore we examined the effect of physiological hyperleptinemia by performing TRAP-Seq on diet-induced obese (DIO) mice. LepRbeGFP-L10a mice were weaned onto a high fat diet (40%kcal from fat) at 21 days, and maintained on this diet for at least 8 weeks; at dissection high fat diet fed mice weighed significantly more than controls (29.4+/- 2.1 vs 22.1+/-0.72 grams, p=.0018). Hypothalamic extracts were obtained from 11-12 week old mice, and TRAP isolation performed. The resultant mRNA was used to generate multiplexed sequencing libraries for analysis by RNA-seq.

We sequenced 3 independent samples, each comprised of hypothalamic extracts from pooled groups of 4-6 adult LepRbeGFP-L10a mice. For RNA-seq differential expression analysis, chow fed LepRbeGFP-L10a animals that received 10 hours of vehicle treatment from our previous experiment were used as the control group.

Only 70 genes were found to be differentially expressed in LepRb neurons of

DIO LepRbeGFP-L10a mice versus chow fed controls. Of these, less than 50 were enriched in LepRb neurons. The large majority of the genes regulated in DIO mice had already been identified as regulated either in response to leptin treatment or in LepRbeGFP-

L10a;ob/ob (Table 4.3, top). Primary among them are Socs3, the Serpina3 family, and a number of neuropeptides (Table 4.3, bottom). Many of the genes that had been identified in previous experiments as acute response genes, including Arid5a, Etv6,

Rasl11a and others were not changed in response to DIO, suggesting that the slow increase in leptin levels of DIO mice may not trigger the same response in LepRb neurons as bolus leptin treatment. Transcripts from a few acute phase response genes however, such as Atf3 and Junb were also elevated in response to DIO, suggesting that these genes are a direct target of leptin signaling independent of cellular activation.

Many neuropeptides were also highly regulated by DIO (Table 4.3, bottom) consistent with the interpretation that these genes are far more sensitive to chronic, rather than acute, changes in leptin levels.

Relatively few transcripts in LepRb neurons were altered only in response to DIO and not to other treatments. In order to compare the effects of different treatment modalities on LepRb gene expression, we plotted fold change against fold change for multiple treatment conditions (Figure 4.2). When compared to other treatments, such as the inverse of LepRbeGFP-L10a;ob/ob (which might best recapitulate a “high leptin” state), the effect of DIO on transcripts enriched in LepRb neurons most closely resembled 10 hour leptin treatment (slope=0.51, r2=0.46 for DIO vs 10hr leptin), and was not correlated with the results observed in LepRbeGFP-L10a;ob/ob (slope=.0065, r2=.00005 for

DIO vs inverse ob; slope=0.14, r2=0.065 for inverse ob vs 10 hour leptin). This is consistent with the notion that the effects of DIO on hypothalamic gene transcription can be primarily assigned to the effects of hyperleptinemia, rather than a specific effect of high fat diet per se.

Five of the genes enriched in LepRb neurons that were altered only in response to DIO are neuropeptides important for the regulation of energy balance. These include thyrotropin releasing hormone (TRH), Neuromedin-S (NMS), VGF, Corticotropin releasing hormone (CRH), and neuropeptide W (NPW) (Table 4.3, bottom). Each of these neuropeptides inhibit feeding following central injection, suggesting that they may play important roles in the anorexic response to high fat feeding [47-52]. Although these neuropeptides are clearly essential for the appropriate regulation of energy homeostasis, that they are not suppressed in LepRb neurons of LepRbeGFP-L10a;ob/ob animals suggests that they may represent transcripts that are not solely leptin dependent, but rather are being coordinately regulated by multiple adiposity signals in the DIO model.

Regulation of the LepRb transcriptome by STAT3

Our analysis of the LepRb transcriptome revealed over 200 genes modulated by changes in leptin availability. This analysis did not allow us to assess the mechanisms by which leptin may be regulating these transcripts, however. Multiple signaling pathways engaged by LepRb terminate in the nucleus, and the gene targets of these pathways have not been comprehensively assayed. In order to determine which genes are specifically regulated by LepRb-STAT3 signaling, and thus may be most important for the control of energy homeostasis by leptin, we performed TRAP-Seq on hypothalamic extracts from mice in which STAT3 was specifically ablated from LepRb neurons.

LepRbeGFP-L10a mice were crossed to STAT3flox mice to generate

Leprcre/creSTAT3flox/flox (STAT3LepRbKO) and Leprcre/creSTAT3+/+ (STAT3LepRWT) mice, both of which are also homozygous for the Rosa26eGFP-L10a allele. In the presence of

Leprcre, exons 18-20 of STAT3, which encode the SH2 domain, are specifically deleted, resulting in ablation of STAT3 from all LepRb expressing cells (Figure 4.3 A). In order to confirm successful deletion of STAT3 from LepRb neurons, STAT3LepRKO and

STAT3LepRWT mice were treated with vehicle or leptin (5mg/kg i.p. for 90 minutes), and pSTAT3-IR in eGFP-labeled LepRb neurons was assessed. Leptin induced pSTAT3 is a specific marker of LepRb neurons in the hypothalamus, and thus robust pSTAT3-IR was detected in eGFP positive LepRb neurons in STAT3LepRWT mice following leptin treatment. Leptin did not induce pSTAT3-IR in STAT3LepRKO mice however, confirming that these mice lack functional LepRb-STAT3 signaling in LepRb neurons (Figure 4.3

B). Commensurate with their lack of functional LepRb-STAT3 signaling, STAT3LepRKO mice are obese (36.5+/-2.0 g for STAT3LepRKO vs 20.4+/-0.95 g for STAT3LepRWT, p<.0001), and have significantly increased adiposity and decreased lean mass compared to controls, as well as markedly elevated leptin and insulin levels (Figure 4.3

C-E).

Pooled hypothalami from adult STAT3LepRKO mice and STAT3LepRWT controls were subjected to TRAP isolation and subsequent RNA sequencing as described above. A total of 3 independent samples from each genotype were sequenced, and differentially expressed genes were examined. Over 150 genes were differentially regulated in STAT3LepRKO mice compared to controls, and more than 100 of these genes were enriched in LepRb neurons either at baseline or following STAT3 ablation.

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Surprisingly, the initial RNA-seq analysis did not reveal a decrease in Stat3 transcripts in the STAT3LepRKO mice. Closer inspection of the reads mapping to the STAT3 gene however revealed that while the number of reads mapping to exons 1-17 was essentially unchanged between STAT3LepRKO mice and STAT3LepRWT mice, the floxed exons (18-20) revealed a >50% decrease in reads mapping to this region.

STAT3 activation is a marker of leptin-LepRb signaling in the hypothalamus, but

STAT3 is also activated by multiple cytokine signaling pathways and thus may control the transcription of multiple genes independent of leptin action [26, 53-54]. Similarly, multiple LepRb pathways terminate in the nucleus and may regulate gene expression independently of STAT3 signaling. Therefore, to determine which transcripts were specifically LepRb-STAT3 regulated, we focused our analysis on those genes that were concordantly or discordantly regulated between leptin-deficient LepRbeGFP-L10a;ob/ob and STAT3LepRKO mice. We therefore compared the fold changes observed in

STAT3LepRKO and LepRbeGFP-L10aob/ob mice, as well as directly examining differential expression between these two models (Figure 4.4, Table 4.5). Those transcripts that were differentially expressed in one model relative to their respective control group, but not in comparison to the other model in which that transcript was unchanged, were excluded from this analysis since it is unclear whether they represent meaningful differences or merely biological variation in the given control groups.

The genes that were coordinately regulated in both models (Figure 4.4 A, B) are most likely LepRb-STAT3 dependent, since for this group the loss of LepRb-STAT3 signaling mimics the total loss of leptin signaling represented by the LepRbeGFP-

L10a;ob/ob model . Many of the neuropeptides previously suggested to be LepRb-STAT3

101 regulated, including Pomc, Agrp, Npy, Nts, and Cartpt fell in this group. So too did a number of genes predicted to be important for leptin’s regulation of energy balance, including Npy2r and Serpina3n. Npy2r and Serpina3n however were more suppressed in the LepRbeGFP-L10a;ob/ob model than in the STAT3LepRKO model, suggesting that they are also partially regulated by leptin via STAT3-independent mechanisms. Other genes identified in this analysis, including Fam159a, Ccl17, Tmem176a, and Rbp4 have not been linked to the central control of energy balance, although the identification of the adipokine RBP4 in the central nervous system may provide new insight into the function and sites of action of this protein [55].

The genes altered only in ob/ob but not STAT3LepRKO mice (Figure 4.4, C,D) are likely leptin dependent, but controlled by STAT3-independent pathways. These include the neuropeptides Ghrh and Tac1, and cell signaling proteins such as Atg7, Plagl1, and

Socs3. ATG7 has previously been shown to be important for the function of both POMC and AgRP neurons, while Plagl1 is an imprinted gene that is altered in the offspring of obese mothers [56-59]. As a STAT3 responsive gene, it is highly surprising to find that

Socs3 is not repressed in the STAT3LepRKO mice as it is in the LepRbeGFP-L10a;ob/ob model. It should be noted, however, that Socs3 transcription is also directly activated by

STAT1, which was repressed in the LepRbeGFP-L10a;ob/ob mice, but induced in

STAT3LepRKO mice [60-61]. Thus it is likely that compensatory STAT1 signaling is responsible to restoring Socs3 levels to baseline.

The expression of a final group of genes was significantly altered in

STAT3LepRKO mice, but not in LepRbeGFP-L10a;ob/ob (Figure 4.4 F, Table 4.5), suggesting that they are STAT3 regulated independent of leptin action. Many of these

102 genes have been shown to be induced by STAT1 activation, including Irf1, Psmb8,

Tap1 and others [62]. STAT3 is a critical negative regulator of STAT1, and Stat1 transcript levels were increased in STAT3LepRKO mice[26, 63]. Thus it appears that loss of STAT3 inhibition of STAT1 may be driving increased transcription of these genes, although they may also represent genes that are specifically repressed by STAT3 [64].

Regulation of the LepRb transcriptome in Leprs/s vs STAT3LepRKO mice

Although the LepRbeGFP-L10a;ob/ob model aids in interpreting the results from the

STAT3LepRKO, a limitation of the STAT3LepRbKO model is that it does not allow us to isolate the effects of LepRb-STAT3 signaling disruption from the effects of loss of

s/s STAT3 signaling in toto. Lepr mice contain a mutation at Tyr1138 that prevents the recruitment of STAT3 to the leptin receptor, while leaving global STAT3 signaling intact.

We therefore isolated RNA from the arcuate nucleus of ob/ob and controls (n=10 per group, males only), STAT3LepRbKO and STAT3LepRbWT (n=9-10 per group, mixed sex), and Leprs/s and Lepr+/+ controls (n=8 per group, females only), and examined changes in gene expression of target genes by RT-qPCR.

As expected, those genes most significantly changed in our TRAP-seq dataset were most likely to be observed to change in the qPCR analysis (Table 4.6). Our analysis also confirm that Stat1 is elevated in STAT3LepRKO mice, but not Leprs/s mice; consistently, STAT1-induced transcripts (such as Psmb8 and Irf1) are induced in the

STAT3LepRKO mice, but not in Leprs/s mice. Thus the large upregulation of interferon- gamma target gene expression observed in the TRAP-seq data from STAT3LepRKO

103 mice can be more definitively assigned to loss of negative inhibition of STAT1, rather than loss of LepRb-STAT3 signaling specifically [26, 63-65].

Discussion

We examined the regulation of the LepRb transcriptome in response to exogenous leptin, leptin deficiency, diet induced obesity, and LepRb-specific STAT3 ablation, revealing hundreds of genes coordinately and individually regulated by leptin, adiposity, and LepRb-STAT3 signaling. This analysis, combined with our previous data, allowed us to identify transcription factors that respond briefly and transiently to leptin stimulation, their presumed second and third order target genes, including neuropeptides, cell signaling proteins, and protease inhibitors, and the LepRb-STAT3 responsive genes that may be most critical for the control of energy balance by leptin.

Although cell-specific TRAP is in many ways superior to other mechanisms of cell isolation in neuronal cells that respond poorly to cellular dispersion and FACS sorting techniques, TRAP isolation still suffers from the issue of cross-contamination from surrounding tissue. Therefore we chose to focus our analysis on those transcripts that were enriched in LepRb neurons at baseline or became enriched in response to one of our treatment paradigms. We also chose to focus on those genes which were seen to respond to more than one treatment paradigm, as these presumably represent those genes which are most sensitive to alterations in leptin signaling. Since the majority of our study paradigms also had unique control groups, this also increased the power of our analysis to identify truly leptin regulated transcripts, since those transcripts

104 that were observed to change in more than one treatment are less likely to be false positives resulting from natural biological variation in a given control group.

TRAP-seq isolation is specific for those transcripts that are bound into the ribosomal active site, and thus reflect changes in translations as well as changes in transcription. This was particularly evident when assessing those transcripts observed to be downregulated at both 3 and 10 hours after leptin treatment, all of which are preferentially expressed in AgRP neurons. Since TRAP-seq isolation is dependent on ongoing active translation, if leptin inhibits translation in the AgRP (or other) LepRb populations, the transcriptional responses of these neurons to leptin will likely be underrepresented in our data set. Understanding the regulation of AgRP neurons (or other leptin-inhibited neurons) may either require AgRP specific TRAP-isolation, or fasting/refeeding paradigms, rather than leptin treatment.

Our analysis of animals treated acutely (for 3 hours) or sub-acutely (for 10 hours) with leptin revealed that in the short term leptin induces a number of immediate response genes, but does not regulate neuropeptide expression. Surprisingly, many transcription factors observed to be upregulated after 3 hours of leptin treatment, including Atf3, Junb, Etv6, and Arid5, were still undergoing increased translation 10 hours after leptin treatment, long after phospho-STAT3 is no longer detectable in the nucleus [66]. This suggests that their transcriptional programs may be regulating a second, third, or nth phase response to leptin over an extended time scale. It may also suggest that these first and second order transcription factors may be the critical regulators of neuropeptide expression that responds to chronic leptin (in the form of

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DIO) or chronic leptin deficiency (in the case of LepRbeGFP-L10aob/ob mice), but not to acute leptin.

The finding that the majority of neuropeptide transcription was not responsive to leptin treatment was unexpected. Many hypothalamic neuronal populations that express neuropeptides also express LepRb, which has led to speculation that leptin signaling directly regulates neuropeptide expression in these neurons. This is especially true for

Pomc and Agrp transcription, where leptin-induced pSTAT3 is thought to stimulate or repress their respective transcription via direct promoter binding [31-32]. Although Pomc expression increased mildly (~1.5fold) following leptin treatment of LepRbeGFP-L10a mice,

Agrp was totally unchanged. Furthermore, neither transcript responded to leptin treatment in LepRbeGFP-L10a;ob/ob mice. It is also possible however, that LepRb-STAT3 signaling is only partially responsible for the regulation of transcription of these genes.

This is consistent with reports that other transcriptional regulators such as FOXO1,

JUNB, and FOS may also impact transcription from these promoters [30, 32]. The requirement for the activation of multiple transcription factors has been observed in other neuropeptides, such as CART, which has overlapping STAT3/CREB/AP-1 sites, and neurotensin, which has functional AP-1, CREB, ATF, and STAT3 binding elements in its promoter [67-71]. Thus it appears that LepRb-STAT3 may be necessary, but not sufficient, for the regulation of neuropeptide transcription in LepRb neurons.

Our analysis also revealed a number of genes that may play important roles in controlling the physiology of LepRb neurons. A recent GWAS study has linked mutations in ASB4 to an obesity phenotype, highlighting the potential importance of this transcript for the control of energy balance [72]. ASB4 is a SOCS-box containing protein

106 that has been shown to target IRS4 for degradation [73]. Since the IRS proteins are thought to link LepRb to PI3K signaling, upregulation of ASB4 in response to leptin may be critical for negative inhibition of the LepRb-PI3K pathway. ASB4 may also be critical for mediating crosstalk between leptin and insulin signaling in LepRb neurons. Future studies examining the expression of ASB4 in LepRb neurons, as well as its functions in these neurons, may shed light onto the mechanisms by which the mutations identified in the GWAS study may result in obesity.

The finding that Stat3 transcription is leptin sensitive is perhaps surprising, as leptin is thought to engage a negative feedback loop mediated by STAT3/SOCS3.

STAT3 has been shown to be autoregulatory in cooperation with still-unidentified cAMP- response element (CRE) binding partners in other tissues however, although this has never been demonstrated in hypothalamic neurons [74]. Unphosphorylated STAT3 (U-

STAT3) has also been shown to have gene regulatory functions in certain contexts, however, and it is thus also possible that increased levels of newly synthesized U-

STAT3 may counterbalance the effects of pSTAT3, and thus act as a negative feedback mechanism[75-76]. Regardless, the decrease in Stat3 expression in ob/ob mice may underlie the delay in acute gene transcription observed in this model.

The Serpina3 genes comprise another intriguing cluster of leptin-regulated transcripts, especially given the neuropeptidergic nature of many LepRb neuronal populations. The SERPINA3 family of protease inhibitors falls generally into the anti- alpha-1-chymotrypsin family, but the individual proteases which they preferentially target have not been well studied. The various SERPINA3 family members may serve multiple functions in LepRb neurons; SERPINA3H and SERPINA3I do not contain an

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NH3-terminal secretion sequence, and thus may be involved in intracellular protease inhibition for example, whereas SERPINA3N is secreted and thus may be involved in maintenance of neuropeptides in the extracellular space, or in remodeling of the ECM

[77]. Furthermore, SERPINA3N, which was one of the most highly expressed and highly regulated Serpina3 genes in LepRb neurons, contains a reactive center loop that is most similar to the human SERPINA3, but which is completely different from the other murine Serpina3 genes, suggesting a unique protease specificity for this isoform [40].

Recently various groups have determined that Serpina3n may specifically inhibit granzyme B which has important roles in extracellular matrix remodeling, as well as in inflammatory and apoptotic responses [78-79]. The role of granzyme B, as well as its potential cleavage targets, in the hypothalamus may thus be an important point of further study.

High dose exogenous leptin treatment and leptin-deficient ob/ob mice are useful tools for examining the gross effects of leptin on the LepRb transcriptome, but they do not provide insight into the role of leptin at physiologically relevant concentrations. Thus we performed TRAP-seq isolations on diet-induced obese LepRbeGFP-L10a mice. The vast majority of genes (including many neuropeptides) altered in response to either exogenous leptin in LepRbeGFP-L10a or in LepRbeGFP-L10a;ob/ob animals were also changed in the DIO animals, confirming that leptin also regulates the expression of these transcripts at physiologically relevant levels. The transcripts that did not respond to DIO were generally those that appear to be acute response genes, including transcription factors (Etv6, Arid5a, Tbx19) and cytokine signaling inhibitors (Cish,

Socs2). A number of transcripts in these categories did respond to DIO, however,

108 including Atf3, JunB, Asb4 and Socs3, suggesting that the expression of these transcripts may be closely linked to changes LepRb signaling as opposed to general neuronal activation.

When compared to multiple treatment paradigms, our studies of DIO suggest that the transcriptome of high fat fed mice most closely resembles that of mice treated transiently with superphysiologic leptin. This finding contrasts sharply with the concept of ‘leptin resistance’ that has argued for an inability of leptin to act appropriately in the hypothalamus of obese rodents or humans. While rodents fed a high fat diet fail to increase pSTAT3 in response to exogenous leptin treatment to the same degree as lean controls, an effect that is attributed to increases in SOCS3, our data argue that there remains sufficient signaling in LepRb neurons to still respond appropriately to increased levels of endogenous leptin, at least at the transcriptional/translational level

[80-81]. Leptin is known to have multiple fast actions either in depolarizing or hyperpolarizing target neurons, however, and thus it remains possible that while the transcriptional effects of leptin may be maintained in DIO mice, the fast actions of leptin may no longer be functioning appropriately[82-83].

Multiple signaling pathways engaged by LepRb terminate in the nucleus, but

LepRb-STAT3 signaling appears to be most critical for the control of energy homeostasis. To determine which genes are specifically regulated by LepRb-STAT3 signaling, and thus may be most important for the control of energy homeostasis by leptin, we performed TRAP-Seq on hypothalamic extracts from mice in which STAT3 was specifically ablated from LepRb neurons. A large number of transcripts previously predicted to be STAT3 regulated, including Pomc, Agrp, Npy, Cartpt, and Nts were

109 found to be concordantly regulated in both LepRbeGFP-L10a;ob/ob and STAT3LepRKO mice, confirming that LepRb-STAT3 signaling is necessary for the cell-autonomous regulation of these transcripts by leptin. Six transcripts were also revealed by this analysis that have not been previously associated with hypothalamic leptin signaling, including Maff, Ccl17, Fam159a, Gdpd3, RBP4, and Tmem176a. In the future, it will be important to determine where these transcripts are expressed in the hypothalamus, and how their regulation by leptin may affect energy balance.

Atf3, which we have shown to be as responsive as Socs3 to increases in leptin signaling, was observed to change neither in the LepRbeGFP-L10aob/ob nor STAT3LepRKO conditions. In the case of the LepRbeGFP-L10aob/ob mice, this is perhaps unsurprising, since as an acute response gene, ATF3 is maintained at very low levels in the absence of stimulus, and hypoleptinemic ob/ob mice represent an absence of stimulus for LepRb neurons. Atf3 was significantly upregulated in DIO mice however, suggesting ATF3 expression in LepRb neurons may be responsive to hyperleptinemia. Thus the finding that Atf3 is unchanged in the hyperleptinemic STAT3LepRKO mice may indicate that this gene is in fact STAT3 regulated. Thus ATF3 may still be a critical component of the transcriptional programs engaged by leptin to regulate energy balance. More work will clearly be necessary to evaluate the role of STAT3 in the regulation of ATF3, as well as to determine the necessity of ATF3 for leptin action.

Our analysis also revealed a number of genes that appear to be STAT3 regulated, but independent of leptin signaling. These genes, which were observed to change in STAT3LepRKO mice, but not LepRbeGFP-L10a;ob/ob mice, include many known

STAT1- induced target genes. Unfortunately, it is not currently feasible to perform

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TRAP-Seq in Leprs/s animals (which have disrupted LepRb-STAT3 signaling, but normal

cre STAT3 expression), as the need for a non-Tyr1138 mutant Lepr allele to drive cell specific expression of the eGFP-tagged L10a ribosomal subunit precludes homozygous expression of the Leprs/s allele. Therefore to confirm that the changes observed in the

STAT3LepRKO were due to disinhibition of STAT1, and not loss of LepRb-STAT3 signaling specifically, we compared the expression of these transcripts in RNA isolated from the whole arcuate nuclei of STAT3LepRKO mice, ob/ob, and Leprs/s mice. This analysis confirmed that Stat1 levels were increased only in STAT3LepRKO and not

Leprs/s mice, paralleling the increases in Irf1 and Psmb8 observed only in the

STAT3LepRKO and not Leprs/s model. While increases in Stat1 in the STAT3LepRbKO model can be attributed to loss of STAT3 inhibition, the reasons for the decrease in

Stat1 observed in ob/ob mice is not immediately clear, and thus the role of STAT1 in hypothalamic leptin action may require further investigation.

Overall, by examining the responses of the LepRb transcriptome to exogenous leptin, to genetic leptin deficiency, to diet induced obesity, and to loss of STAT3 signaling, we have identified a cohort of new genes that may be important for our understanding of LepRb neuron biology, as well as the physiology of leptin action and obesity in the central nervous system. We have also defined which genes require the presence of LepRb-STAT3 signaling for their expression, and have confirmed previously identified LepRb targets, as well as identifying new genes that may be critical for the control of energy homeostasis by leptin. Together, this data provides a resource for new exploration into the physiology of leptin action in the control of body weight and other metabolic, reproductive, and neuroendocrine functions.

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Materials and Methods

Animals: Rosa26eGFP-L10a mice were generated as previously described [84]. The generation of Leprcre mice has also been previously described [85]. Leprcre mice were crossed to RosaeGFP-L10a mice to generate Leprcre/+;Rosa26eGFP-L10a/+ mice which were subsequently intercrossed to generate double homozygous Leprcre/cre;RosaeGFP-L10a/eGFP-

L10a (LepRbeGFP-L10a) study animals. Ob/ob mice on the C57Bl6 background and C57Bl6 control mice were from Jackson labs (stock #000632 and #000664, respectively).

Double homozygous LepReGFP-L10a mice were backcrossed to ob/ob mice until

Leprcre/cre;RosaeGFP-L10a/eGFP-L10a;Ob/+ mice were obtained. These mice were subsequently intercrossed to generate Leprcre/cre;RosaeGFP-L10a/eGFP-L10a;Ob/Ob

(LepRbeGFP-L10aob/ob) and Leprcre/cre;RosaeGFP-L10a/eGFP-L10a;+/+ (LeprRbeGFP-L10a) mice for study. Pomc-dsRed transgenic mice were from Dr. Malcolm J. Low, and were generated as described [86]. Npy-GFP transgenic mice were from Jackson (stock

#006417). STAT3flox mice were also from Jackson (stock # 016923) and were backcrossed to LepRbeGFP-L10a mice to generate LepRbeGFP-L10a;STAT3Flox/+ mice. These mice were subsequently intercrossed to generate Leprcre/cre;RosaeGFP-L10a/eGFP-

L10a;STAT3Flox/Flox (STAT3LepRKO) and Leprcre/cre;RosaeGFP-L10a/eGFP-L10a;STAT3+/+

(STAT3LepRWT) controls for study. Leprs/s and controls were generated as previously described[21]. Experiments described used mixed groups of male and female mice except where explicitly noted. All procedures were approved by the University of

Michigan University Committee on the Use and Care of Animals in accordance with

AAALAC and NIH guidelines. Animals were bred at the University of Michigan and

112 maintained in a 12 hr light/12 hr dark cycle with ad libitum access to food and water except as noted in experimental protocols.

Hypothalamic dissections for TRAP-seq and RT-PCR: Adult homozygous mice were anesthetized and their brains removed to a mouse coronal brain matrix (1mm sections).

For whole hypothalamic dissections, a 3x3x3mm block was dissected from the ventral diencephalon immediately caudal to the optic chiasm and immediately homogenized for

TRAP-seq analysis. For arcuate specific dissections, 3 consecutive 1mm sections were removed immediately caudal to the optic chiasm, and arcuate nuclei were dissected bilaterally by hand from the mediobasal hypothalamus of each section, pooled and snap frozen for later processing.

Immunoprecipitation of ribosomes (TRAP): Messenger RNA was isolated from eGFP- tagged ribosomes, as well as from the eGFP-depleted fraction, as previously described

[87-88]. RNA was assessed for quality using the TapeStation (Agilent, Santa Clara,

CA). Samples with RINs (RNA Integrity Numbers) of 7.5 or greater were prepped using the Illumina TruSeq mRNA Sample Prep v2 kit (Catalog #s RS-122-2001, RS-122-

2002) (Illumina, San Diego, CA), where 0.1-3ug of total RNA was converted to mRNA using a polyA purification. The mRNA was fragmented via chemical fragmentation and copied into first strand cDNA using reverse transcriptase and random primers. The 3’ ends of the cDNA were adenylated, and 6-nucleotide-barcoded adapters ligated. The products were purified and enriched by PCR to create the final cDNA library. Final libraries were checked for quality and quantity by TapeStation (Agilent) and qPCR using

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Kapa’s library quantification kit for Illumina Sequencing platforms (catalog # KK4835)

(Kapa Biosystems,Wilmington MA). They were clustered on the cBot (Illumina) and sequenced 4 samples per lane on a 50 cycle single end run on a HiSeq 2000 (Illumina) using version 2 reagents according to manufacturer’s protocols.

RNA-seq analysis: 50bp single-end reads underwent QC analysis prior to alignment to mouse genome build mm9 using TopHat and Bowtie alignment software [89].

Differential expression was determined using Cufflinks Cuffdiff analysis, with thresholds for differential expression set to fold change >1.5 or <0.66 and a false discovery rate of

Uniprot Database for gene ontology and protein class analysis [91].

Arcuate dissections and RT-PCR: Ad libitum fed adult male ob/ob and c57bl6 controls

(n=10 per group), female Leprs/s and wild type controls (n=8 per group), and mixed male and female STAT3LepRKO and STAT3LepRWT mice (n=10 per genotype) were dissected at the midpoint of the light cycle. RNA was extracted from microdissected arcuate nuclei using Trizol (Invitrogen) according to manufacturer’s protocol, and subsequently converted to cDNA using iScript cDNA synthesis kit (Biorad # 170-8891) for use in reverse transcriptase PCR. cDNA was analyzed in duplicate by quantitative real time-

PCR on an Applied Biosystems StepOnePlus Real-Time PCR System for Tbp

(endogenous control) and each of the following: Socs3, Serpina3N, Agrp, Stat1, Npy,

Irf9, Stat3, Gch1, Tmema176a, Cartpt, Pomc, Psmb8, and Irf1. All Taqman assays were acquired from Applied Biosystems (Foster City, CA).

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Leptin treatment and high fat diet for Trap-seq: 10-13 week old LepRbeGFP-L10a and ob/ob

LepRbeGFP-L10a mice had food removed at the onset of the light cycle. Animals were immediately treated with metreleptin (5 mg/kg, i.p) (a generous gift from AstraZenica,

Inc.) or vehicle (0.9% saline) and hypothalami were dissected 10 hours post-treatment.

For diet-induced obesity (DIO) experiments, cohorts of LepRbeGFP-L10a mice were weaned at 21 days onto high fat chow (Research Diets D12492, 60% kcal from fat).

Mice were maintained on high fat diet for at least 8 weeks, and were dissected at 11-12 weeks of age. DIO animals were compared to the same controls as the leptin treatment group (vehicle treated LepRbeGFP-L10a) that had been raised on standard chow (Purina

Lab Diet 5001)).

Animal phenotyping of STAT3LepRKO and STAT3LepRWT mice: Analysis of body fat and lean mass was performed between 8-9 weeks of age using an NMR-based analyzer

(Minispec LF90II, Bruker Optics). Animals were dissected within 3 days of body composition analysis, and terminal serum analyzed for insulin and leptin concentrations.

Insulin was assessed using a double-antibody radioimmunoassay using an 125I-Human insulin tracer (EMD Millipore), a rat insulin standard (Novo), a guinea pig anti-rat insulin first antibody (EMD Millipore), and a sheep anti-guinea pig gamma globulin-PEG second antibody (MDRTC). Leptin was assayed by commercial ELISA (EMD Millipore).

Perfusion and immunohistochemistry: For phospho-STAT3 (pSTAT3) staining, mice were fasted overnight and treated with leptin (5mg/kg i.p) or vehicle 60 minutes prior to

115 perfusion. For perfusion, mice were anesthetized with a lethal dose of intraperitoneal pentobarbital and transcardially perfused with phosphate buffered saline followed by

10% neutral buffered formalin. Brains were removed, post-fixed overnight, and dehydrated in 30% sucrose before coronal sectioning (30 µm) using a freezing microtome (Leica). Immunostaining was performed as previously described [92] using primary antibodies for GFP (Aves Labs #GFP1020, chicken, 1:1000), SerpinA3N (R&D

Biotech # AF4709, 1:1000), DsRed (Clontech, # 632496 1:1000), and phospho-STAT3

(Cell Signaling Tech #9131S). All antibodies were reacted with species-specific Alexa

Fluor-488 or -568 conjugated (Invitrogen, 1:200) secondary antibodies or processed via avidin-biotin/diaminobenzidine (DAB) method (ABC kit, Vector Labs; DAB reagents,

Sigma), and imaged as previously described [93]. DAB images were pseudocolored using Photoshop software.

Statistics: RT-qPCR data are reported as mean fold change vs normalized vehicle.

Statistical analysis of RT-qPCR data and animal phenotyping data was performed using

Prism (version 6.0) software. Unpaired t-tests were used to compare results between groups of two. p < 0.05 was considered statistically significant.

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Gene Enrichment FPKM 3hr Leptin 10hr Leptin ob/ob ob/ob + Leptin Socs3 3.11 3.61 4.78 3.14 0.25 10.00 Serpina3h 6.01 4.71 2.19 6.15 0.32 7.33 Serpina3i 10.59 1.05 1.91 2.48 0.13 10.46 Serpina3n 7.35 95.53 0.96 2.06 0.26 5.06 Asb4 6.57 71.31 1.24 1.74 0.61 1.64 Stat3 2.47 39.29 1.36 1.62 0.52 2.33 Irf9 1.98 9.12 1.37 1.83 0.38 3.01 Tuba1c 0.12 0.50 1.32 1.52 0.37 2.48 Atf3 4.70 3.22 4.97 4.58 1.02 2.39 Junb 2.13 27.06 2.01 1.91 0.97 1.67 Rasl11a 0.96 12.57 1.68 1.57 0.74 1.83 Arid5a 0.56 1.47 4.52 1.30 1.00 2.50 Etv6 0.75 2.52 2.65 1.23 0.96 1.59 Socs2 1.01 3.43 1.92 1.19 0.68 1.68 Drd1a 1.03 2.04 1.80 0.72 0.66 1.66 Cish 1.21 5.22 1.79 1.14 0.67 1.65 Sbno2 1.08 3.93 1.70 1.46 0.74 1.78 Serpina3m 7.31 3.75 1.05 3.92 0.52 2.79 Gpr151 0.64 1.55 1.36 2.30 0.77 2.33 Cd24a 2.25 22.02 1.17 1.94 0.77 2.30 Tbx19 7.87 2.41 1.33 1.89 0.73 1.84 Lck 2.23 4.27 1.21 1.58 0.86 1.70 Prokr2 1.32 1.19 1.79 2.29 0.61 1.51 Flt1 4.23 1.89 0.54 0.41 1.23 0.92 Podxl 2.18 2.36 0.53 0.57 1.12 0.78 Slco1a4 2.32 3.54 0.52 0.40 1.10 0.95 Fn1 4.19 1.97 0.49 0.61 1.24 0.75 Ptprb 1.92 0.85 0.48 0.34 1.24 0.71 Tek 2.83 0.75 0.47 0.26 1.14 1.01 Cdh5 2.92 0.93 0.45 0.29 1.00 0.84 Eltd1 2.76 1.21 0.45 0.45 1.30 0.81 Abcb1a 4.27 1.69 0.43 0.43 1.35 0.99 Ly6c1 1.89 8.72 0.37 0.42 1.42 0.89

Table 4.1: Fold change in LepRb-enriched genes in leptin treated and ob/ob mice. TRAP-Seq was performed on LepRbeGFP-L10a mice treated for 3 or 10 hours with leptin

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(5mg/kg i.p) or vehicle, or on LepRbeGFP-L10a;ob/ob mice treated for 10 hours with leptin (5mg/kg i.p) or vehicle. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also changed, were included in this analysis. Enrichment and expression (FPKM) values displayed were from mice treated with vehicle for 3 hours. Fold change values for 3 hour and 10 hour leptin treatments in LepRbeGFP-L10a or LepRbeGFP-L10aob/ob are versus vehicle treated controls, ob/ob is versus 10 hour vehicle treated LepRbeGFP-L10a. n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult animals. p<.05 for values in bold and italics. FPKM: Fragments Per Kilobase of exon per Million reads mapped.

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Figure 4.1: SERPINA3N colocalization with LepRb, POMC, and NPY in the arcuate nucleus. Representative images showing colocalization of Serpina3n-IR (green) with (A-C) pSTAT3-IR (red) or (D-E) dsRed-IR (red) in POMC-dsRed mice treated for 60 minutes with leptin (5mg/kg i.p.). (G-I) Colocalization of SERPINA3N-IR (green) was also detected in NPY neurons of Npy-GFP mice (Npy-GFP-IR; pseudocolored red). Filled arrowheads indicate colocalized neurons; empty arrowheads indicate isolated Serpina3n positive neurons. Scale bar = 100M.

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Neuropeptides Enrichment FPKM 3hr Leptin 10hr Leptin ob/ob ob/ob + Leptin Agrp 41.89 260.9 0.69 1.19 5.55 0.94 Npy 21.55 1326.6 0.80 1.29 4.17 0.89 Pomc 21.07 975.6 0.96 1.56 0.16 1.35 Cartpt 9.38 976.0 0.93 1.12 0.28 0.81 Ghrh 8.60 99.1 0.65 0.94 2.66 0.96 Nts 6.30 124.7 0.88 1.36 0.41 1.10 Tac1 4.67 285.2 0.86 1.34 0.57 1.05 Gal 3.46 159.4 0.82 1.60 1.01 0.98 Kiss1 2.20 5.9 0.84 3.29 0.46 0.97 Tac2 1.52 96.7 0.76 2.25 0.99 1.02 Nmb 0.99 3.77 0.85 0.99 4.14 0.77

Table 4.2: Fold change in neuropeptides enriched in LepRb neurons. TRAP-Seq was performed on LepRbeGFP-L10a mice treated for 3 or 10 hours with leptin (5mg/kg i.p) or vehicle, or on LepRbeGFP-L10aob/ob mice treated for 10 hours with leptin (5mg/kg i.p) or vehicle. Neuropeptides enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also changed, were included in this analysis. Enrichment and expression (FPKM) values displayed were from mice treated with vehicle for 3 hours. Fold change values for 3 hour and 10 hour leptin treatments in LepRbeGFP-L10a or LepRbeGFP-L10aob/ob are versus vehicle treated controls, ob/ob is versus 10 hour vehicle treated LepRbeGFP-L10a. n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult mice. p<.05 for values in bold and italics. FPKM: Fragments Per Kilobase of exon per Million reads mapped.

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3hr 10hr ob/ob + Gene Enrichment FPKM Leptin Leptin ob/ob Leptin DIO Socs3 3.11 3.61 4.78 3.14 0.25 10.00 2.56 Serpina3h 6.01 4.71 2.19 6.15 0.32 7.33 2.32 Serpina3n 7.35 95.53 0.96 2.06 0.26 5.06 1.89 Asb4 6.57 71.31 1.24 1.74 0.61 1.64 1.62 Serpina3i 10.59 1.05 1.91 2.48 0.13 10.46 1.48 Atf3 4.70 3.22 4.97 4.58 1.02 2.39 6.26 Junb 2.13 27.06 2.01 1.91 0.97 1.67 1.87 Prokr2 1.32 1.19 1.79 2.29 0.61 1.51 2.04 Gpr151 0.64 1.55 1.36 2.30 0.77 2.33 2.15 Serpina3m 7.31 3.75 1.05 3.92 0.52 2.79 2.14 Tbx19 7.87 2.41 1.33 1.89 0.73 1.84 1.47 Cd24a 2.25 22.02 1.17 1.94 0.77 2.30 1.14 Lck 2.23 4.27 1.21 1.58 0.86 1.70 1.12 Rasl11a 0.96 12.57 1.68 1.57 0.74 1.83 1.44 Arid5a 0.56 1.47 4.52 1.30 1.00 2.50 1.50 Socs2 1.01 3.43 1.92 1.19 0.68 1.68 1.17 Drd1a 1.03 2.04 1.80 0.72 0.66 1.66 0.84 Cish 1.21 5.22 1.79 1.14 0.67 1.65 1.24 Etv6 0.75 2.52 2.65 1.23 0.96 1.59 1.14 Pomc 21.07 975.7 0.96 1.56 0.16 1.35 2.17 Nts 6.30 124.7 0.88 1.36 0.41 1.10 1.57 Cartpt 9.38 976.0 0.93 1.12 0.28 0.81 1.53 Npy 21.55 1326.7 0.80 1.29 4.17 0.89 0.60 Agrp 41.89 260.9 0.69 1.19 5.55 0.94 0.57 Npw 8.26 3.72 1.02 1.26 0.80 1.08 2.67 Trh 0.98 39.93 0.69 1.26 0.95 0.95 2.10 Nms 3.08 3.78 0.98 1.47 0.67 0.95 1.97 Crh 3.37 15.04 0.81 1.37 1.27 1.00 1.96 Vgf 2.35 207.72 0.90 1.19 1.43 1.13 1.72 Gal 3.46 159.46 0.82 1.60 1.01 0.98 1.77 Tac2 1.52 96.76 0.76 2.25 0.99 1.02 1.65 Tac1 4.67 285.16 0.86 1.34 0.57 1.05 1.26 Ghrh 8.60 99.17 0.65 0.94 2.66 0.96 1.13 Nmb 0.99 3.77 0.85 0.99 4.14 0.77 1.08 Kiss1 2.20 5.90 0.84 3.29 0.46 0.97 2.75

Table 4.3: Fold change in LepRb-enriched genes in diet-induced obese (DIO), leptin treated, and ob/ob mice. TRAP-Seq was performed on LepRbeGFP-L10a mice treated for 3 or 10 hours with leptin (5mg/kg i.p) or vehicle, on LepRbeGFP-L10aob/ob mice treated for 10 hours with leptin (5mg/kg i.p) or vehicle or on LepRbeGFP-L10a mice fed a

121 high fat diet for 8-9 weeks. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also significantly changed, were included in this analysis. Enrichment and expression (FPKM) values displayed were from mice treated with vehicle for 3 hours. Fold change values for 3 hour and 10 hour leptin treatments in LepRbeGFP-L10a or LepRbeGFP-L10aob/ob are versus vehicle treated controls, ob/ob and DIO is versus 10 hour vehicle treated LepRbeGFP-L10a. n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult animals. p<.05 for values in bold and italics. FPKM: Fragments Per Kilobase of exon per Million reads mapped.

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Figure 4.2: Fold change in LepRb-enriched genes in diet-induced obese (DIO), leptin treated, and ob/ob mice. n.b. A subset of this data is displayed in Table 4.3.

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TRAP-Seq was performed on LepRbeGFP-L10a mice treated for 10 hours with leptin (5mg/kg i.p) or vehicle, on LepRbeGFP-L10aob/ob mice or on LepRbeGFP-L10a mice fed a high fat diet for 8-9 weeks. (Top) Fold change in DIO mice vs fold change in 10 hour leptin treatment. (Middle) Inverse fold change of ob/ob vs fold change in 10 hour leptin treatment. (Bottom) Fold change in DIO vs inverse fold change of ob/ob. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also significantly changed, were included in this analysis. Fold change values for 10 hour leptin treatment in LepRbeGFP-L10a, for ob/ob, and for DIO, is versus 10 hour vehicle treated LepRbeGFP-L10a. For ease of interpretation, the inverse of the fold change in ob/ob is plotted here. Dashed lines are at FC=1.5 and FC=0.667 for all axes. n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult animals.

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Figure 4.3 Conditional ablation of STAT3 in LepRb neurons. (A) Leprcre mediates the excision of exons 18-20 of the STAT3 gene, resulting in STAT3 ablation from LepRb neurons (STAT3LepRKO mice). (B) Representative images showing colocalization of pSTAT3-IR (red) and GFP-IR (green) in the PMv of STAT3LepRKO and STAT3LepRWT (both of which also contain Rosa26eGFP-L10a) mice treated with leptin (5mg/kg i.p.) for 90

125 minutes. Arrows indicate colocalized neurons. (C) STAT3LepRKO and STAT3LepRWT underwent body composition analysis prior to dissection. Terminal serum from fed STAT3LepRKO and STAT3LepRWT mice was assayed for leptin (D) and insulin (E). (n=15 per group). Mean-/+ SEM is shown for (C-E). ***p<0.001 by t-test. ****p<.0001 by t-test.

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Figure 4.4: Fold change in LepRb-enriched genes in STAT3LepRKO vs ob/ob mice. A subset of this data, with corresponding values appears in Table 4.4 (A-E) and Table 4.5 (F) TRAP-Seq was performed on STAT3LepRKO and STAT3LepRWT and LepRbeGFP- L10aob/ob mice. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also significantly changed, were included in this analysis. Fold change values for STAT3LepRKO mice were versus STAT3LepRWT controls. Fold change values for LepRbeGFP-L10aob/ob are versus 10 hour vehicle treated LepRbeGFP-L10a. n=3-4 samples per treatment group. Dashed lines are at FC=1.5 and FC=0.667 for both axes. Each sample was comprised of pooled hypothalami of 4-6 adult animals. p<.05 for values in bold and italics.

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STAT3 vs Gene Enrichment FPKM ob/ob Stat3LepRKO ob/ob Figure 4.4 Agrp 75.34 229.60 5.55 3.73 0.73 Npy 24.98 1152.79 4.17 3.39 0.86 Fam159a 4.79 6.09 2.94 2.25 0.88 Rbp4 2.05 10.30 1.92 1.74 0.93 A Ccl17 1.73 1.81 2.62 4.32 0.85 Maff 1.47 1.37 1.88 2.18 0.83 Gdpd3 1.34 4.79 1.54 2.06 0.90 Pomc 37.76 539.53 0.16 0.28 1.39 Cartpt 11.37 661.42 0.28 0.35 1.07 Nts 5.39 113.14 0.41 0.37 0.93 Tmem176a 3.92 32.57 0.40 0.57 1.38 B Serpina3n 13.18 69.85 0.26 0.51 1.97 Npy2r 3.72 18.83 0.44 0.61 1.78 Tuba1c 1.42 10.46 0.37 0.15 0.21 Fosl2 0.95 2.31 3.26 1.50 0.65 Fosb 1.32 1.05 3.20 1.31 0.38 Ctla2a 0.82 2.47 2.97 1.19 0.52 Ghrh 11.83 56.26 2.66 1.08 0.36 C Procr 4.30 1.18 2.45 0.42 0.20 Gem 1.76 1.91 1.67 1.12 0.50 Camk1g 2.41 31.35 1.53 1.15 0.64 Serpina3i 31.59 0.58 0.13 1.37 6.18 Socs3 2.51 1.94 0.25 1.05 3.65 Atg7 8.52 61.48 0.42 0.68 1.83 Stat3 2.61 31.53 0.52 0.87 1.63 D Vwa5a 2.27 6.88 0.52 0.99 2.04 Plagl1 2.97 33.71 0.55 0.85 1.54 Tac1 4.55 301.96 0.57 0.83 1.72 Yeats2 5.46 37.38 0.62 0.78 1.51 Stat1 1.43 5.84 0.56 3.49 6.02 E Irf9 2.19 7.68 0.38 2.20 5.69

Table 4.4: Fold change in LepRb-enriched genes in STAT3LepRKO and ob/ob mice. TRAP-Seq was performed on STAT3LepRKO and STAT3LepRWT and LepRbeGFP- L10aob/ob mice. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also significantly

128 changed, were included in this analysis. Enrichment and expression (FPKM) values displayed are from STAT3LepRWT mice. Fold change values for STAT3LepRKO mice were versus STAT3LepRWT controls (Column 5), or against LepRbeGFP-L10aob/ob (Column 6). Fold change values for LepRbeGFP-L10aob/ob (Column 4) are versus 10 hour vehicle treated LepRbeGFP-L10a. (Column 7) designates the corresponding section of Figure 4.4 n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult animals. p<.05 for values in bold and italics. FPKM: Fragments Per Kilobase of exon per Million reads mapped.

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STAT3 vs Figure Gene Enrichment FPKM ob/ob Stat3LepRKO ob/ob 4.4 Bst2 1.11 1.45 1.30 49.14 47.84 Ifi47 2.23 0.42 0.48 49.06 152.81 Gm4951 0.44 0.09 1.26 39.21 37.92 Iigp1 0.81 0.23 0.60 33.53 90.96 Isg15 0.42 1.03 0.53 31.03 51.26 Serpina3f 2.56 0.20 0.27 29.80 33.49 Ifit1 0.72 0.67 0.92 29.69 39.38 Gbp3 0.82 0.53 0.54 27.82 90.13 Rtp4 0.64 0.68 1.02 25.99 21.21 Oasl2 1.31 1.40 1.04 23.57 24.70 Irgm2 0.98 0.65 0.59 22.91 51.99 H2-Q6 17.57 0.48 0.34 19.77 60.79 H2-Q7 2.44 0.73 0.41 16.87 23.98 Mpa2l 1.63 0.63 0.61 16.03 30.60 Igtp 1.83 1.76 0.64 15.40 25.13 Ifi44 1.70 0.13 0.91 15.12 28.50 Gbp4 1.58 0.21 0.38 14.57 48.45

Psmb8 2.37 3.10 0.80 13.81 17.82

Usp18 1.32 0.48 0.57 13.53 20.30

Gm4841 0.54 0.12 1.03 11.99 13.57

Psmb9 1.11 1.50 0.61 11.95 15.98 F H2-K1 1.15 4.13 0.81 10.96 16.51

H2-Gs10 1.31 1.53 0.98 9.50 11.07 Parp10 0.86 0.53 0.95 9.23 8.64 Apol9a 1.87 0.14 1.75 8.08 6.57 Tap1 2.04 0.83 0.91 7.28 9.08 Irgm1 1.10 5.68 1.05 7.24 7.65 H2-Q8 6.44 0.62 0.65 6.64 5.66 Ifit3 0.51 1.43 1.07 6.37 6.49 B2m 1.77 60.49 0.82 6.09 10.66 Irf7 1.66 0.97 1.09 5.68 4.33 Il18bp 0.32 0.65 2.28 5.62 4.37 Gbp9 2.58 0.46 0.51 5.45 17.25 Gbp2 1.24 0.51 1.00 5.41 9.18 Uba7 1.87 0.28 0.55 5.31 12.35 Ifi35 1.84 2.22 0.79 5.23 6.16 Xaf1 0.39 1.44 0.81 4.96 8.68 Apol6 0.94 0.37 1.12 4.82 3.92 Oas1b 1.73 0.45 1.06 4.66 5.14 Rsad2 2.19 0.34 1.22 4.59 3.04

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Samd9l 0.65 0.34 1.51 4.44 5.80 Casp1 0.54 0.37 0.90 4.39 4.66 9230105E10Rik 0.40 0.34 1.61 4.28 4.97 Irf8 0.40 0.59 0.97 4.21 5.71 Trim21 1.30 1.35 0.68 4.08 4.67 Gbp6 0.95 0.48 0.69 3.99 6.93 Cd274 2.51 2.52 0.84 3.87 5.49 H2-T23 1.90 6.47 1.31 3.65 2.87 Eif2ak2 1.20 2.51 1.15 3.52 3.06 I830012O16Rik 0.60 0.95 0.87 3.42 3.76 Dtx3l 1.28 0.96 0.85 3.33 5.22 H2-Aa 0.44 0.76 0.95 3.32 5.27 F Dhx58 0.68 0.51 0.83 3.26 3.13 Ube2l6 1.33 6.04 1.05 2.96 2.84 Ifit2 0.64 3.66 1.14 2.93 2.93 Irf1 1.88 4.24 0.71 2.91 3.76 Ddx58 1.11 0.89 1.25 2.81 2.76 H2-D1 2.22 17.38 0.91 2.77 3.19 Zc3hav1 1.33 0.59 0.81 2.42 3.11 Gm6548 1.33 2.75 1.13 2.06 1.91 Nmi 0.96 2.21 0.76 2.02 2.65 Psmb10 1.48 45.60 1.02 1.93 1.56 Lgals3bp 2.57 8.76 0.90 1.89 2.14 Trim25 1.59 1.68 1.00 1.82 1.78 Cish 1.25 3.78 0.67 1.78 2.06 Samhd1 1.36 7.23 1.06 1.78 1.75 Stat2 0.81 3.08 0.91 1.67 1.79 Tapbp 1.75 10.48 0.95 1.63 1.82 Psme2 2.11 102.53 0.87 1.59 1.79 Pcsk1n 2.40 401.14 0.98 0.62 0.49 Gm5779 1.71 2.17 0.52 0.11 0.14

Table 4.5: Fold change in LepRb-enriched genes in STAT3LepRKO and ob/ob mice. This data also appears in Figure 4.4 as group (F). TRAP-Seq was performed on STAT3LepRKO and STAT3LepRWT and LepRbeGFP-L10aob/ob mice. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also significantly changed, were included in this analysis. Enrichment and expression (FPKM) values displayed are from STAT3LepRWT mice. Fold change values for STAT3LepRKO mice were versus STAT3LepRWT controls (Column 5),

131 or against LepRbeGFP-L10aob/ob (Column 6). Fold change values for LepRbeGFP-L10aob/ob (Column 4) are versus 10 hour vehicle treated LepRbeGFP-L10a. n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult animals. p<.05 for values in bold and italics. FPKM: Fragments Per Kilobase of exon per Million reads mapped.

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Fold Change in TRAP Fold Change in Arcuate

Gene Enrichment ob/ob STAT3LepRKO ob/ob STAT3LepRKO Leprs/s Agrp 75.34 5.55 3.73 1.75 3.28 3.87 Stat1 1.43 0.56 3.49 0.73 1.46 0.96 Stat3 2.61 0.52 0.87 0.79 0.94 0.89 Psmb8 2.37 0.80 13.81 1.74 4.65 1.4 Irf1 1.88 0.71 2.91 0.98 1.51* 0.78 Irf9 2.19 0.38 2.20 0.66 1.2 0.67 Gch1 8.99 0.51 0.81 0.57 1.04 1.03 Socs3 2.51 0.25 1.05 0.48 0.76 1.34

Table 4.6: Fold change in ob/ob, STAT3LepRKO, and Leprs/s mice as determined by TRAP or qPCR. TRAP-Seq was performed on STAT3LepRKO and STAT3LepRWT and LepRbeGFP-L10aob/ob mice. Genes enriched (FPKM in TRAP/FPKM in TRAP-depleted >1.5) at baseline, or that became enriched in a condition in which they were also significantly changed, were included in this analysis. Enrichment and expression (FPKM) values displayed are from STAT3LepRWT mice. Fold change values as determined by RNA-Seq for STAT3LepRKO mice were versus STAT3LepRWT controls (Column 4), Fold change values as determined by RNA-Seq for LepRbeGFP-L10aob/ob (Column 3) are versus 10 hour vehicle treated LepRbeGFP-L10a. n=3-4 samples per treatment group. Each sample was comprised of pooled hypothalami of 4-6 adult animals. Whole RNA was also isolated from the arcuate nuclei of adult ob/ob and c57bl6 controls, Leprs/s and Lepr+/+ controls, and STAT3LepRKO and STAT3LepRWT mice (n=8-10 mice per group). Changes in transcript expression were assayed by RT-qPCR using ABI Taqman assays for the listed genes, and Tbp as an endogenous control. p<.05 for values in bold and italics. *p=0.053.

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CHAPTER FIVE

CONCLUSIONS AND FUTURE DIRECTIONS

Novel populations of LepRb neurons

The work presented in Chapter 2 of this dissertation identified a score of new populations of LepRb neurons that may be critical for the control of energy balance by leptin. We explored the metabolic function of one of these populations, LepRbPdyn neurons, and determined that these neurons contributed to the regulation of energy expenditure by leptin. Other newly identified LepRb populations may also be essential for functions of leptin not otherwise discussed in this dissertation, including the regulation of growth, glucose homeostasis, reproduction, or stress. Thus these data provide new routes for exploration in parsing LepRb subpopulations, as well as identifying the outputs they collectively and individually control.

One limitation of the TRAP-Seq approach is that it fails to identify those transcripts which are present in LepRb neurons but are broadly expressed in multiple hypothalamic or brainstem populations. For example, while studies have identified both

Vgat and Nos1 neurons as critical LepRb subpopulations for the control of energy balance, neither of these transcripts was found to be enriched in hypothalamic or brainstem LepRb neurons[1, 2]. One solution to identifying broadly expressed transcripts which are also expressed in LepRb neurons might be to translationally profile a variety of hypothalamic cell types. The ability to perform multiple comparisons would

139 allow for identification of broadly expressed transcripts that are nonetheless preferentially expressed in LepRb neurons (or other neurons of interest). For example, if we were to perform TRAP-Seq on SF-1 (i.e. VMH) neurons, which are glutamatergic and do not express Nos1, both Vgat and Nos1 may enrich in hypothalamic LepRb neurons by comparison. In the future, the generation of a “TRAP-seq database” composed of translational profiling data from multiple CNS populations (both neuronal and non-neuronal) may be highly valuable for exploring the unique transcriptomic identities of cell populations throughout the hypothalamus and brainstem which may be critical for regulating multiple aspects of physiology.

There is a small subset of genes identified as enriched in LepRb neurons in the hypothalamus and hindbrain that I believe are particularly worthy of further exploration, including those LepRb neurons expressing Growth Hormone Releasing Hormone

(GHRH). Leptin is known to be a permissive signal for linear growth, but while ob/ob mice are short, Leprs/s mice are long-bodied [3-5]. The mechanisms for these divergent phenotypes are unclear, and highlight the need to improve our understanding of leptin’s regulation of growth and development. GHRH was highly enriched in hypothalamic

LepRb neurons, and thus LepRbGhrh neurons may represent a critical site for the integration of leptin signaling with other determinants of linear growth. Unfortunately, many of the tools required for the exploration of novel LepRb subpopulations remain to be generated. As a mouse lab, our work is biased toward the use of genetic mouse models to explore these questions, although multiple routes could be taken to explore the function of these LepRb subpopulations. In order to start addressing the physiology of GHRH and leptin, I have generated a line of mice expressing Cre recombinase only

140 in GHRH neurons (GhrhCre). It is my hope that this mouse line may be useful for further explorations of leptin signaling, as it may be used to both identify GHRH expressing neurons (which are challenging to identify by IHC techniques), as well as to ablate

LepRb specifically from GHRH neurons.

Another novel population that may be worthy of further exploration are those

LepRb neurons that express neuropeptide W (NPW). NPW has long been known to be an anorexigenic neuropeptide, and has been shown to be expressed in the VMH, LHA, and Arc, as well as a number of hindbrain regions [6]. Our data reveals that Npw is enriched in both hypothalamic and brainstem LepRb neurons, although the sites of colocalization are unclear. Our data also revealed that Npw is upregulated in hypothalamic LepRb neurons in DIO mice, although it is not clear if Npw is responding to increases in leptin specifically, or to other adiposity signals. The potential roles for both brainstem and hypothalamic NPW-expressing LepRb neurons is also intriguing given the association shown between NPW and stress [7, 8]. Leptin is an anxiolytic and thus NPW neurons may represent a site for the integrated control of metabolic and stress responses by leptin [9, 10]. In the future, generation of NPWcre mice may aid in identifying where NPW is expressed throughout the central nervous system, as well as exploring the role of leptin in regulating these neurons.

My hope is that other researchers, both inside and outside of our research group, will use the list of LepRb enriched and de-enriched genes identified in this dissertation to further our knowledge of leptin action, not only in the hypothalamus, but also in less well studied brainstem LepRb neurons. The rapid adoption of CRISPR/Cas9 technologies may accelerate our collective abilities to answer these questions, and may

141 also allow higher throughput explorations of the multiple subpopulations of LepRb neurons we have identified here.

Acute responses to leptin and the role of ATF3

Whereas the work presented in Chapter 2 allowed us to define new populations of LepRb neurons, the work presented in Chapters 3 allowed us to identify and confirm new gene targets of acute leptin/LepRb signaling. In particular, we identified a number of cytokine signaling inhibitors that may be critical for negative inhibition of LepRb signaling, as well as a number of transcription factors that may mediate the early phase cellular responses to leptin signaling. Some of these transcription factors, including Atf3,

Arid5a, and Etv6, have not previously been reported to be induced by leptin, and thus represent novel leptin response genes. This data also revealed that most neuropeptide transcription/translation is not altered in response to short term leptin treatment.

One limitation of acute pharmacological leptin treatment is that it does not accurately recapitulate the physiology of leptin signaling either in dosage or in signaling duration. Thus it is possible that many of the genes observed to be upregulated following bolus leptin treatment (which achieves concentrations 1000-fold higher than normal physiology) are a form of cellular stress response rather than an accurate response to physiological leptin [11]. While there is no easy replacement for pharmacological studies, in the future it may be valuable to investigate the transcriptional effects of lower dose leptin treatment in free feeding mice. It may also help to look at the effects of low dose, short term leptin infusion in ob/ob mice, as this may best recapitulate physiological leptin signaling.

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Another limitation of our studies of leptin treatment is the necessity of evaluating all hypothalamic LepRb neurons en masse. Not only do multiple LepRb populations respond very differently to leptin (with leptin-activated POMC and leptin-inhibited AgRP neurons being the prototypical examples), but additionally different LepRb subpopulations produce unique gene products (whether neuropeptides or fast neurotransmitters) which may also be differentially regulated by leptin. Thus is it unclear if the transcripts that we observe to change in response to leptin treatment in hypothalamic LepRb neurons are modestly altered in all LepRb subpopulations, or robustly altered in one particular LepRb subpopulation. This ambiguity was demonstrated in our own studies, as we observed leptin induced upregulation of ATF3 in the Arc and DMH, but not the VMH or PMv, suggesting that ATF3 is not a ‘universal’ leptin response gene, despite its robust response to leptin as measured in our TRAP-

Seq data. In the future, it may be necessary to repeat these experiments in individual hypothalamic nuclei (by microdissection), or in individual cell populations (by using different Cre-drivers to induce eGFP-tagged ribosome expression) in order to determine the full extent of hypothalamic leptin action.

Despite the limitations described above, our TRAP-Seq data, combined with IHC analysis, nonetheless identified Atf3 as a novel leptin response gene. Given the preferential expression of ATF3 in the Arc and DMH, hypothalamic nuclei thought to be most critical for the regulation of body weight by leptin, the role of ATF3 in leptin action is certainly worthy of pursuit [1, 12, 13]. To this end, we have obtained Atf3flox mice from

Dr. Tsonwin Hai, and are currently working to ablate ATF3 from all LepRb neurons through use with our Leprcre model (ATF3LepRKO mice). This analysis will allow us to

143 determine the necessity of ATF3 for the control of energy balance by leptin.

Furthermore, we are working to generate ATF3LepRKO mice that also contain the

Rosa26eGFP-L10a allele, to allow us to determine how loss of ATF3 may affect gene expression in hypothalamic LepRb neurons. I expect ATF3LepRKO mice to be obese and hyperphagic, although not to the same degree as STAT3LepRKO mice. If this hypothesis is supported by our studies, it may also be valuable to perform ChIP experiments against both ATF3 and STAT3 to determine which genes may be concordantly regulated by these two transcription factors.

Chronic responses to leptin

The experiments described in Chapter 4 allowed us to examine the full extent of regulation of the LepRb transcriptome, by observing changes in response to physiological and pharmacological leptin excess, as well as to leptin deficiency. These experiments also allowed us to assess the necessity of LepRb-STAT3 signaling for the expression of leptin target genes. Together, this analysis revealed that leptin regulates neuropeptide expression over an extended time course, and that this regulation is

STAT3 dependent. Once again, these experiments had certain limitations, including the use of pharmacological leptin and the examination of hypothalamic LepRb neurons in toto, solutions for which are discussed above. Despite these reservations however, our analysis revealed a cohort of new gene targets of leptin which may be critical mediators of leptin action, including the Serpina3 family, transcription factors Atf3 and JunB

(discussed above), and intracellular proteins such as Asb4.

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The Serpina3 family of genes remains intriguing for many of the reasons outlined in Chapter 4. As a set of genes potently regulated by leptin and by LepRb-STAT3 signaling specifically, there is a high likelihood that these genes are relevant for the control of energy balance. Their identity as protease inhibitors, as well as their coexpression in peptidergic POMC and AgRP neurons, may indicate that these genes are critical regulators of neuropeptide production, secretion, or maintenance (in the intracellular or extracellular space), and thus may be ideal targets for pharmacotherapy development. Future efforts will hopefully be directed at developing mice in which certain key isoforms (in particular Serpina3h, Serpina3i and Serpina3n) are floxed, allowing for LepRb specific deletion of these potentially critical genes, and assessment of the resulting metabolic phenotype.

Asb4 is particularly intriguing due to its possible associations with human obesity.

Not only ASB4 has been linked to obesity via GWAS, our data suggests it is highly regulated by leptin [14]. It is also known to be expressed in a variety of cell types important for the control of metabolism, including POMC and AgRP neurons [15-17].

Similar to ATF3, although ASB4 was not observed to change in STAT3LepRKO mice, its induction in hyperleptinemic DIO but not STAT3LepRKO mice may indicate that it is in fact a LepRb-STAT3 regulated gene. Furthermore, ASB4 has the potential to regulate

LepRb neuron biology in interesting ways. As a substrate-recognition subunit of the E3- ubiquitin ligase complex, ASB4 may target multiple intracellular proteins for degradation

[18]. Thus induction of ASB4 by leptin may impinge on a number of intracellular signaling pathways to regulate overall LepRb neuron physiology. We are currently working to generate Asb4Flox mice in order to achieve LepRb specific ablation of ASB4.

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The role of STAT3 in regulation of the LepRb transcriptome

In addition to identifying a cohort of genes regulated cell-autonomously by leptin, the work presented in Chapter 4 also allowed us to start querying the mechanisms for this regulation. The TRAP-Seq data from STAT3LepRKO mice, combined with our previous data from LepRbeGFP-L10a;ob/ob mice allowed us to identify those genes which require LepRb-STAT3 signaling for their expression. Many of these genes had previously been identified as STAT3-responsive, including Pomc, Agrp, Npy and Cartpt, but our methods allowed us to confirm that this regulation was cell autonomous and occurs in vivo. Our studies also allowed us to identify new genes which have not been reported to be LepRb-STAT3 regulated, such as Ccl17, Gdpd3 and Tmem176a, which may be important for the control of energy balance by leptin. The role of these genes, including their expression patterns and functional targets, may be of considerable interest to future researchers.

While the STAT3LepRKO data revealed the necessity of STAT3 signaling for the expression of certain gene targets, the use of the STAT3LepRKO mice did not allow us to determine if multiple signaling pathways might be required for expression of a given target gene in response to leptin. Other studies have demonstrated, for example, that

Agrp and Npy which show increased expression in STAT3LepRKO mice, also require

PI3K signaling for their regulation by leptin, although it is not clear if the requirement for

PI3K signaling is cell autonomous[19]. Thus, the question still remains, is LepRb-STAT3 signaling sufficient to rescue the obese phenotype of db/db mice, and if so, what are the target genes necessary to achieve this effect?

146

From a technical point of view, it has been challenging to address this question, as use of the mouse models with homozygous mutant LepR alleles (Leprs/s, Leprl/l and

Leprf/f) preclude the expression of cre from the LepRb locus. Recent developments in

CRISPR/Cas9 technology however would allow us to introduce these site-specific mutations into the LeprCre allele, and thus perform LepRb-specific TRAP-seq in these various models. Leprs-cre/s-cre mice would allow us to examine the role of LepRb-STAT3 signaling specifically, without the confounding effects on gene transcription (attributed to

STAT1) observed by the total ablation of STAT3 in the STAT3LepRKO model.

Furthermore, use of both the Leprl-cre/l-cre and Leprf-cre/f-cre (or, even better, a combined

Leprlf-cre/lf-cre) mice would be of particular utility for addressing the sufficiency of LepRb-

STAT3 signaling for the maintenance of many of these gene targets. As an added benefit, double mutant Leprlf-cre/lf-cre mice would not be expected to be hyperleptinemic at baseline, and thus leptin-induced gene expression could also be evaluated in this model. This work would allow us to finally link the proximal signaling pathways engaged by leptin with the distal gene targets they control, and thus provide considerable insight into the physiology of LepRb neurons.

Conclusions

Since the discovery of leptin more than 20 years ago, we have made considerable progress in our understanding of leptin biology. On the level of the whole animal, we understand why leptin is secreted, where it acts, and what aspects of physiology it helps control. On a tissue level however, we are still working to identify which cells respond to leptin, how they communicate with each other, and what neural networks may be engaged by leptin action. Similarly, on a cellular level, we are still

147 working to understand the physiology of the leptin receptor, what signaling pathways it engages, and what target genes are modulated by these pathways. Thus despite the breadth of our understanding of leptin’s control physiology, we still lack the depth of knowledge necessary to assign cellular and molecular mechanisms for this control.

My hope is that the data presented in this dissertation, by defining new populations of LepRb neurons, by identifying many of the receptors, neuropeptides, ion channels, and intracellular signaling proteins expressed in these neurons, and by revealing those proteins whose expression is specifically regulated by leptin, provides future researchers with multiple avenues of exploration to further our understanding of the cellular and molecular mechanisms of leptin action.

References

1. Vong, L., et al., Leptin Action on GABAergic Neurons Prevents Obesity and Reduces Inhibitory Tone to POMC Neurons. Neuron, 2011. 71(1): p. 142-54. 2. Leshan, R.L., et al., Leptin action through hypothalamic nitric oxide synthase-1-expressing neurons controls energy balance. Nat Med, 2012. 3. Bates, S.H., et al., STAT3 signaling is required for leptin regulation of energy balance but not reproduction. Nature, 2003. 421: p. 856-859. 4. Bates, S.H. and M.G. Myers, Jr., The role of leptin receptor signaling in feeding and neuroendocrine function. Trends Endocrinol.Metab, 2003. 14(10): p. 447-452. 5. Bates, S.H., et al., LRb-STAT3 signaling is required for the neuroendocrine regulation of energy expenditure by leptin. Diabetes, 2004. 53(12): p. 3067-3073. 6. Takenoya, F., et al., Distribution of neuropeptide W in the rat brain. Neuropeptides, 2010. 44(2): p. 99-106. 7. Takenoya, F., et al., Neuropeptide W-Induced Hypophagia is Mediated Through Corticotropin- Releasing Hormone-Containing Neurons. J Mol Neurosci, 2015. 8. Beck, B., C. Bossenmeyer-Pourie, and G. Pourie, Association of neuropeptide W, but not obestatin, with energy intake and endocrine status in Zucker rats. A new player in long-term stress-feeding interactions. Appetite, 2010. 55(2): p. 319-24. 9. Niimi, M. and K. Murao, Neuropeptide W as a stress mediator in the hypothalamus. Endocrine, 2005. 27(1): p. 51-4.

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10. Lu, X.Y., et al., Leptin: a potential novel antidepressant. Proc.Natl.Acad.Sci.U.S.A, 2006. 103(5): p. 1593-1598. 11. Faouzi, M., et al., Differential Accessibility of Circulating Leptin to Individual Hypothalamic Sites. Endocrinology, 2007. 12. Rezai-Zadeh, K., et al., Leptin receptor neurons in the dorsomedial hypothalamus are key regulators of energy expenditure and body weight, but not food intake. Mol Metab, 2014. 3(7): p. 681-93. 13. Krashes, M.J., et al., An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature, 2014. 507(7491): p. 238-42. 14. Locke, A.E., et al., Genetic studies of body mass index yield new insights for obesity biology. Nature, 2015. 518(7538): p. 197-206. 15. Li, J.Y., et al., Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits. J Neuroendocrinol, 2005. 17(6): p. 394-404. 16. Li, J.Y., et al., Expression of ankyrin repeat and suppressor of cytokine signaling box protein 4 (Asb-4) in proopiomelanocortin neurons of the arcuate nucleus of mice produces a hyperphagic, lean phenotype. Endocrinology, 2010. 151(1): p. 134-42. 17. Li, J.Y., et al., Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci, 2011. 12: p. 95. 18. Andresen, C.A., et al., Protein interaction screening for the ankyrin repeats and suppressor of cytokine signaling (SOCS) box (ASB) family identify Asb11 as a novel endoplasmic reticulum resident ubiquitin ligase. J Biol Chem, 2014. 289(4): p. 2043-54. 19. Morrison, C.D., et al., Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am.J Physiol Endocrinol.Metab, 2005. 289(6): p. E1051-E1057.

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APPENDIX 1

Highly enriched genes in hypothalamic LepRb neurons

Hypothalamic blocks from 4 cohorts (4-6 mice per cohort) of adult LepRbeGFP-L10a mice were subjected to TRAP. TRAP-enriched and depleted RNA fractions were converted to cDNA libraries prior to running on the Illumina HiSeq2000 sequencing platform. Reads were mapped to mouse genome mm9 for analysis. Transcript levels were compared between the TRAP-enriched and depleted fractions to determine fold enrichment in the LepR-specific (TRAP) fraction. The table shows the gene symbol, expression (in FPKM) in TRAP-enriched (LepR) and TRAP-depleted (Non-LepR) fraction, fold enrichment, and NCBI gene ID for all genes enriched greater than 1.5-fold in the TRAP (LepRb) fraction. Some reads were incorrectly mapped to miRNAs; these were excluded from the analysis. Note that ribosomal and mitochondrial genes may be overrepresented in the data set due to their high levels of transcript turnover (which may increase the likelihood of non-specific TRAP enrichment).

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Agrp 254.20 6.07 41.89 11604 Prlh 99.25 3.73 26.58 623503 Npy 1328.71 61.67 21.55 109648 Pomc 1066.65 50.62 21.07 18976 Xist 6.24 0.52 11.95 213742 Galp 0.65 0.06 11.37 232836 Ucn 22.05 2.05 10.74 22226 Serpina3i 1.07 0.10 10.59 628900 Gsx1 13.05 1.25 10.41 14842 Cartpt 1039.21 110.82 9.38 27220 4732456N10Rik 10.48 1.17 8.96 239673 Ghrh 100.17 11.65 8.60 14601 Npw 3.49 0.42 8.26 381073 Tbx19 2.45 0.31 7.87 83993 Serpina3n 98.25 13.36 7.35 20716 Serpina3m 4.02 0.55 7.31 20717 Cited1 211.84 29.43 7.20 12705 Fam150b 1.78 0.25 7.05 100294583 Zar1 9.78 1.44 6.81 317755 Asb4 75.49 11.49 6.57 65255 Serpina3c 0.96 0.15 6.47 16625 Nts 136.55 21.67 6.30 67405 Nags 0.99 0.16 6.14 217214 Tbx3 8.31 1.38 6.02 21386 Serpina3h 4.79 0.80 6.01 546546 Nr5a1 19.09 3.26 5.86 26423 Gm5868 1.62 0.28 5.71 545758 Nr5a2 1.38 0.24 5.66 26424 Sprr1a 1.52 0.27 5.59 20753 Fam83g 0.36 0.07 5.44 69640 Lepr 1.97 0.36 5.44 16847 Cd36 2.54 0.48 5.25 12491 Nr0b1 3.20 0.62 5.15 11614 Procr 2.05 0.41 4.99 19124 Gch1 11.09 2.25 4.94 14528 En1 8.78 1.78 4.93 13798 Bahcc1 12.00 2.44 4.91 268515

151

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Il2rg 0.55 0.11 4.90 16186 3930402G23Rik 0.92 0.19 4.89 665306 Lamb3 6.24 1.28 4.88 16780 Slc6a3 45.90 9.41 4.88 13162 Mc3r 8.20 1.70 4.82 17201 Fam159b 7.01 1.46 4.82 77803 Atf3 3.76 0.80 4.70 11910 Tac1 302.36 64.70 4.67 21333 Irs4 60.98 13.33 4.57 16370 Klf14 0.77 0.17 4.55 619665 Atg7 21.70 4.81 4.51 74244 1700028B04Rik 0.55 0.12 4.47 70001 Nup62cl 2.29 0.51 4.47 279706 Mbnl3 0.76 0.17 4.45 171170 Gucy2c 1.98 0.45 4.34 14917 Abcb1a 1.67 0.39 4.27 18671 Irx6 3.15 0.74 4.25 64379 Flt1 1.91 0.45 4.23 14254 Fn1 1.95 0.47 4.19 14268 H2-Q2 26.43 6.32 4.18 15013 Gpc4 8.88 2.15 4.13 14735 Rnaset2b 1.05 0.25 4.12 68195 AI467606 7.14 1.74 4.10 101602 Rpl10a 2.56 0.63 4.09 19896 Sct 1.91 0.48 4.03 20287 Rrad 28.61 7.12 4.02 56437 Gm833 1.47 0.37 4.01 330004 Calcr 14.88 3.72 4.00 12311 Guca1a 1.27 0.32 3.98 14913 Cacng6 0.45 0.11 3.96 54378 Tnrc18 28.39 7.18 3.95 231861 Foxa1 7.41 1.90 3.90 15375 Mmrn2 0.45 0.12 3.85 105450 Sult5a1 0.76 0.20 3.83 57429 Adh7 0.76 0.20 3.82 11529 Pitx3 3.13 0.83 3.76 18742 Tnfrsf8 4.14 1.11 3.73 21941 Traf3ip3 1.13 0.30 3.71 215243 Slc10a4 11.25 3.04 3.70 231290

152

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Cdhr4 0.80 0.22 3.67 69398 Ddc 129.13 35.28 3.66 13195 Chrna6 7.39 2.02 3.65 11440 Fgf20 0.58 0.16 3.63 80857 Prr19 3.42 0.95 3.60 623131 Tmem8c 1.30 0.36 3.59 66139 Hmx2 5.26 1.47 3.57 15372 Th 162.82 46.73 3.48 21823 Gpx3 456.33 131.38 3.47 14778 Otp 50.10 14.46 3.47 18420 Gal 164.56 47.52 3.46 14419 Elovl3 0.31 0.09 3.46 12686 Nmu 1.24 0.36 3.46 56183 Npy2r 15.28 4.43 3.45 18167 Esyt3 8.61 2.50 3.44 272636 Serpina3f,Serpina 2.54 0.74 3.43 #N/A 3g Tmem176a 41.51 12.21 3.40 66058 Foxd2 2.96 0.87 3.39 17301 Crh 16.33 4.85 3.37 12918 Rps28 2.74 0.82 3.34 54127 Plek2 1.71 0.51 3.32 27260 Prdm12 5.54 1.67 3.32 381359 Corin 0.48 0.15 3.30 53419 Stbd1 2.61 0.81 3.24 52331 Pdyn 53.06 16.46 3.22 18610 Robo4 0.74 0.23 3.22 74144 Btg3 0.80 0.25 3.21 12228 Scpppq1 1.42 0.45 3.17 100271704 Ntn5 0.36 0.11 3.17 243967 Fam183b 86.44 27.42 3.15 75429 2010204K13Rik 406.58 129.53 3.14 68355 Nkx2-4 5.35 1.71 3.12 228731 Hist1h4d 4.45 1.42 3.12 319156 Fam159a 9.30 2.98 3.12 545667 Grp 14.59 4.70 3.11 225642 Socs3 3.61 1.16 3.11 12702 Adcyap1 91.38 29.47 3.10 11516 Nms 4.49 1.46 3.08 433292 Resp18 1561.25 517.25 3.02 19711

153

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID A930018P22Rik 0.36 0.12 3.01 68243 Trpc6 1.49 0.50 2.99 22068 Prok2 3.12 1.04 2.99 50501 Yeats2 23.59 7.91 2.98 208146 Usp51 5.57 1.88 2.97 635253 Gm14492 1.54 0.52 2.97 677289 Spint2 117.67 39.90 2.95 20733 A230065H16Rik 92.77 31.64 2.93 380787 Vill 2.04 0.70 2.92 22351 Cdh5 0.94 0.32 2.92 12562 Mir369,Mir410, 33.86 11.64 2.91 #N/A Mir412,Mirg Tie1 0.80 0.28 2.90 21846 Aldh3b2 13.39 4.62 2.90 621603 Arhgap36 69.83 24.12 2.90 75404 Msln 1.44 0.50 2.88 56047 C1qtnf7 0.57 0.20 2.88 109323 Col5a3 0.47 0.16 2.86 53867 Chrnb3 2.33 0.82 2.85 108043 Tek 0.75 0.26 2.83 21687 Aldh1a7 2.10 0.75 2.82 26358 Krt18 4.75 1.69 2.81 16668 Foxa2 1.85 0.66 2.81 15376 Slc18a2 24.55 8.76 2.80 214084 Gna14 0.68 0.24 2.80 14675 Ghsr 2.35 0.84 2.79 208188 D830015G02Rik 3.30 1.19 2.78 791403 Bcl3 1.49 0.54 2.77 12051 Eltd1 1.19 0.43 2.76 170757 U46068 0.97 0.35 2.75 228801 Slc26a10 0.68 0.25 2.75 216441 Pi16 6.95 2.53 2.75 74116 Pdzd3 1.81 0.66 2.73 170761 Hmx3 7.60 2.79 2.72 15373 Kir3dl2 0.60 0.22 2.72 245615 Slc38a4 1.22 0.45 2.71 69354 Gck 21.11 7.80 2.71 103988 Six6 4.76 1.76 2.70 20476 Cdkn3 2.00 0.74 2.70 72391 Tnfrsf11b 1.31 0.49 2.70 18383

154

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID D330045A20Rik 0.90 0.33 2.69 102871 Ecel1 70.84 26.34 2.69 13599 2310007B03Rik 0.30 0.11 2.69 71874 Ube2t 2.07 0.77 2.69 67196 Prokr1 0.39 0.15 2.68 58182 Pirt 0.79 0.29 2.68 193003 Sema3g 0.66 0.25 2.68 218877 Gse1 28.81 10.79 2.67 382034 Fgf17 1.21 0.45 2.67 14171 Cpa4 1.69 0.64 2.66 71791 Gstp2 1.02 0.39 2.65 14869 AU021092 0.43 0.16 2.65 239691 Gast 3.82 1.45 2.64 14459 Cngb1 2.88 1.09 2.64 333329 Ly6h 737.15 279.88 2.63 23934 Vwf 0.46 0.17 2.63 22371 Sstr5 1.90 0.72 2.63 20609 Rspo4 1.17 0.45 2.62 228770 P2rx2 0.52 0.20 2.61 231602 Bcor 7.78 2.99 2.60 71458 Acbd7 4.37 1.68 2.60 78245 Bcam 18.66 7.21 2.59 57278 Col16a1 2.57 0.99 2.58 107581 Acvrl1 1.20 0.47 2.58 11482 Plagl1 36.92 14.32 2.58 22634 Pcbd1 247.62 96.07 2.58 13180 Rln1 3.14 1.22 2.57 19773 Rbp4 11.88 4.65 2.55 19662 Igbp1b 1.26 0.49 2.55 50540 Tmem176b 72.42 28.43 2.55 65963 Rpl35 5.24 2.06 2.54 66489 Atp6v1c2 4.28 1.69 2.54 68775 Pgr15l 3.66 1.46 2.52 245526 Gabrq 9.77 3.89 2.51 57249 Fezf1 18.22 7.25 2.51 73191 Umodl1 0.65 0.26 2.51 52020 Dmrtc1a 27.22 10.87 2.50 70887 Gnb3 1.53 0.61 2.50 14695 Glp1r 2.08 0.83 2.50 14652

155

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Nnmt 1.91 0.76 2.50 18113 Hnf1b 4.34 1.74 2.50 21410 Cdh29,Uba7 0.87 0.35 2.49 #N/A 2310007A19Rik 25.27 10.20 2.48 66353 Efcab10 19.04 7.70 2.47 75040 BC020535 1.05 0.42 2.47 228788 Cd93 0.32 0.13 2.47 17064 Stat3 39.73 16.08 2.47 20848 Gstm6 51.15 20.70 2.47 14867 Serpine1 1.72 0.70 2.46 18787 Isl1 26.87 10.93 2.46 16392 Dlk1 106.59 43.44 2.45 13386 Ntf3 1.37 0.56 2.45 18205 Bsx 11.59 4.73 2.45 244813 Ret 14.95 6.10 2.45 19713 Ankrd2 3.07 1.26 2.45 56642 Kcna5 12.54 5.13 2.44 16493 H2-Q6 1.20 0.49 2.44 110557 Cldn23 0.34 0.14 2.44 71908 Tm4sf5 6.14 2.52 2.44 75604 Egflam 1.44 0.60 2.42 268780 Col11a2 1.70 0.70 2.42 12815 Ano7 1.14 0.47 2.41 404545 Vwa5a 7.20 3.00 2.40 67776 Ptx4 0.39 0.16 2.40 68509 Crabp1 38.79 16.18 2.40 12903 Stap2 0.49 0.21 2.40 106766 Barhl1 2.81 1.18 2.39 54422 Lipg 0.40 0.17 2.39 16891 Bmp3 0.57 0.24 2.38 110075 Fxyd2 31.23 13.10 2.38 11936 Rtp2 0.61 0.26 2.35 224055 Vgf 210.14 89.39 2.35 381677 BC048546 4.07 1.73 2.35 232400 Ghr 8.27 3.53 2.34 14600 Sncg 218.43 93.33 2.34 20618 Izumo4 47.47 20.33 2.34 71564 Jph2 1.65 0.71 2.34 59091 Cd274 3.38 1.45 2.33 60533

156

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Dgkk 22.96 9.85 2.33 331374 1700045I19Rik 9.98 4.29 2.33 74264 C1qtnf2 3.01 1.29 2.33 69183 Lst1 7.50 3.23 2.33 16988 Slco1a4 3.62 1.56 2.32 28250 She 0.55 0.24 2.31 214547 Rtp1 0.89 0.39 2.31 239766 Il17b 1.15 0.50 2.29 56069 Apoa2 14.28 6.25 2.28 11807 Prlhr 1.09 0.48 2.28 226278 Syt5 168.56 73.99 2.28 53420 4930588N13Rik 0.98 0.43 2.28 75860 Pecam1 1.65 0.72 2.27 18613 1700023E05Rik 0.35 0.15 2.27 71868 Rplp2 327.59 144.58 2.27 67186 Ndn 1694.98 748.37 2.26 17984 H2-Q7 1.26 0.56 2.26 15018 Rsph9 35.30 15.61 2.26 75564 Gem 3.97 1.76 2.26 14579 Rgs2 92.12 40.86 2.25 19735 Rps21 1275.10 566.57 2.25 66481 Cd24a 22.91 10.18 2.25 12484 Cd1d1 1.61 0.72 2.25 12479 Crip2 141.87 63.15 2.25 68337 Mir682 13311.40 5946.01 2.24 751556 Lck 4.30 1.93 2.23 16818 Gpr6 3.75 1.69 2.22 140741 Upk3a 1.17 0.53 2.22 22270 Lmx1b 2.44 1.10 2.22 16917 1700125D06Rik 2.27 1.02 2.22 68233 Vat1 281.86 127.29 2.21 26949 Acta1 12.32 5.57 2.21 11459 6330403K07Rik 1560.50 708.97 2.20 103712 Rpl36a 2.10 0.96 2.20 19982 Kiss1 5.96 2.71 2.20 280287 Camk1g 42.97 19.54 2.20 215303 Zcchc12 511.83 232.89 2.20 72693 Lamb2 1.64 0.75 2.20 16779 Magel2 17.16 7.83 2.19 27385

157

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Gpr101 16.83 7.70 2.19 245424 Rai14 1.92 0.88 2.18 75646 Ngb 139.38 63.87 2.18 64242 Podxl 2.38 1.09 2.18 27205 A930005I04Rik 3.70 1.70 2.18 403174 Snhg12 141.21 64.88 2.18 100039864 Nr4a2 14.01 6.44 2.18 18227 Hk2 1.91 0.88 2.18 15277 Rnf183 0.39 0.18 2.17 76072 Adamts2 5.93 2.73 2.17 216725 Snord104 1324.66 611.09 2.17 100216537 Prss30 0.85 0.39 2.16 30943 Atp1a1 102.61 47.61 2.16 11928 Celf6 99.22 46.04 2.15 76183 LOC100042049 119.63 55.53 2.15 100042049 Crhr2 2.28 1.06 2.15 12922 Rprml 28.45 13.21 2.15 104582 Erg 0.70 0.33 2.15 13876 Cmbl 51.44 23.99 2.14 69574 Irx3 5.96 2.78 2.14 16373 Nop10 223.49 104.39 2.14 66181 S100a10 108.06 50.64 2.13 20194 Junb 35.38 16.58 2.13 16477 Tinagl1 1.30 0.61 2.13 94242 Tgm3 0.45 0.21 2.13 21818 Zfp92 9.76 4.59 2.13 22754 Rpl30 124.70 58.79 2.12 19946 Lhfpl5 29.77 14.05 2.12 328789 A730017C20Rik 347.50 164.23 2.12 225583 Nxnl2 2.12 1.00 2.11 75124 Barx2 6.40 3.03 2.11 12023 A4galt 1.07 0.51 2.11 239559 Arhgap6 27.61 13.11 2.11 11856 Cdhr2 1.69 0.80 2.11 268663 Brs3 2.57 1.22 2.11 12209 1110038B12Rik 56.98 27.06 2.11 68763 Gm4598 1.05 0.50 2.10 100043706 Gpc3 10.30 4.90 2.10 14734 2210013O21Rik 224.68 107.03 2.10 0

158

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Flt3l 1.73 0.82 2.10 14256 Pam16 1.83 0.87 2.10 66449 Rpl41 421.24 201.08 2.09 67945 AW551984 62.35 29.76 2.09 244810 Pcsk1 13.61 6.51 2.09 18548 Fxyd6 311.19 148.87 2.09 59095 Ooep 2.32 1.11 2.09 67968 Scg2 364.81 174.95 2.09 20254 Gchfr 5.29 2.54 2.08 320415 Fgl2 1.32 0.63 2.08 14190 Crhbp 15.84 7.61 2.08 12919 Bex2 1546.42 743.18 2.08 12069 Bex4 35.72 17.18 2.08 406217 Rps18 87.71 42.22 2.08 20084 Rpl37 65.65 31.63 2.08 67281 Hes6 48.55 23.40 2.07 55927 Mpo 0.32 0.15 2.07 17523 Dkkl1 1.31 0.63 2.07 50722 Avpr1a 2.60 1.26 2.07 54140 Mesdc2 37.80 18.30 2.07 67943 Gabre 4.29 2.08 2.06 14404 Etnk2 16.11 7.81 2.06 214253 Chrm5 2.54 1.23 2.06 213788 Chrna5 0.34 0.16 2.06 110835 Sec61b 78.42 38.10 2.06 66212 Maff 3.27 1.59 2.05 17133 Rpl11 1.77 0.86 2.05 67025 Gm1673 264.17 128.76 2.05 381633 Rpl36 37.08 18.10 2.05 54217 Lgals3bp 10.98 5.37 2.04 19039 Styk1 0.54 0.26 2.04 243659 BC051628 0.81 0.40 2.04 332713 Fam167a 4.21 2.06 2.04 219148 Gm4987 2.17 1.06 2.04 245405 Nup98 7.41 3.63 2.04 269966 Pam 86.25 42.29 2.04 18484 Car12 2.05 1.01 2.04 76459 Cldn5 6.10 2.99 2.04 12741 Ly6e 117.34 57.61 2.04 17069

159

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Rps14 681.72 335.57 2.03 20044 Cyb5r1 27.72 13.65 2.03 72017 Hcrtr2 3.08 1.52 2.03 387285 Klhdc8b 38.37 18.93 2.03 78267 Sec14l3 1.71 0.84 2.03 380683 Wdr89 84.23 41.57 2.03 72338 Nodal 0.68 0.34 2.02 18119 Marveld3 0.92 0.45 2.02 73608 Rps19 126.02 62.31 2.02 20085 Kncn 0.61 0.30 2.02 654462 Rpl35a 116.77 57.80 2.02 57808 Hspa1b 1.30 0.64 2.02 15511 Cldn2 0.28 0.14 2.02 12738 Hsd17b2 1.06 0.52 2.02 15486 Npy5r 3.93 1.95 2.02 18168 4933436C20Rik 2.87 1.43 2.01 71296 Sag 0.76 0.38 2.01 20215 Ass1 33.90 16.85 2.01 11898 Slco1c1 3.58 1.78 2.01 58807 Gm5424 86.22 42.92 2.01 432466 Krt77 7.49 3.73 2.01 406220 Gm13253 2.32 1.15 2.01 664903 Hn1 101.99 50.90 2.00 15374 Rpl23 79.61 39.76 2.00 65019 Spag16 0.77 0.39 2.00 66722 Nme2 218.99 109.54 2.00 18103 C2cd4c 8.34 4.18 1.99 237397 Aadat 0.39 0.20 1.99 23923 Ccdc160 11.20 5.62 1.99 434778 Mmp23 1.05 0.53 1.99 26561 Tbca 341.18 171.47 1.99 21371 Pafah1b3 119.35 60.02 1.99 18476 Ntsr1 5.87 2.96 1.99 18216 Rps5 1116.93 562.77 1.98 20103 Pcsk4 4.76 2.40 1.98 18551 Gm766 0.32 0.16 1.98 330440 Psme1 135.44 68.31 1.98 19186 Sox3 8.93 4.50 1.98 20675 Tmem9 100.69 50.83 1.98 66241

160

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Sspo 0.34 0.17 1.98 243369 Irf9 9.59 4.84 1.98 16391 Cryga 1.43 0.72 1.98 12964 Efnb1 6.54 3.30 1.98 13641 Pld3 289.06 146.04 1.98 18807 Qpct 24.25 12.26 1.98 70536 Nucb1 47.01 23.77 1.98 18220 Gsbs 15.20 7.69 1.98 19051 Scml4 4.95 2.51 1.97 268297 Txndc17 73.42 37.22 1.97 52700 Rpl39 115.71 58.70 1.97 67248 Bex1 393.29 199.57 1.97 19716 Rnf128 18.50 9.39 1.97 66889 Rpl22l1 220.47 111.99 1.97 68028 Cmtm8 3.92 1.99 1.97 70031 Rps10 52.46 26.66 1.97 67097 H2-Gs10 1.43 0.73 1.97 15015 Casr 0.33 0.17 1.97 12374 Rxrg 24.86 12.66 1.96 20183 Gm4926 0.35 0.18 1.96 237749 Pltp 12.14 6.21 1.96 18830 Caly 426.04 217.85 1.96 68566 Capsl 5.16 2.64 1.95 75568 Tstd1 3.25 1.67 1.95 226654 Rpl32 612.35 314.68 1.95 19951 Hyi 65.72 33.81 1.94 68180 Gaa 269.65 138.90 1.94 14387 Igsf9 2.02 1.04 1.94 93842 BC029722 260.02 134.21 1.94 613262 Rpl37a 98.89 51.09 1.94 19981 Gzmk 2.52 1.30 1.94 14945 Calca 12.75 6.59 1.93 12310 Palmd 5.23 2.70 1.93 114301 Ctxn1 341.73 177.15 1.93 330695 BC055111 0.30 0.15 1.93 242602 Rpl18 81.43 42.28 1.93 19899 Glra3 4.79 2.49 1.92 110304 Prss12 2.50 1.30 1.92 19142 Aprt 87.56 45.51 1.92 11821

161

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID 1700037H04Rik 252.59 131.31 1.92 67326 Cidea 9.15 4.75 1.92 12683 Smpdl3b 1.75 0.91 1.92 100340 Pla2g2c 0.78 0.41 1.92 18781 Scgn 3.58 1.87 1.92 214189 Ptprb 0.87 0.45 1.92 19263 Il13ra1 5.38 2.82 1.91 16164 Prom1 0.80 0.42 1.91 19126 Krtcap3 2.33 1.22 1.91 69815 2310033P09Rik 37.86 19.85 1.91 67862 Ap1s2 26.21 13.75 1.91 108012 Psme2 24.97 13.10 1.91 19188 Nptx2 28.67 15.06 1.90 53324 Prpf8 36.60 19.24 1.90 192159 C130026L21Rik 3.31 1.74 1.90 330164 Col11a1 1.08 0.57 1.90 12814 Trpc4 4.57 2.41 1.90 22066 Chodl 4.45 2.35 1.90 246048 Fam110a 18.45 9.73 1.90 73847 Pak6 25.88 13.66 1.89 214230 BC037703 0.30 0.16 1.89 242125 Romo1 502.88 265.60 1.89 67067 0610011F06Rik 122.02 64.51 1.89 68347 Rps15 1351.77 714.82 1.89 20054 Ccdc137 23.00 12.17 1.89 67291 Efcab6 1.20 0.63 1.89 77627 Ly6c1 8.53 4.52 1.89 17067 Tceal3 241.25 127.95 1.89 594844 Spint1 1.27 0.67 1.89 20732 Rps8 139.54 74.02 1.88 20116 Magee2 19.22 10.21 1.88 272790 Nme3 125.21 66.51 1.88 79059 Aard 20.27 10.77 1.88 239435 Doc2b 46.53 24.73 1.88 13447 Npy1r 8.81 4.68 1.88 18166 Rac3 126.51 67.29 1.88 170758 Morn2 65.07 34.62 1.88 378462 Rnasek 628.10 334.37 1.88 52898 F2rl2 1.58 0.84 1.88 14064

162

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Baiap3 142.98 76.20 1.88 545192 Rnase6 1.32 0.70 1.88 78416 Gm1027 4.11 2.19 1.88 381538 2210404O07Rik 2.34 1.25 1.87 72273 Diap3 0.51 0.27 1.87 56419 Grhl1 9.09 4.85 1.87 195733 Fam70a 61.22 32.72 1.87 245386 Nradd 1.89 1.01 1.87 67169 4930515G01Rik 0.63 0.34 1.87 67642 Rps25 18.29 9.79 1.87 75617 Ngfrap1 958.23 513.12 1.87 12070 Rpl19 8.66 4.64 1.87 19921 Cnpy2 79.42 42.59 1.86 56530 Zfp575 8.42 4.51 1.86 101544 2310061J03Rik 15.92 8.55 1.86 66391 Rpl9 1.78 0.96 1.86 20005 Arl10 57.47 30.87 1.86 56795 Rxfp3 2.79 1.50 1.86 239336 Bnc2 0.29 0.15 1.86 242509 Rpl38 68.41 36.83 1.86 67671 Pgrmc1 456.73 245.92 1.86 53328 Oaz1 49.73 26.80 1.86 18245 Sox14 3.03 1.64 1.85 20669 Armcx6 7.06 3.81 1.85 278097 Bdnf 10.70 5.77 1.85 12064 Hrh1 0.44 0.24 1.85 15465 1700001L19Rik 21.39 11.55 1.85 69315 Atp2c2 0.61 0.33 1.85 69047 Ssr4 141.91 76.69 1.85 20832 Egr4 4.47 2.42 1.85 13656 Agtr1a 1.04 0.57 1.85 11607 Ctla2a 1.17 0.63 1.85 13024 Rps20 314.67 170.66 1.84 67427 Esam 3.52 1.91 1.84 69524 Atic 35.71 19.41 1.84 108147 H2- 23.49 12.77 1.84 #N/A D1,LOC547349 Rpl34,Rpl34-ps1 151.55 82.45 1.84 68436 Fsip1 1.06 0.58 1.84 71313 Rfx8 0.36 0.20 1.84 619289

163

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID H2-Bl 12.43 6.77 1.84 14963 Tbc1d10c 0.63 0.35 1.83 108995 Rps11 673.97 367.79 1.83 27207 Fchsd1 6.23 3.40 1.83 319262 Rpl26 238.89 130.58 1.83 19941 Rps26 388.33 212.29 1.83 27370 As3mt 38.08 20.84 1.83 57344 Ggnbp1 1.78 0.97 1.83 70772 Wnt5a 4.29 2.35 1.83 22418 BC017612 14.51 7.95 1.83 170748 Scarf2 2.98 1.63 1.82 224024 Tbx6 0.42 0.23 1.82 21389 Meig1 8.80 4.83 1.82 104362 Slc35f2 1.85 1.02 1.82 72022 Rspo1 23.00 12.63 1.82 192199 Rerg 27.35 15.03 1.82 232441 Vgll2 0.70 0.38 1.82 215031 Ppp1r11 159.17 87.46 1.82 76497 Crem 16.92 9.31 1.82 12916 Epb4.1l4a 10.45 5.75 1.82 13824 Erh 6.40 3.52 1.82 13877 Apex1 50.84 28.02 1.81 11792 Hap1 581.05 320.43 1.81 15114 Ets2 46.74 25.79 1.81 23872 Tnfaip8l3 4.91 2.71 1.81 244882 Mcfd2 85.15 47.07 1.81 193813 Adra2a 10.80 5.98 1.81 11551 Rps7 60.49 33.47 1.81 20115 Nop16 40.21 22.25 1.81 28126 Mrpl54 151.14 83.63 1.81 66047 Rps6 4.60 2.55 1.80 20104 Nov 6.63 3.68 1.80 18133 Amigo2 33.46 18.55 1.80 105827 Tmsb10 224.31 124.50 1.80 19240 Itm2c 512.31 284.41 1.80 64294 Tmem158 53.43 29.67 1.80 72309 Psmb8 6.11 3.39 1.80 16913 Rpl8 1230.80 684.25 1.80 26961 Ldha 192.77 107.39 1.80 16828

164

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID 9130206I24Rik 0.48 0.26 1.79 100040736 Nol3 39.11 21.80 1.79 78688 Oxtr 2.95 1.64 1.79 18430 Rpl13a 601.37 335.41 1.79 22121 B3gnt7 1.29 0.72 1.79 227327 Cyb561 55.77 31.13 1.79 13056 Hint1 1111.18 620.29 1.79 15254 D630023F18Rik 5.57 3.11 1.79 98303 Rwdd2a 111.72 62.42 1.79 69519 Rcn1 29.26 16.35 1.79 19672 Tmem35 78.56 43.96 1.79 67564 Polr1d 119.95 67.15 1.79 20018 2610019F03Rik 23.83 13.34 1.79 72148 Sema4f 19.02 10.65 1.79 20355 Tpbg 6.02 3.37 1.79 21983 Vat1l 211.25 118.41 1.78 270097 Ubl5 138.83 77.85 1.78 66177 Rplp1 1078.20 604.78 1.78 56040 Mc4r 1.72 0.97 1.78 17202 1700086L19Rik 50.14 28.14 1.78 74284 Nos3 1.55 0.87 1.78 18127 Cstad 7.27 4.08 1.78 78617 Cpne8 10.72 6.02 1.78 66871 Dnahc1 3.01 1.69 1.78 110084 Slc37a1 3.30 1.85 1.78 224674 1500012F01Rik 175.65 98.74 1.78 68949 Spcs1 174.54 98.13 1.78 69019 Morf4l2 254.72 143.31 1.78 56397 Tceb2 372.28 209.45 1.78 67673 Pgpep1l 2.98 1.67 1.78 78444 Twist2 1.31 0.74 1.78 13345 Srp14 467.11 263.31 1.77 20813 BC006779 0.31 0.17 1.77 229003 Rnaset2b 2.79 1.57 1.77 68195 Gpr149 5.20 2.93 1.77 229357 Tmem66 219.56 123.86 1.77 67887 Mst1r 0.48 0.27 1.77 19882 9130024F11Rik 0.62 0.35 1.77 329160 Tmem130 357.97 202.48 1.77 243339

165

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Nme5 23.49 13.29 1.77 75533 Gbp9 0.41 0.23 1.77 236573 Pdia6 51.63 29.23 1.77 71853 Mrpl52 301.57 170.90 1.76 68836 Panx1 12.13 6.88 1.76 55991 Shisa2 2.97 1.69 1.76 219134 St8sia4 6.16 3.50 1.76 20452 Prtn3 4.44 2.52 1.76 19152 Vim 26.24 14.91 1.76 22352 1110058L19Rik 110.16 62.62 1.76 68002 Pon3 0.61 0.35 1.76 269823 Rab26 45.45 25.85 1.76 328778 Rps12 2.61 1.49 1.76 20042 B630019K06Rik 143.18 81.43 1.76 102941 Mrgpre 10.02 5.70 1.76 244238 Rpl6 87.57 49.87 1.76 19988 Col5a2 0.28 0.16 1.75 12832 Prss35 0.77 0.44 1.75 244954 Rps16 66.30 37.79 1.75 20055 Lmx1a 3.41 1.94 1.75 110648 Rpl3 9.66 5.51 1.75 27367 Capns1 415.82 237.21 1.75 12336 Vstm2l 131.45 75.04 1.75 277432 Marcksl1 153.24 87.50 1.75 17357 Slc2a1 20.61 11.77 1.75 20525 Fam179a 4.85 2.77 1.75 320159 Slc39a11 10.05 5.74 1.75 69806 Tmem106a 0.96 0.55 1.75 217203 2410015M20Rik 210.70 120.39 1.75 224904 Rps3a 63.64 36.36 1.75 20091 Ntn1 3.57 2.04 1.75 18208 Fbll1 97.11 55.53 1.75 237730 Mir703 3052.41 1745.61 1.75 735265 Rps29 17.07 9.76 1.75 20090 Tusc3 142.24 81.35 1.75 80286 Sub1 190.08 108.72 1.75 20024 Rsad2 0.51 0.29 1.75 58185 2610002D18Rik 0.60 0.34 1.75 69885 Lrrc9 1.07 0.61 1.75 78257

166

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Entpd6 47.34 27.11 1.75 12497 Tmem59l 285.16 163.31 1.75 67937 Ptprn 198.65 113.77 1.75 19275 Trim15 1.28 0.74 1.75 69097 Vkorc1 30.16 17.28 1.75 27973 Cenpt 15.36 8.80 1.74 320394 Nenf 246.49 141.28 1.74 66208 Nme4 3.94 2.26 1.74 56520 1810035L17Rik 331.21 189.94 1.74 380773 Cxxc1 30.18 17.32 1.74 74322 Tll1 0.57 0.33 1.74 21892 B2m 81.10 46.57 1.74 12010 Glt8d2 1.16 0.67 1.74 74782 Apoa1 1.60 0.92 1.74 11806 Eng 1.95 1.12 1.74 13805 Rpusd1 60.35 34.72 1.74 106707 Irf1 6.01 3.46 1.74 16362 Ptger2 0.54 0.31 1.74 19217 Hspa5 108.22 62.33 1.74 14828 Stk32a 7.04 4.06 1.74 269019 Rps27 13.12 7.56 1.73 57294 Dnttip2 17.22 9.93 1.73 99480 Epdr1 88.64 51.15 1.73 105298 3110040N11Rik 33.85 19.54 1.73 67290 Lhfpl1 0.44 0.26 1.73 237091 Irx5 2.89 1.67 1.73 54352 Eid2b 35.40 20.44 1.73 434156 Tap2 3.39 1.96 1.73 21355 Rpl24 1.34 0.78 1.73 68193 Magohb 19.96 11.54 1.73 66441 Hebp2 9.72 5.62 1.73 56016 Txnrd3 7.68 4.44 1.73 232223 Tmem179 139.02 80.42 1.73 104885 Gng3 466.56 269.92 1.73 14704 Ptpro 14.08 8.15 1.73 19277 Mapk15 1.58 0.91 1.73 332110 Sec61g 6.25 3.62 1.73 20335 Tmem114 4.12 2.38 1.73 74720 Ifltd1 1.20 0.70 1.73 74071

167

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Rpl36al 112.59 65.26 1.73 66483 Pnma1 8.53 4.94 1.73 70481 Nrsn2 535.15 310.68 1.72 228777 Sdf2 77.86 45.25 1.72 20316 Lrrc6 3.90 2.26 1.72 54562 Sgcz 2.08 1.21 1.72 244431 Cyb5r3 79.19 46.05 1.72 109754 Rab11fip1 0.95 0.55 1.72 75767 Krtap17-1 2.06 1.20 1.72 77914 Atpif1 1119.69 652.23 1.72 11983 Polr2k 19.50 11.36 1.72 17749 Tspan6 23.16 13.52 1.71 56496 Slc39a6 18.65 10.89 1.71 106957 Aldh1a1 76.73 44.84 1.71 11668 Sdc2 35.26 20.61 1.71 15529 Tmed4 75.56 44.17 1.71 103694 Nop58 29.71 17.37 1.71 55989 Dad1 264.92 154.94 1.71 13135 Prr12 24.29 14.21 1.71 233210 Nrl 2.21 1.29 1.71 18185 Ap2s1 593.95 348.26 1.71 232910 Mpa2l 0.30 0.17 1.71 100702 Cetn2 87.94 51.57 1.71 26370 Sst 587.90 344.86 1.70 20604 Myh7 4.03 2.37 1.70 140781 Txn1 192.91 113.21 1.70 22166 Snrpd2 216.07 126.80 1.70 107686 Kcnk2 25.36 14.89 1.70 16526 Atn1,Grcc10 787.16 462.10 1.70 #N/A Timm17b 34.28 20.12 1.70 21855 Rab27a 13.50 7.93 1.70 11891 Mn1 5.22 3.07 1.70 433938 Magee1 122.11 71.80 1.70 107528 Tmem50a 109.91 64.63 1.70 71817 Usp27x 18.82 11.07 1.70 54651 Sstr1 10.90 6.41 1.70 20605 Wbp5 288.09 169.46 1.70 22381 Nme1 414.27 243.70 1.70 18102 Gabra5 33.48 19.70 1.70 110886

168

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Foxq1 0.99 0.58 1.70 15220 Fxyd7 311.61 183.42 1.70 57780 Rps27a 6.41 3.77 1.70 78294 Gm11110 2.05 1.20 1.70 100169874 Myh6 1.81 1.06 1.70 17888 Sec11a 34.28 20.19 1.70 56529 Ttc22 1.53 0.90 1.70 230576 Hspe1 116.59 68.69 1.70 15528 Cyb561d2 13.84 8.15 1.70 56368 Tap1 1.27 0.75 1.70 21354 Qrfpr 3.11 1.83 1.70 229214 Igsf1 14.38 8.48 1.70 209268 Pdlim7 61.03 36.03 1.69 67399 Mrpl33 149.89 88.52 1.69 66845 Lrrn4 0.69 0.41 1.69 320974 Rpl14 178.60 105.58 1.69 67115 Doc2a 43.55 25.74 1.69 13446 Pnma3 28.16 16.65 1.69 245468 Wdr54 24.84 14.69 1.69 75659 Pnck 278.44 164.66 1.69 93843 Ctsl 54.18 32.05 1.69 13039 2210016F16Rik 17.02 10.07 1.69 70153 N4bp2l1 56.83 33.64 1.69 100637 Phf1 62.94 37.27 1.69 21652 Spata24 19.33 11.46 1.69 71242 Mrap2 31.58 18.73 1.69 244958 Slc12a7 0.89 0.53 1.68 20499 F2r 8.95 5.31 1.68 14062 Trappc2l 272.41 161.88 1.68 59005 Lrpap1 78.74 46.80 1.68 16976 Pnmal1 105.63 62.85 1.68 71691 Gstm4 21.74 12.94 1.68 14865 Tmed3 42.03 25.04 1.68 66111 Tm2d3 88.67 52.84 1.68 68634 Mum1l1 5.09 3.03 1.68 245631 Rph3al 7.03 4.19 1.68 380714 Imp3 87.76 52.35 1.68 102462 Cbln1 99.97 59.69 1.67 12404 Ostc 74.83 44.70 1.67 66357

169

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Mboat7 70.76 42.29 1.67 77582 Plcz1 1.27 0.76 1.67 114875 Gm8773 3.77 2.25 1.67 667705 Syt4 82.68 49.48 1.67 20983 Sf3b5 146.83 87.92 1.67 66125 2900062L11Rik 67.69 40.55 1.67 76976 Polr3gl 32.26 19.33 1.67 69870 Wnt7b 7.80 4.67 1.67 22422 Snrpf 33.06 19.81 1.67 69878 Pfdn5 373.46 223.90 1.67 56612 C4b 1.56 0.93 1.67 12268 Maged1 548.70 329.04 1.67 94275 Krtcap2 145.65 87.35 1.67 66059 Rps24 78.16 46.89 1.67 20088 4933433P14Rik 48.00 28.80 1.67 66787 Fosb 3.93 2.36 1.67 14282 Wdr83 51.70 31.04 1.67 67836 Ezr 11.27 6.76 1.67 22350 Btg2 10.05 6.03 1.66 12227 Abcc4 1.06 0.63 1.66 239273 Gpr150 1.59 0.96 1.66 238725 Btg1 8.19 4.92 1.66 12226 Cd200 108.44 65.18 1.66 17470 Sdf2l1 13.71 8.25 1.66 64136 Wdr6 234.14 140.85 1.66 83669 Tmem91 133.99 80.62 1.66 320208 Snrpg 28.83 17.35 1.66 68011 Fam131a 94.36 56.85 1.66 78408 Polr2f 291.33 175.51 1.66 69833 Fank1 8.07 4.86 1.66 66930 Gpx7 5.01 3.02 1.66 67305 Mrpl14 118.37 71.32 1.66 68463 Manf 32.90 19.84 1.66 74840 Itm2a 11.36 6.86 1.66 16431 Rps4y2 65.56 39.57 1.66 66184 1700008P20Rik 1.33 0.81 1.66 69301 Itga2b 1.85 1.11 1.66 16399 Mrps34 135.60 81.90 1.66 79044 Phf13 15.25 9.21 1.66 230936

170

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Ccdc72 38.36 23.20 1.65 66167 Tesc 42.96 25.99 1.65 57816 Dctn1 176.59 106.86 1.65 13191 Gng10 71.61 43.34 1.65 14700 2900010J23Rik 467.85 283.31 1.65 72931 Cited4 8.59 5.20 1.65 56222 Tctex1d2 45.25 27.43 1.65 66061 Stk19 41.74 25.32 1.65 54402 Mmp11 1.53 0.93 1.65 17385 Tomm7 133.69 81.18 1.65 66169 Tmem200a 3.93 2.39 1.65 77220 Snhg1 72.76 44.19 1.65 83673 Ssr2 64.27 39.05 1.65 66256 Rpl28 3.24 1.97 1.65 19943 Hcrtr1 5.94 3.61 1.64 230777 Anxa1 3.53 2.15 1.64 16952 BC056474 77.01 46.88 1.64 414077 Ocln 0.76 0.46 1.64 18260 Ndufa7 546.29 332.76 1.64 66416 Erp29 170.83 104.15 1.64 67397 Rps3 378.89 231.00 1.64 27050 Pcsk1n 679.78 414.61 1.64 30052 Myoc 2.11 1.29 1.64 17926 Ttc9b 109.17 66.59 1.64 73032 Npdc1 211.95 129.31 1.64 18146 Apex2 4.18 2.55 1.64 77622 6720456B07Rik 264.20 161.23 1.64 101314 Cxx1c 602.65 367.81 1.64 72865 Pcdh20 5.54 3.38 1.64 219257 Gm6251 86.77 52.98 1.64 621697 Pvrl3 5.20 3.18 1.64 58998 Rpl18a 32.07 19.58 1.64 76808 Gm6788 5.10 3.12 1.64 627788 Nkx2-1 17.23 10.52 1.64 21869 Trpm4 3.45 2.11 1.64 68667 Creld2 12.50 7.63 1.64 76737 Ajap1 28.04 17.14 1.64 230959 A530016L24Rik 0.37 0.23 1.64 319942 Nudt11 56.75 34.69 1.64 58242

171

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Nat14 55.62 34.04 1.63 269854 Htatip2 16.03 9.81 1.63 53415 Rbm43 25.95 15.89 1.63 71684 Uqcr11 411.99 252.38 1.63 66594 Mfap2 3.34 2.05 1.63 17150 Cd151 18.32 11.23 1.63 12476 Crlf2 16.03 9.83 1.63 57914 Eef1b2 197.20 121.03 1.63 55949 Rps4x 110.98 68.12 1.63 20102 Tmem22 33.66 20.67 1.63 245020 Irak2 7.08 4.35 1.63 108960 Hint2 124.91 76.72 1.63 68917 Mrps14 51.87 31.88 1.63 64659 Tro 48.53 29.85 1.63 56191 Pfdn4 73.75 45.38 1.63 109054 Tomm6 297.39 183.02 1.62 66119 Prss23 0.91 0.56 1.62 76453 Hyou1 28.89 17.80 1.62 12282 Col4a5 0.36 0.22 1.62 12830 Rps27l 52.90 32.61 1.62 67941 Cdh15 2.30 1.42 1.62 12555 Svop 78.57 48.43 1.62 68666 Ctsz 21.45 13.22 1.62 64138 Tceal1 147.98 91.23 1.62 237052 Chchd4 102.54 63.22 1.62 72170 Selm 422.66 260.73 1.62 114679 Entpd3 6.95 4.28 1.62 215446 Ccdc151 3.96 2.45 1.62 77609 Atp6v0c 102.66 63.36 1.62 11984 Pnp2 1.67 1.03 1.62 667034 Xbp1 73.77 45.54 1.62 22433 4930526I15Rik 15.22 9.40 1.62 75135 Bola2 403.40 249.10 1.62 66162 Lemd1 8.29 5.12 1.62 213409 Cd44 2.04 1.26 1.62 12505 Atp6v1f 393.28 243.00 1.62 66144 Med29 9.87 6.10 1.62 67224 Gpr27 13.14 8.12 1.62 14761 Lsmd1 185.97 114.97 1.62 78304

172

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Rgl2 12.03 7.44 1.62 19732 Rpl29 31.07 19.21 1.62 19944 Gm1060 9.77 6.04 1.62 381738 Sh3yl1 16.57 10.25 1.62 24057 Pear1 0.41 0.25 1.62 73182 Rrp7a 78.41 48.52 1.62 74778 Isyna1 49.67 30.74 1.62 71780 Stub1 256.64 158.84 1.62 56424 Gm5617 20.99 12.99 1.62 434402 Dnajc12 61.63 38.16 1.62 30045 Dcun1d5 71.89 44.52 1.61 76863 Ptrh1 10.00 6.19 1.61 329384 Rnaseh2c 73.79 45.74 1.61 68209 Rps9 538.69 333.97 1.61 76846 Cisd3 59.88 37.13 1.61 217149 Drd2 17.57 10.90 1.61 13489 Ssbp4 153.74 95.41 1.61 76900 Atg9b 9.28 5.76 1.61 213948 Cthrc1 11.02 6.84 1.61 68588 Ormdl3 47.00 29.20 1.61 66612 Xdh 0.71 0.44 1.61 22436 2310030G06Rik 4.99 3.10 1.61 66952 Tmie 28.94 18.00 1.61 20776 Rps2 89.44 55.62 1.61 16898 2410006H16Rik 141.18 87.81 1.61 69221 Sntg2 2.74 1.71 1.61 268534 Iscu 157.73 98.16 1.61 66383 Tppp3 341.27 212.44 1.61 67971 Rasal1 22.75 14.16 1.61 19415 Tacstd2 1.33 0.83 1.61 56753 Rpl27a 99.52 62.00 1.61 26451 Snd1 40.39 25.16 1.60 56463 Cadps2 24.79 15.44 1.60 320405 2610029G23Rik 11.96 7.46 1.60 67683 Ccnjl 1.30 0.81 1.60 380694 Elof1 128.59 80.18 1.60 66126 Fhad1 5.66 3.54 1.60 329977 Gja4 1.47 0.92 1.60 14612 C1ql2 14.62 9.13 1.60 226359

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Dmrta2 2.49 1.56 1.60 242620 Nol12 26.95 16.87 1.60 97961 Nol4 33.43 20.93 1.60 319211 Gpr125 3.84 2.41 1.60 70693 Vit 1.64 1.03 1.60 74199 Med31 35.28 22.11 1.60 67279 Slc1a4 19.15 12.00 1.60 55963 Irf2bp1 49.78 31.22 1.59 272359 Fut4 1.24 0.78 1.59 14345 Sgsm1 67.87 42.58 1.59 52850 D17Wsu104e 67.16 42.14 1.59 28106 Rnf208 258.85 162.43 1.59 68846 Ccdc107 43.14 27.08 1.59 622404 6530401D17Rik 24.68 15.49 1.59 76219 Fsd1 55.42 34.79 1.59 240121 Cebpb 9.52 5.98 1.59 12608 1600002K03Rik 8.24 5.18 1.59 69770 Rhov 34.12 21.44 1.59 228543 1810046J19Rik 191.84 120.53 1.59 103742 Zcchc18 367.73 231.16 1.59 66995 Sumf1 15.30 9.62 1.59 58911 Mif 224.35 141.08 1.59 17319 3110082I17Rik 12.41 7.80 1.59 73212 Bola1 42.73 26.88 1.59 69168 Ddost 54.17 34.07 1.59 13200 Ndufv3 527.02 331.52 1.59 78330 Slc16a8 1.77 1.12 1.59 57274 Naca 75.36 47.47 1.59 17938 Magoh 107.65 67.82 1.59 17149 Timm22 31.37 19.76 1.59 56322 Pdia3 76.31 48.10 1.59 14827 Kdr 0.50 0.31 1.59 16542 Timm8b 429.53 270.77 1.59 30057 Hpcal4 154.69 97.56 1.59 170638 Arhgdig 174.72 110.22 1.59 14570 Hax1 15.61 9.85 1.59 23897 Gm16379 35.88 22.66 1.58 100040259 Zbtb12 10.76 6.80 1.58 193736 Hn1l 8.36 5.28 1.58 52009

174

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Tmem43 16.93 10.69 1.58 74122 Dpy30 103.95 65.65 1.58 66310 B3gnt2 13.68 8.64 1.58 53625 Pfdn1 181.73 114.80 1.58 67199 Nudt8 21.44 13.55 1.58 66387 C2cd4d 2.04 1.29 1.58 271944 H2-Ke6 60.50 38.24 1.58 14979 Gng4 73.23 46.29 1.58 14706 Acsl5 19.61 12.40 1.58 433256 Trappc4 132.62 83.87 1.58 60409 2310003F16Rik 320.41 202.63 1.58 67693 Cdh13 27.47 17.38 1.58 12554 Tspyl3 23.81 15.07 1.58 241732 Cdh22 14.96 9.47 1.58 104010 Ube2l6 5.43 3.44 1.58 56791 Kremen2 1.65 1.04 1.58 73016 Gm13889 45.11 28.58 1.58 620695 Rab3b 42.35 26.83 1.58 69908 Dos 305.18 193.37 1.58 100503659 Nudt15 0.44 0.28 1.58 214254 Ache 102.70 65.09 1.58 11423 Med30 51.68 32.76 1.58 69790 Rasgef1c 11.01 6.99 1.58 74563 Slc25a39 159.33 101.09 1.58 68066 Gcnt2 2.34 1.48 1.58 14538 Egfl7 25.33 16.07 1.58 353156 Hmgn1 51.50 32.71 1.57 15312 Nudt10 43.07 27.37 1.57 102954 Tmem185b 10.91 6.93 1.57 226351 Slc9a2 1.29 0.82 1.57 226999 Katnal2 3.29 2.09 1.57 71206 Klhl1 10.27 6.53 1.57 93688 Sv2c 3.93 2.50 1.57 75209 Rpl4 778.06 494.99 1.57 67891 Fkbp11 6.31 4.02 1.57 66120 Lancl3 5.05 3.22 1.57 236285 Eef1a1 903.07 575.21 1.57 13627 Ucp2 27.65 17.62 1.57 22228 Plin5 1.42 0.91 1.57 66968

175

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Zcchc17 134.17 85.49 1.57 619605 Cxcl12 6.53 4.16 1.57 20315 Mmp17 21.06 13.42 1.57 23948 Vip 7.65 4.88 1.57 22353 B3galnt1 37.81 24.12 1.57 26879 Ttc25 1.22 0.78 1.57 74407 Gpr165 14.36 9.17 1.57 76206 Bid 22.13 14.13 1.57 12122 Gm561 143.21 91.49 1.57 228715 Ndufa6 636.49 406.92 1.56 67130 Acpl2 5.24 3.35 1.56 235534 Thyn1 92.07 58.98 1.56 77862 Tmem215 3.66 2.35 1.56 320500 Dpm3 98.74 63.29 1.56 68563 Ifi35 3.03 1.94 1.56 70110 2310044H10Rik 159.16 102.04 1.56 69683 Ushbp1 0.53 0.34 1.56 234395 1110001A16Rik 29.19 18.72 1.56 68554 Mrpl41 43.70 28.03 1.56 107733 Rpl7 107.92 69.26 1.56 19989 Hspa1a 2.78 1.78 1.56 193740 Sssca1 92.97 59.67 1.56 56390 Ndufb2 483.34 310.40 1.56 68198 Arl2 310.86 199.71 1.56 56327 Cst6 1.30 0.83 1.56 73720 Shfm1 406.96 261.69 1.56 20422 Leprel4 20.62 13.26 1.55 66180 Pim2 44.32 28.52 1.55 18715 Susd2 11.12 7.15 1.55 71733 Adamtsl2 1.50 0.97 1.55 77794 Ydjc 32.44 20.88 1.55 69101 Fau 24.66 15.87 1.55 14109 Tapbp 12.25 7.88 1.55 21356 Gm2694 21.40 13.78 1.55 100040294 Ergic3 152.29 98.09 1.55 66366 Mgmt 2.92 1.88 1.55 17314 Vamp4 87.73 56.53 1.55 53330 Fam173a 134.98 86.98 1.55 214917 2410022L05Rik 177.29 114.25 1.55 66423

176

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Ltk 3.92 2.52 1.55 17005 5133401N09Rik 34.01 21.93 1.55 75731 Aamp 230.07 148.35 1.55 227290 Ppap2c 11.62 7.49 1.55 50784 Tagln2 13.78 8.89 1.55 21346 Cd320 5.93 3.83 1.55 54219 Nedd8 588.23 380.13 1.55 18002 Chchd1 146.77 94.88 1.55 66121 Mrpl20 231.14 149.47 1.55 66448 Gbgt1 1.06 0.69 1.55 227671 2810004N23Rik 27.87 18.03 1.55 66523 Cdhr3 0.66 0.43 1.55 68764 Ccdc19 4.65 3.01 1.55 71870 Btf3 76.73 49.65 1.55 218490 Sgsm2 33.53 21.70 1.55 97761 Bcas2 136.96 88.65 1.55 68183 Spag4 4.12 2.66 1.54 245865 Eif3m 54.71 35.42 1.54 98221 Chchd2 177.94 115.23 1.54 14004 Hpcal1 145.70 94.36 1.54 53602 Tmem14c 72.59 47.03 1.54 66154 Zrsr1 75.19 48.72 1.54 22183 Gspt2 26.47 17.16 1.54 14853 Mrps28 51.32 33.27 1.54 66230 Med28 37.26 24.18 1.54 66999 Atp6v0b 304.26 197.46 1.54 114143 Rpl17 3.11 2.02 1.54 319195 Selk 154.65 100.43 1.54 80795 Cetn4 10.36 6.73 1.54 207175 Larp7 13.73 8.92 1.54 28036 Znhit1 110.39 71.72 1.54 70103 Klf5 3.55 2.31 1.54 12224 Calb1 164.92 107.25 1.54 12307 1110017D15Rik 11.61 7.56 1.54 73721 Prkar2b 47.32 30.80 1.54 19088 Cdh18 13.63 8.87 1.54 320865 Unc119 99.44 64.76 1.54 22248 Rps6ka2 15.02 9.78 1.54 20112 Peg10 76.05 49.55 1.53 170676

177

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID BC022687 20.33 13.25 1.53 217887 1700010I14Rik 1.96 1.28 1.53 66931 Cox7a2l 155.57 101.37 1.53 20463 Wdr34 45.90 29.91 1.53 71820 Ndufa1 419.79 273.67 1.53 54405 Als2cr4 23.44 15.28 1.53 381259 Eid2 95.30 62.15 1.53 386655 Atp5j2 786.16 512.77 1.53 57423 Wbp11 82.62 53.90 1.53 60321 Gmip 3.56 2.32 1.53 78816 Vwa5b1 7.36 4.80 1.53 75718 6430531B16Rik 1.96 1.28 1.53 381933 Nes 0.69 0.45 1.53 18008 Pdcd5 23.26 15.19 1.53 56330 Chchd7 83.43 54.47 1.53 66433 3110001D03Rik 79.69 52.06 1.53 66928 Ethe1 23.34 15.25 1.53 66071 Blcap 191.50 125.11 1.53 53619 Rai2 15.01 9.81 1.53 24004 Podxl2 231.24 151.10 1.53 319655 Brd9 41.03 26.81 1.53 105246 Psd 68.88 45.01 1.53 73728 Ccdc60 1.00 0.65 1.53 269693 Sqstm1 632.71 413.70 1.53 18412 Syngr3 187.11 122.38 1.53 20974 Pnmal2 276.21 180.69 1.53 434128 Best1 2.36 1.54 1.53 24115 Npm3 5.13 3.36 1.53 18150 Ropn1l 1.71 1.12 1.53 252967 Srpr 26.56 17.40 1.53 67398 1700003E16Rik 22.45 14.71 1.53 71837 Pa2g4 52.98 34.70 1.53 18813 Rpl13 20.37 13.35 1.53 270106 Dctn3 216.91 142.13 1.53 53598 D430019H16Rik 31.33 20.53 1.53 268595 Cfdp1 209.42 137.36 1.52 23837 Cdc45 1.69 1.11 1.52 12544 Rabl5 147.56 96.82 1.52 67286 BC002163 295.43 193.85 1.52 170658

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Mrps12 163.01 106.97 1.52 24030 Gpr173 5.53 3.63 1.52 70771 Pemt 5.62 3.69 1.52 18618 Wscd2 8.31 5.46 1.52 320916 Tsen34 71.08 46.69 1.52 66078 rp9 62.13 40.82 1.52 55934 C530028O21Rik 136.69 89.82 1.52 319352 Pop5 91.60 60.20 1.52 117109 Gm6548 21.46 14.11 1.52 625054 Stk33 3.57 2.35 1.52 117229 Tac2 98.55 64.80 1.52 21334 Th1l 46.14 30.34 1.52 57314 Tmem28 11.11 7.31 1.52 620592 1500011H22Rik 135.04 88.83 1.52 68948 Gldn 1.57 1.04 1.52 235379 Tcirg1 3.33 2.19 1.52 27060 Man1b1 32.09 21.12 1.52 227619 Uqcrh 926.15 609.74 1.52 66576 Fbxl6 16.24 10.70 1.52 30840 Rab24 72.51 47.76 1.52 19336 Ccdc28a 60.46 39.83 1.52 215814 B4galt3 24.05 15.85 1.52 57370 Amn1 17.47 11.51 1.52 232566 Dph2 10.13 6.67 1.52 67728 Rtn1 1004.01 661.89 1.52 104001 Macrod1 23.31 15.37 1.52 107227 Tubb2a 483.08 318.64 1.52 22151 Plxnc1 15.31 10.10 1.52 54712 Edf1 375.28 247.63 1.52 59022 Nagk 78.17 51.59 1.52 56174 Nhp2l1 17.62 11.63 1.51 20826 Ccdc67 1.91 1.26 1.51 234964 Slc16a11 10.17 6.72 1.51 216867 Kcnj5 3.72 2.45 1.51 16521 Cgref1 40.66 26.86 1.51 68567 Sumo1 57.97 38.31 1.51 22218 Tubb5 493.12 326.09 1.51 22154 Zfp553 19.41 12.83 1.51 233887 Insm1 5.39 3.56 1.51 53626

179

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Dak 9.89 6.54 1.51 225913 Ern2 0.66 0.43 1.51 26918 Ctxn2 70.14 46.44 1.51 381418 Habp4 211.14 139.84 1.51 56541 Shisa5 48.34 32.02 1.51 66940 1700003M02Rik 1.07 0.71 1.51 69329 Celf3 63.55 42.12 1.51 78784 Dkk3 18.06 11.98 1.51 50781 1500009L16Rik 49.04 32.53 1.51 69784 0610040B10Rik 16.91 11.22 1.51 67672 Nhlrc1 28.36 18.83 1.51 105193 Rbmxrt 8.97 5.96 1.51 19656 Mrps33 21.59 14.34 1.51 14548 Zdhhc12 5.08 3.38 1.51 66220 Mrpl34 109.52 72.77 1.51 94065 Ifnar2 12.48 8.29 1.50 15976 Ormdl1 7.96 5.29 1.50 227102 ORF19 20.33 13.51 1.50 68767 Alkbh7 56.52 37.58 1.50 66400 Sod1 731.41 486.49 1.50 20655 Slc7a3 11.64 7.75 1.50 11989 2310036O22Rik 188.75 125.58 1.50 68544 1700025K23Rik 32.62 21.70 1.50 66337 1810022K09Rik 29.74 19.79 1.50 69126 Rpp21 101.37 67.49 1.50 67676 Myl6 102.78 68.43 1.50 17904 Hyal2 6.55 4.36 1.50 15587 Fam136a 33.60 22.37 1.50 66488 Icam5 15.03 10.01 1.50 15898 0610007C21Rik 80.75 53.78 1.50 381629 Suv39h1 26.75 17.82 1.50 20937 Lmna 35.93 23.94 1.50 16905 Exosc9 38.92 25.93 1.50 50911 Tmem106c 52.84 35.22 1.50 380967 Tomm34 72.52 48.34 1.50 67145 Atox1 263.67 175.75 1.50 11927 Ndufc1 408.59 272.35 1.50 66377 Vps72 49.83 33.22 1.50 21427

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APPENDIX 2

Highly enriched genes in brainstem LepRb neurons

Brainstem sections from 3 cohorts (6-8 mice per cohort) of adult LepRbeGFP-L10a mice were subjected to TRAP. TRAP-enriched and depleted RNA fractions were converted to cDNA libraries prior to running on the Illumina HiSeq2000 sequencing platform. Reads were mapped to mouse genome mm9 for analysis. Transcript levels were compared between the TRAP-enriched and depleted fractions to determine fold enrichment in the LepRb-specific (TRAP) fraction. The table shows the gene symbol, expression (in FPKM) in TRAP-enriched (LepRb) and TRAP-depleted (Non-LepRb) fraction, fold enrichment, and NCBI gene ID for all genes enriched greater than 1.5-fold in the TRAP (LepRb) fraction. Some reads were incorrectly mapped to miRNAs; these were excluded from the analysis. Note that ribosomal and mitochondrial genes may be overrepresented in the data set due to their high levels of transcript turnover (which may increase the likelihood of non-specific TRAP enrichment).

181

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Npw 9.16 0.36 25.46 381073 Xist 16.97 0.70 24.37 213742 Ucn 187.48 9.01 20.81 22226 Vip 110.43 5.48 20.14 22353 Prok2 1.40 0.08 16.77 50501 Cartpt 1404.28 96.13 14.61 27220 Trim54 0.95 0.08 11.69 58522 4732456N10Rik 0.89 0.08 11.34 239673 Col7a1 0.85 0.08 10.41 12836 Flt1 3.76 0.41 9.20 14254 Abcb1a 3.46 0.39 8.81 18671 Abcc6 0.26 0.03 8.22 27421 Slc6a3 65.76 8.15 8.07 13162 Cdhr4 0.72 0.10 7.55 69398 Mmrn2 0.68 0.09 7.35 105450 Gkn1 0.65 0.09 7.16 66283 Gm4598 0.48 0.07 6.92 100043706 Eltd1 2.53 0.38 6.71 170757 Th 244.10 36.41 6.70 21823 Pitx3 6.53 0.98 6.65 18742 Tie1 1.96 0.30 6.65 21846 Casr 0.31 0.05 6.60 12374 Foxa1 5.35 0.84 6.40 15375 Tek 1.42 0.23 6.18 21687 Pth2 6.67 1.10 6.09 114640 Gucy2c 1.67 0.28 6.01 14917 Robo4 1.50 0.25 5.91 74144 Gpr116 2.52 0.44 5.72 224792 Tmem207 0.26 0.05 5.65 100043057 AI467606 1.65 0.31 5.27 101602 Vwf 1.11 0.21 5.24 22371 Sult5a1 1.12 0.22 5.18 57429 Ntsr1 5.10 0.98 5.18 18216 Fn1 2.62 0.52 5.03 14268 Cdh5 1.63 0.33 4.95 12562 Cldn5 15.70 3.18 4.93 12741 Sema3g 1.13 0.24 4.82 218877 Icam2 1.06 0.23 4.73 15896 Podxl 3.12 0.68 4.62 27205

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Ano2 0.57 0.13 4.55 243634 Krt19 0.93 0.21 4.48 16669 Slco1a4 7.78 1.79 4.34 28250 Chrna6 11.37 2.65 4.29 11440 BC020535 1.90 0.44 4.28 228788 Col24a1 0.27 0.06 4.26 71355 Ly6c1 21.20 4.98 4.26 17067 Adcy4 0.61 0.14 4.22 104110 Sigirr 0.57 0.13 4.22 24058 Ret 24.03 5.70 4.21 19713 3930402G23Rik 0.38 0.09 4.19 665306 Il2rg 0.25 0.06 4.17 16186 Mc3r 1.40 0.34 4.17 17201 Col5a3 0.96 0.23 4.13 53867 Slco1c1 6.43 1.58 4.06 58807 Dlk1 71.05 17.54 4.05 13386 Ace2 0.25 0.06 4.03 70008 Pecam1 2.56 0.64 3.97 18613 Col11a2 3.27 0.83 3.97 12815 Adcyap1 53.18 13.46 3.95 11516 Col16a1 3.77 0.96 3.93 107581 Acvrl1 2.12 0.54 3.89 11482 Bhlhe23 1.45 0.38 3.87 140489 Col5a2 0.56 0.15 3.86 12832 Trpv1 0.25 0.06 3.86 193034 Ly6a 15.69 4.08 3.84 110454 Msln 0.25 0.07 3.82 56047 Tinagl1 3.02 0.81 3.72 94242 BC051628 0.52 0.14 3.69 332713 Slc26a10 1.13 0.31 3.62 216441 Yeats2 22.45 6.22 3.61 208146 Avpr1a 1.03 0.29 3.61 54140 Notum 1.16 0.32 3.60 77583 Klrg1 0.40 0.11 3.59 50928 Postn 1.38 0.38 3.59 50706 Slc10a4 22.58 6.29 3.59 231290 Slc39a4 0.95 0.27 3.57 72027 Chrnb3 4.45 1.25 3.57 108043 Tmem149 7.38 2.07 3.57 101883

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Kdr 0.79 0.22 3.52 16542 Pou4f3 2.34 0.67 3.51 18998 Nxnl2 1.27 0.37 3.48 75124 Col4a5 0.65 0.19 3.46 12830 Lmx1a 2.70 0.78 3.44 110648 Foxa2 3.50 1.02 3.42 15376 Col5a1 0.94 0.28 3.38 12831 Ager 0.52 0.16 3.33 11596 Ddc 155.67 46.78 3.33 13195 Fam167a 3.26 0.98 3.32 219148 Fxyd2 21.64 6.55 3.30 11936 Cck 301.01 91.40 3.29 12424 AU021092 0.64 0.19 3.28 239691 Slc38a5 0.39 0.12 3.27 209837 Tac1 126.29 38.84 3.25 21333 Prr19 1.26 0.39 3.24 623131 Atg7 26.93 8.38 3.21 74244 Lamb2 2.80 0.87 3.21 16779 Ferd3l 0.69 0.21 3.20 114712 Pear1 1.05 0.33 3.15 73182 Tle2 20.85 6.62 3.15 21886 Emcn 1.72 0.55 3.14 59308 Dnase1l2 2.67 0.85 3.14 66705 Grp 17.38 5.56 3.13 225642 Nes 0.80 0.25 3.13 18008 Osmr 0.34 0.11 3.12 18414 Lamc3 0.46 0.15 3.10 23928 Fgf15 0.47 0.15 3.09 14170 Ptprb 1.57 0.51 3.09 19263 2010204K13Rik 164.63 53.30 3.09 68355 Anxa1 3.91 1.28 3.05 16952 En1 34.57 11.35 3.05 13798 Col11a1 1.48 0.49 3.02 12814 Itm2a 19.49 6.45 3.02 16431 Tac2 8.19 2.74 2.99 21334 Ano7 0.88 0.30 2.97 404545 Fst 1.19 0.40 2.97 14313 Ghsr 1.16 0.40 2.93 208188 Slc16a11 31.02 10.64 2.91 216867

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID H2-Q2 7.29 2.51 2.91 15013 Tm4sf5 2.72 0.94 2.90 75604 Wfdc1 3.69 1.27 2.89 67866 Cpne7 19.91 6.93 2.87 102278 Slc2a1 36.37 12.70 2.86 20525 C4b 1.87 0.66 2.85 12268 Bmp1 24.42 8.58 2.85 12153 Xdh 1.87 0.66 2.85 22436 Serpine1 0.54 0.19 2.84 18787 Jph2 1.04 0.37 2.81 59091 Ghrh 3.69 1.31 2.81 14601 Gdf5 0.36 0.13 2.80 14563 Ushbp1 1.08 0.38 2.80 234395 Hspg2 0.51 0.18 2.79 15530 Adamts10 12.66 4.57 2.77 224697 Eng 3.17 1.14 2.77 13805 Flt3l 2.86 1.03 2.77 14256 Tbxa2r 0.32 0.11 2.77 21390 Ntf3 3.13 1.14 2.75 18205 Trpc6 0.94 0.34 2.75 22068 Rrad 4.13 1.51 2.74 56437 Arap3 1.04 0.38 2.72 106952 Iqca 0.32 0.12 2.72 74918 Drd2 31.24 11.48 2.72 13489 Brs3 0.23 0.08 2.72 12209 Pglyrp1 5.49 2.02 2.72 21946 BC006779 0.45 0.17 2.71 229003 Aldh3b2 2.45 0.91 2.70 621603 Rsph9 18.09 6.70 2.70 75564 Stap2 7.36 2.73 2.70 106766 Lat 0.55 0.20 2.69 16797 Trpa1 0.28 0.10 2.68 277328 Atp2c2 0.51 0.19 2.67 69047 A230065H16Rik 44.41 16.64 2.67 380787 Mfsd7c 0.45 0.17 2.67 217721 Tstd1 6.47 2.45 2.64 226654 Pi16 2.06 0.78 2.63 74116 Myoc 3.26 1.25 2.62 17926 Gm14378 4.24 1.62 2.61 100044509

185

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Syt5 75.91 29.06 2.61 53420 Ido1 0.62 0.24 2.61 15930 Fgf20 0.77 0.30 2.61 80857 H2-Q8 1.30 0.50 2.60 15019 Slc38a4 0.36 0.14 2.60 69354 BC089491 5.51 2.12 2.60 280621 Sncg 377.77 145.60 2.59 20618 Kcp 0.40 0.15 2.59 333088 Ngb 71.34 27.53 2.59 64242 Thbd 1.64 0.63 2.59 21824 Gbp9 0.43 0.17 2.59 236573 Twist2 1.56 0.60 2.58 13345 Tm6sf2 0.87 0.34 2.58 107770 Lsr 2.02 0.78 2.58 54135 Sstr5 0.56 0.22 2.58 20609 Mmp23 3.58 1.39 2.58 26561 Nptx2 16.61 6.46 2.57 53324 Zar1 3.89 1.52 2.56 317755 Fsd2 0.47 0.18 2.55 244091 Ndc80 0.47 0.18 2.54 67052 Pltp 23.40 9.24 2.53 18830 Ly6h 334.00 131.89 2.53 23934 Gpx3 87.41 34.53 2.53 14778 Gpc3 3.48 1.38 2.52 14734 Slc19a3 0.76 0.30 2.52 80721 Cyyr1 0.40 0.16 2.51 224405 Socs3 1.78 0.71 2.51 12702 Chrm5 1.04 0.42 2.51 213788 Sox17 1.23 0.49 2.50 20671 Rxfp3 1.96 0.78 2.50 239336 Notch4 1.07 0.43 2.50 18132 Gldn 0.46 0.18 2.50 235379 Cd93 0.63 0.25 2.48 17064 Izumo4 37.15 15.01 2.48 71564 Sp100 0.58 0.23 2.47 20684 Nos3 2.39 0.97 2.47 18127 Pcbd1 129.18 52.34 2.47 13180 Resp18 652.14 264.29 2.47 19711 Prss23 1.25 0.51 2.46 76453

186

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Ankrd2 1.50 0.61 2.45 56642 Pou4f1 9.64 3.95 2.44 18996 Gch1 12.11 4.97 2.44 14528 Mamdc4 0.69 0.28 2.44 381352 Fmo2 0.62 0.26 2.43 55990 Cdkn3 1.48 0.61 2.43 72391 She 0.51 0.21 2.43 214547 Tmprss6 0.35 0.14 2.42 71753 Gpr149 3.51 1.45 2.42 229357 6330403K07Rik 945.86 390.81 2.42 103712 Abcg2 4.42 1.83 2.42 26357 Slc18a2 38.54 15.94 2.42 214084 Tacr3 6.82 2.83 2.41 21338 Mfsd10 10.76 4.47 2.41 68294 Dnahc1 1.25 0.52 2.41 110084 Il13ra1 3.44 1.43 2.40 16164 Esam 6.49 2.70 2.40 69524 Nostrin 1.94 0.81 2.40 329416 Ggt5 0.35 0.15 2.39 23887 Atp10a 1.81 0.76 2.39 11982 Fbln2 0.52 0.22 2.39 14115 Stbd1 1.79 0.75 2.38 52331 AW551984 28.76 12.09 2.38 244810 Odf3l2 0.53 0.22 2.38 382384 Kcnj8 1.20 0.50 2.37 16523 Ggnbp1 2.52 1.07 2.37 70772 Glra3 4.39 1.87 2.35 110304 F12 0.52 0.22 2.35 58992 Erg 0.99 0.42 2.35 13876 Spint2 52.30 22.27 2.35 20733 Nr4a2 10.43 4.45 2.34 18227 Gtf2a1l 0.67 0.29 2.33 71828 Kcna5 3.13 1.35 2.33 16493 Col27a1 2.08 0.90 2.32 373864 Cnga3 0.33 0.14 2.32 12790 Defb1 3.34 1.44 2.31 13214 Mgst2 2.65 1.15 2.31 211666 Gabrq 2.47 1.07 2.31 57249 Zfp366 0.31 0.13 2.30 238803

187

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Fgf17 0.95 0.42 2.29 14171 Tmem26 0.48 0.21 2.29 327766 Rgl2 17.34 7.59 2.29 19732 Insm1 4.98 2.18 2.29 53626 Naalad2 0.32 0.14 2.29 72560 Zcchc12 228.45 100.01 2.28 72693 Sostdc1 3.72 1.63 2.28 66042 Kank3 8.01 3.51 2.28 80880 Islr2 3.93 1.73 2.28 320563 Fxyd6 196.19 86.60 2.27 59095 Gm694 1.66 0.73 2.26 277744 E130309F12Rik 4.98 2.20 2.26 272031 Dmrta2 1.71 0.75 2.26 242620 Sstr2 7.69 3.40 2.26 20606 Abcc9 0.81 0.36 2.26 20928 Pgr15l 0.89 0.40 2.25 245526 Ctxn2 75.17 33.42 2.25 381418 Myo15 0.45 0.20 2.25 17910 Klhl1 22.37 9.95 2.25 93688 Leng8 37.70 16.81 2.24 232798 Ltbp4 6.11 2.73 2.24 108075 Acer2 2.28 1.02 2.23 230379 Efcab10 8.14 3.65 2.23 75040 Myh7 3.31 1.49 2.22 140781 Hfe2 2.74 1.24 2.21 69585 Cfh 1.24 0.56 2.21 12628 Rcn1 11.43 5.17 2.21 19672 Baiap3 68.53 31.23 2.19 545192 Ctxn1 150.10 68.57 2.19 330695 Dgkk 11.54 5.29 2.18 331374 Vat1 118.97 54.57 2.18 26949 Itih5 2.69 1.23 2.18 209378 Gdpd2 3.13 1.44 2.18 71584 Zap70 2.51 1.16 2.17 22637 Celf6 40.56 18.71 2.17 76183 Klf2 3.90 1.80 2.17 16598 Adam33 1.28 0.59 2.16 110751 Spnb2 60.62 28.09 2.16 20742 Krtap17-1 1.68 0.78 2.16 77914

188

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Epha2 0.29 0.13 2.15 13836 Mapk15 1.00 0.47 2.15 332110 Serpina3g 3.92 1.82 2.15 20715 2310046K01Rik 0.49 0.23 2.15 69698 Tmem91 113.61 52.88 2.15 320208 Rspo1 9.63 4.48 2.15 192199 Cd44 0.82 0.38 2.15 12505 Lmx1b 2.39 1.12 2.14 16917 Pou4f2 8.23 3.85 2.14 18997 Prr22 0.84 0.39 2.14 100504446 Scn3b 30.40 14.23 2.14 235281 Col6a1 0.97 0.45 2.13 12833 Uts2r 0.71 0.33 2.13 217369 Glra2 17.57 8.27 2.12 237213 Pgrmc1 336.26 158.46 2.12 53328 Npffr1 1.14 0.54 2.12 237362 Rxrg 9.96 4.70 2.12 20183 Gpr6 1.14 0.54 2.11 140741 Aldh1a7 1.52 0.72 2.11 26358 Vtn 17.92 8.50 2.11 22370 2810008D09Rik 34.93 16.59 2.11 76972 Pcdh8 9.58 4.55 2.11 18530 Snca 233.59 111.01 2.10 20617 Dmrtb1 4.25 2.02 2.10 56296 Bnc2 1.04 0.49 2.10 242509 Pam 65.15 31.05 2.10 18484 Abcc4 1.79 0.85 2.10 239273 Lhfpl5 22.89 10.92 2.10 328789 Col2a1 0.36 0.17 2.09 12824 Ecel1 10.75 5.15 2.09 13599 Ramp2 22.64 10.86 2.08 54409 Apcdd1 5.05 2.43 2.07 494504 A730017C20Rik 177.31 85.57 2.07 225583 Asb4 4.96 2.40 2.07 65255 Styk1 0.29 0.14 2.06 243659 Myh6 1.32 0.64 2.06 17888 Gja4 2.38 1.16 2.06 14612 Gipr 1.06 0.52 2.05 381853 Fam183b 16.43 8.01 2.05 75429

189

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Myh11 0.58 0.28 2.05 17880 Syt17 23.68 11.56 2.05 110058 Vill 0.83 0.41 2.05 22351 Arhgap36 13.49 6.60 2.05 75404 Panx1 11.54 5.64 2.04 55991 Eln 1.34 0.66 2.04 13717 Galr1 0.54 0.26 2.03 14427 Xlr3b 0.99 0.49 2.03 574437 Prss50 1.05 0.52 2.02 235631 Oas1b 0.54 0.27 2.02 23961 Ttc22 1.17 0.58 2.02 230576 Ak8 2.85 1.41 2.02 68870 Rhbdf2 0.30 0.15 2.02 217344 Il17rd 0.45 0.22 2.02 171463 Klhl14 2.90 1.44 2.01 225266 Fgf13 95.44 47.39 2.01 14168 Tnfrsf8 0.99 0.49 2.01 21941 Slc7a3 10.72 5.33 2.01 11989 C1qtnf2 1.53 0.76 2.01 69183 1700086L19Rik 9.30 4.64 2.00 74284 C2cd4c 3.47 1.73 2.00 237397 Ptger2 0.49 0.24 2.00 19217 Fank1 3.77 1.88 2.00 66930 Atp13a5 1.46 0.73 2.00 268878 Dkkl1 2.65 1.33 2.00 50722 Pgpep1l 2.84 1.42 2.00 78444 Rab26 31.80 15.92 2.00 328778 Esr1 0.95 0.47 1.99 13982 Ssh3 10.68 5.36 1.99 245857 Tro 32.31 16.22 1.99 56191 Sema3c 3.67 1.84 1.99 20348 AI428936 4.70 2.37 1.98 233066 Armcx6 13.19 6.65 1.98 278097 Cbln2 35.60 17.98 1.98 12405 Plscr5 1.08 0.55 1.98 100504689 Slc27a3 5.33 2.69 1.98 26568 Bcam 8.38 4.24 1.98 57278 Krtcap3 1.80 0.91 1.98 69815 Fzd6 1.03 0.52 1.97 14368

190

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Arl10 35.21 17.86 1.97 56795 Col4a1 2.45 1.25 1.97 12826 Rprml 22.69 11.51 1.97 104582 Plxnc1 8.14 4.13 1.97 54712 Runx1 0.60 0.31 1.97 12394 Mirg 12.01 6.11 1.97 100040724 Hnf1b 0.90 0.46 1.96 21410 Caly 246.53 125.75 1.96 68566 Tmem130 226.87 115.98 1.96 243339 Bdnf 9.44 4.83 1.95 12064 Stox1 1.12 0.57 1.95 216021 Fam70a 44.52 22.82 1.95 245386 Gaa 149.73 76.80 1.95 14387 Tmem145 41.60 21.36 1.95 330485 Trhr 1.92 0.98 1.95 22045 Cyp26b1 4.05 2.08 1.95 232174 Igsf9 1.10 0.57 1.95 93842 Clic5 0.92 0.48 1.94 224796 Lama5 0.90 0.46 1.94 16776 Oprk1 2.26 1.16 1.94 18387 Sspo 0.27 0.14 1.94 243369 Hap1 300.20 154.86 1.94 15114 Chrd 6.80 3.51 1.94 12667 Best1 2.12 1.09 1.94 24115 Necab1 33.10 17.12 1.93 69352 Scg2 182.04 94.43 1.93 20254 Apold1 1.44 0.75 1.93 381823 Jak3 2.28 1.19 1.93 16453 Tnmd 1.39 0.72 1.92 64103 Rab3b 21.60 11.24 1.92 69908 Col4a2 3.61 1.89 1.92 12827 Moxd1 0.53 0.28 1.91 59012 Map3k6 1.50 0.79 1.91 53608 Tap1 0.91 0.48 1.91 21354 Lrrc45 17.79 9.36 1.90 217366 Snhg12 111.72 58.86 1.90 100039864 Bahcc1 6.03 3.18 1.90 268515 Tmie 14.99 7.91 1.90 20776 Tpd52l1 61.07 32.24 1.89 21987

191

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Rac3 86.32 45.62 1.89 170758 Gm4349 2.51 1.33 1.89 100043305 Syt10 1.42 0.75 1.89 54526 Qpct 14.40 7.63 1.89 70536 1110038B12Rik 37.37 19.82 1.89 68763 Crhbp 10.92 5.79 1.89 12919 Akr1c14 0.74 0.39 1.88 105387 Tspan6 16.52 8.77 1.88 56496 H2-K1 8.12 4.31 1.88 14972 Akap8l 31.42 16.70 1.88 54194 F2r 8.52 4.53 1.88 14062 Prmt2 149.18 79.38 1.88 15468 Barx2 3.38 1.80 1.88 12023 Dkk2 0.47 0.25 1.88 56811 Doc2a 42.07 22.40 1.88 13446 St8sia4 3.84 2.05 1.88 20452 Fam116b 14.44 7.70 1.88 69440 Egflam 1.17 0.63 1.87 268780 Lhx2 14.02 7.48 1.87 16870 Dkk3 28.45 15.19 1.87 50781 Prom1 2.90 1.55 1.87 19126 Slc35f4 11.66 6.23 1.87 75288 Fxyd7 225.62 120.47 1.87 57780 Ebf3 27.22 14.55 1.87 13593 1700001L19Rik 12.90 6.90 1.87 69315 Fosb 2.47 1.32 1.87 14282 Syngr3 154.83 82.95 1.87 20974 Slc5a5 14.16 7.59 1.87 114479 Tcirg1 4.66 2.50 1.87 27060 Nps 4.18 2.24 1.86 100043254 Tusc3 101.93 54.69 1.86 80286 Tekt2 2.23 1.20 1.86 24084 Atg9b 7.44 4.00 1.86 213948 Clec2d 2.65 1.42 1.86 93694 1700045I19Rik 2.69 1.45 1.86 74264 Gabre 0.67 0.36 1.86 14404 D430019H16Rik 10.18 5.48 1.86 268595 Pld3 162.56 87.67 1.85 18807 Srpk3 1.77 0.95 1.85 56504

192

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Zfp692-ps 18.83 10.17 1.85 103836 Lmtk3 31.70 17.15 1.85 381983 Vwa1 10.74 5.82 1.85 246228 Ptpn5 70.60 38.25 1.85 19259 Ubr3 37.34 20.24 1.84 68795 Gm1673 260.70 141.36 1.84 381633 A930038C07Rik 1.39 0.75 1.84 68169 Col20a1 0.83 0.45 1.84 73368 Als2cl 3.24 1.76 1.84 235633 Gpr101 3.49 1.90 1.84 245424 Zfp92 3.26 1.77 1.84 22754 Crip2 55.78 30.41 1.83 68337 Dmrtc1a 9.73 5.31 1.83 70887 Gfra1 13.73 7.51 1.83 14585 Slc12a7 0.73 0.40 1.83 20499 Pdgfrb 2.14 1.17 1.82 18596 Hpcal4 73.42 40.28 1.82 170638 Ajap1 20.19 11.11 1.82 230959 Grm8 6.05 3.33 1.81 14823 Dus3l 42.65 23.50 1.81 224907 Nhedc2 1.06 0.58 1.81 97086 Tmem179 103.61 57.13 1.81 104885 Plxna3 4.36 2.41 1.81 18846 Mrgpre 7.45 4.12 1.81 244238 Npsr1 0.78 0.43 1.81 319239 Vwa5b1 3.13 1.73 1.81 75718 Clec14a 0.54 0.30 1.81 66864 Tacr1 3.96 2.20 1.80 21336 Ccr10 1.65 0.92 1.80 12777 Irs4 4.10 2.27 1.80 16370 Adcy7 5.37 2.98 1.80 11513 Sdc2 26.46 14.70 1.80 15529 Slit1 5.99 3.33 1.80 20562 Akap6 22.08 12.26 1.80 238161 Cd83 59.51 33.10 1.80 12522 Egfl7 25.91 14.43 1.80 353156 Mrap2 31.18 17.38 1.79 244958 Fbxl7 1.01 0.56 1.79 448987 Flna 5.97 3.33 1.79 192176

193

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Hn1 78.81 44.07 1.79 15374 Gbp2 0.72 0.40 1.79 14469 Foxf2 1.22 0.69 1.79 14238 Pitx1 0.87 0.49 1.79 18740 Pnpla6 17.50 9.80 1.79 50767 Adamtsl2 0.70 0.39 1.78 77794 Gm1027 1.55 0.87 1.78 381538 Ppapdc1a 18.53 10.40 1.78 381925 Abhd14b 19.26 10.81 1.78 76491 Pcdh19 4.12 2.32 1.78 279653 Ndn 752.11 423.76 1.77 17984 Chst1 67.80 38.21 1.77 76969 Slc39a6 14.65 8.27 1.77 106957 Leprel2 9.86 5.56 1.77 14789 Ltk 7.32 4.13 1.77 17005 Gpr83 3.21 1.81 1.77 14608 Evx1 2.11 1.19 1.77 14028 Fzd8 2.26 1.28 1.77 14370 C330005M16Rik 1.17 0.66 1.76 101744 Paqr5 0.75 0.43 1.76 74090 Fads3 12.17 6.91 1.76 60527 Spnb3 52.57 29.84 1.76 20743 Rpl37 629.34 357.53 1.76 67281 Pcsk1 6.59 3.74 1.76 18548 Drd5 3.11 1.77 1.76 13492 BC024139 1.77 1.01 1.76 271278 Tmsb10 852.92 484.80 1.76 19240 Vgf 96.99 55.16 1.76 381677 Lgals3bp 5.57 3.17 1.76 19039 Prkch 1.53 0.87 1.76 18755 Trfr2 2.81 1.60 1.75 50765 Spag4 3.97 2.27 1.75 245865 2210013O21Rik 169.64 96.86 1.75 0 Magee1 105.05 60.02 1.75 107528 Cd200 71.00 40.57 1.75 17470 Gpr56 21.24 12.17 1.75 14766 Cyb561 41.31 23.66 1.75 13056 Ets1 1.46 0.84 1.74 23871 Tnxb 0.31 0.18 1.74 81877

194

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Kcna4 4.15 2.38 1.74 16492 Gprasp1 169.61 97.46 1.74 67298 Cbln4 19.02 10.93 1.74 228942 Adcy3 13.41 7.72 1.74 104111 S1pr3 1.69 0.97 1.74 13610 Sema6b 36.16 20.81 1.74 20359 Rpl41 2364.10 1360.85 1.74 67945 Slc35f2 1.93 1.11 1.74 72022 Tubb2a 336.54 193.91 1.74 22151 Sfrp2 7.43 4.28 1.74 20319 Npb 5.67 3.27 1.73 208990 Nop10 190.04 109.68 1.73 66181 Ephb4 0.80 0.46 1.73 13846 Krt77 1.87 1.08 1.73 406220 6330403A02Rik 12.08 6.98 1.73 381310 Megf6 1.05 0.61 1.73 230971 Col25a1 4.47 2.58 1.73 77018 Nrsn2 321.32 185.91 1.73 228777 Cd1d1 1.65 0.96 1.73 12479 Rpl22l1 210.46 121.80 1.73 68028 C530028O21Rik 113.46 65.68 1.73 319352 Trpc4 1.95 1.13 1.73 22066 Chrna4 20.14 11.67 1.73 11438 Foxq1 1.23 0.72 1.73 15220 Sulf1 2.20 1.28 1.72 240725 Bex1 245.12 142.60 1.72 19716 Echdc2 12.39 7.21 1.72 52430 Mal2 38.14 22.21 1.72 105853 Hdac10 5.63 3.28 1.72 170787 Fam179a 1.55 0.90 1.72 320159 Col8a2 0.66 0.38 1.71 329941 Basp1 482.22 281.27 1.71 70350 Adam15 29.68 17.32 1.71 11490 C1ql4 17.11 9.99 1.71 239659 Camkv 93.80 54.84 1.71 235604 Dock6 2.37 1.39 1.71 319899 Rsph4a 2.16 1.26 1.71 212892 Gng4 53.00 31.04 1.71 14706 Dos 237.16 139.13 1.70 100503659

195

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Pcdh18 2.30 1.35 1.70 73173 Slc30a1 5.98 3.51 1.70 22782 Itm2c 287.13 168.88 1.70 64294 Fstl5 9.96 5.86 1.70 213262 Npy2r 1.32 0.78 1.70 18167 Rps15 2171.84 1278.50 1.70 20054 Cldn1 0.88 0.52 1.70 12737 Ptpro 6.94 4.09 1.70 19277 Chrm3 5.96 3.51 1.70 12671 Gng3 361.12 212.70 1.70 14704 Epb4.1l4a 9.42 5.55 1.70 13824 Pdzrn4 3.15 1.86 1.70 239618 Dlk2 16.73 9.87 1.69 106565 Tnrc18 12.80 7.55 1.69 231861 Trim21 1.60 0.95 1.69 20821 Matk 94.18 55.61 1.69 17179 Cdh13 17.84 10.54 1.69 12554 Cspg4 1.31 0.78 1.69 121021 Tmem200a 3.15 1.86 1.69 77220 Slc39a11 8.06 4.76 1.69 69806 Olfm2 60.78 35.98 1.69 244723 Msc 1.79 1.06 1.69 17681 Ptprn 125.82 74.53 1.69 19275 Cxcr7 1.88 1.11 1.69 12778 Entpd6 33.16 19.65 1.69 12497 Magel2 3.71 2.20 1.69 27385 Krt1 10.12 6.00 1.69 16678 Gck 2.68 1.59 1.69 103988 Elfn1 11.08 6.57 1.69 243312 Ecm1 2.44 1.45 1.69 13601 Pthlh 2.67 1.58 1.69 19227 Pnck 163.62 97.17 1.68 93843 Pnmal1 48.89 29.07 1.68 71691 Tpra1 30.36 18.05 1.68 24100 Sema4f 19.87 11.83 1.68 20355 Sox18 3.03 1.81 1.68 20672 Ntrk3 17.57 10.47 1.68 18213 Hcn3 8.96 5.34 1.68 15168 Zcchc18 274.93 163.90 1.68 66995

196

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Lrrc16b 14.72 8.79 1.68 268747 Gm5577 2.31 1.38 1.67 434064 Oxtr 1.42 0.85 1.67 18430 Itga10 0.68 0.41 1.67 213119 Ak5 25.81 15.45 1.67 229949 Rpl37a 872.32 522.66 1.67 19981 Ache 87.83 52.65 1.67 11423 Zbtb8b 3.72 2.23 1.67 215627 Ahi1 232.36 139.31 1.67 52906 BC048546 2.17 1.30 1.67 232400 Atg16l2 6.51 3.90 1.67 73683 Csf2ra 11.14 6.68 1.67 12982 BC002163 39.23 23.55 1.67 170658 Uchl1 1219.04 732.11 1.67 22223 Ehbp1l1 10.94 6.57 1.67 114601 Igsf8 110.43 66.35 1.66 140559 Ppox 13.42 8.07 1.66 19044 Itga7 7.26 4.36 1.66 16404 Psme1 97.51 58.68 1.66 19186 Timp2 204.31 122.98 1.66 21858 Apc2 25.55 15.39 1.66 23805 Stx1a 39.48 23.78 1.66 20907 Rps19 679.19 409.21 1.66 20085 T2 2.85 1.72 1.66 21331 Mesdc2 17.55 10.58 1.66 67943 Lepr 1.29 0.78 1.66 16847 Lrpprc 22.70 13.69 1.66 72416 Palmd 2.75 1.66 1.66 114301 Fbxl6 17.02 10.27 1.66 30840 Abca7 5.67 3.42 1.66 27403 Penk 94.45 57.01 1.66 18619 Oasl2 1.67 1.01 1.66 23962 Tmem158 24.45 14.77 1.66 72309 Usp11 72.00 43.49 1.66 236733 Mboat7 53.72 32.47 1.65 77582 Slc17a6 80.06 48.39 1.65 140919 Pnma3 18.09 10.94 1.65 245468 Rps8 1311.06 792.91 1.65 20116 Cd34 2.95 1.78 1.65 12490

197

LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Atp6v0b 277.73 168.06 1.65 114143 Lrrc3 2.65 1.61 1.65 237387 Kif26a 0.94 0.57 1.65 668303 Rwdd2a 72.22 43.76 1.65 69519 Rpl3 1043.16 632.27 1.65 27367 Ptgis 2.05 1.24 1.65 19223 Ntn1 7.25 4.39 1.65 18208 Alk 2.33 1.42 1.65 11682 Dhcr24 37.10 22.53 1.65 74754 Mc4r 1.44 0.87 1.65 17202 Tm2d3 88.26 53.61 1.65 68634 Slc7a4 10.25 6.23 1.65 224022 Gmip 2.32 1.41 1.65 78816 Aff2 2.32 1.41 1.65 14266 Hyou1 20.56 12.50 1.64 12282 2310007A19Rik 7.63 4.64 1.64 66353 Arsa 11.25 6.84 1.64 11883 Sstr1 8.12 4.94 1.64 20605 Wdr6 129.21 78.74 1.64 83669 Crlf2 14.86 9.05 1.64 57914 Nnat 308.59 188.27 1.64 18111 Odz2 7.40 4.52 1.64 23964 Ass1 19.44 11.88 1.64 11898 Irx6 1.46 0.89 1.64 64379 Blcap 162.39 99.26 1.64 53619 Slc22a8 2.57 1.58 1.63 19879 Prr7 18.68 11.44 1.63 432763 Ocln 1.74 1.07 1.63 18260 Pafah1b3 78.09 47.87 1.63 18476 Irf1 2.91 1.78 1.63 16362 Gpsm3 11.12 6.82 1.63 106512 Rplp1 2776.77 1702.62 1.63 56040 Arhgdig 148.79 91.24 1.63 14570 Pnma1 3.16 1.94 1.63 70481 Pld6 2.58 1.58 1.63 194908 Amn 12.54 7.70 1.63 93835 Ptprz1 8.93 5.49 1.63 19283 Grin2c 29.23 17.97 1.63 14813 A130022J15Rik 4.72 2.90 1.63 101351

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Srpr 35.74 21.98 1.63 67398 Hspa12b 3.69 2.27 1.62 72630 Dll3 2.91 1.79 1.62 13389 2900062L11Rik 49.30 30.35 1.62 76976 Arrdc1 8.48 5.22 1.62 215705 BC029722 167.97 103.43 1.62 613262 Rprm 27.49 16.94 1.62 67874 Rgs12 5.39 3.32 1.62 71729 Ssr4 90.61 55.85 1.62 20832 Ovgp1 3.14 1.94 1.62 12659 Trpv2 9.97 6.15 1.62 22368 Ldha 192.67 118.89 1.62 16828 Dnahc6 0.27 0.17 1.62 330355 Tmem43 13.47 8.31 1.62 74122 Cgref1 26.07 16.09 1.62 68567 Dcaf12l1 41.24 25.48 1.62 245404 Crh 5.55 3.43 1.62 12918 Mum1l1 3.68 2.28 1.62 245631 Scml4 1.85 1.15 1.62 268297 Scn5a 0.46 0.29 1.62 20271 Tspan33 18.06 11.18 1.62 232670 Slc29a1 14.51 8.99 1.61 63959 Rnf112 105.08 65.13 1.61 22671 Lrrc24 26.75 16.58 1.61 378937 Slc26a11 3.59 2.23 1.61 268512 Gpr135 10.24 6.35 1.61 238252 Cxx1c 487.97 302.96 1.61 72865 Lor 28.40 17.64 1.61 16939 Kcnd3 19.76 12.27 1.61 56543 Bex2 805.08 500.09 1.61 12069 Aup1 49.32 30.65 1.61 11993 Slc7a5 18.54 11.52 1.61 20539 3110040N11Rik 23.96 14.90 1.61 67290 Lrrc55 8.62 5.36 1.61 241528 Amotl1 13.24 8.24 1.61 75723 6530401D17Rik 20.55 12.80 1.60 76219 Svop 73.99 46.12 1.60 68666 Nsg2 385.76 240.52 1.60 18197 Rtn1 920.10 573.76 1.60 104001

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Rpl10a 557.06 347.38 1.60 19896 Htr2c 32.58 20.33 1.60 15560 Rps5 812.62 507.04 1.60 20103 Itga2b 1.50 0.93 1.60 16399 Slc22a17 302.16 188.72 1.60 59049 Rps21 2446.80 1528.75 1.60 66481 Rhbdl1 77.71 48.55 1.60 214951 Tmem22 28.79 17.99 1.60 245020 Rgs5 9.74 6.09 1.60 19737 Por 22.97 14.38 1.60 18984 Tmem66 133.99 83.88 1.60 67887 B630019K06Rik 108.36 67.87 1.60 102941 Espn 5.41 3.39 1.60 56226 Cyb561d2 10.43 6.55 1.59 56368 Odz3 7.09 4.45 1.59 23965 Qpctl 9.92 6.23 1.59 67369 Prpf8 32.94 20.67 1.59 192159 Rnasek 468.55 294.49 1.59 52898 2010011I20Rik 19.51 12.27 1.59 67017 Gcnt2 1.96 1.23 1.59 14538 Rpl34-ps1 1259.28 792.48 1.59 619547 Grin3a 4.50 2.83 1.59 242443 Ccdc141 2.40 1.51 1.59 545428 Tspyl2 114.41 72.06 1.59 52808 Adam11 45.04 28.39 1.59 11488 Rpl36a 629.74 397.33 1.58 19982 Gstt2 9.59 6.05 1.58 14872 Fbln1 1.40 0.89 1.58 14114 Evpl 3.14 1.98 1.58 14027 Rps29 2480.53 1565.79 1.58 20090 Rps11 1124.69 709.97 1.58 27207 Vwc2l 5.54 3.50 1.58 320460 Vstm2l 171.75 108.47 1.58 277432 1700037H04Rik 167.02 105.50 1.58 67326 Nid1 1.14 0.72 1.58 18073 Isyna1 30.38 19.19 1.58 71780 Astn1 19.17 12.13 1.58 11899 Fbll1 67.54 42.73 1.58 237730 Rpl19 1308.08 827.79 1.58 19921

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Slco2b1 1.77 1.12 1.58 101488 Alg2 82.15 52.07 1.58 56737 Eid2b 25.99 16.49 1.58 434156 Acsl4 9.47 7.23 1.58 50790 Card9 4.34 2.75 1.57 332579 Ostc 52.05 33.06 1.57 66357 Gpr165 6.39 4.06 1.57 76206 Pde2a 26.55 16.87 1.57 207728 Pom121 15.67 9.97 1.57 107939 Cidea 9.00 5.73 1.57 12683 Fibcd1 5.70 3.63 1.57 98970 Tmem185b 9.48 6.04 1.57 226351 Clptm1l 53.97 34.41 1.57 218335 Akap11 26.31 16.82 1.56 219181 Slc38a3 12.50 7.99 1.56 76257 Mogs 10.62 6.80 1.56 57377 0610011F06Rik 77.82 49.86 1.56 68347 Sv2c 5.40 3.46 1.56 75209 Pygl 2.88 1.85 1.56 110095 Rpl17 1139.88 731.77 1.56 319195 Rps28 1896.23 1217.39 1.56 54127 Ngfrap1 573.79 368.42 1.56 12070 Zwint 415.10 266.54 1.56 52696 Rps6kb2 15.84 10.17 1.56 58988 Pdia6 31.09 19.98 1.56 71853 Tmem176a 8.32 5.34 1.56 66058 Phf1 49.69 31.96 1.55 21652 Cacnb3 65.44 42.09 1.55 12297 Slc1a4 15.05 9.69 1.55 55963 Gabrg1 6.69 4.31 1.55 14405 Slc9a5 9.87 6.35 1.55 277973 Magee2 10.14 6.53 1.55 272790 Oprl1 39.25 25.28 1.55 18389 Polrmt 11.28 7.26 1.55 216151 Grb10 37.32 24.05 1.55 14783 Rpl9 1305.34 841.53 1.55 20005 Plat 9.28 5.99 1.55 18791 Ly6e 67.19 43.34 1.55 17069 Sema4a 16.49 10.66 1.55 20351

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Tmem159 15.59 10.08 1.55 233806 Atp13a2 55.86 36.13 1.55 74772 2900011O08Rik 307.87 199.16 1.55 67254 Tmem132a 43.67 28.25 1.55 98170 Col18a1 4.48 2.90 1.55 12822 Rgag4 19.38 12.54 1.55 331474 Rpl35a 1249.39 809.17 1.54 57808 Rps27a 977.26 633.13 1.54 78294 Sh3rf1 7.32 4.74 1.54 59009 Col9a3 4.80 3.11 1.54 12841 Slc2a6 11.05 7.16 1.54 227659 Rpl27a 558.35 361.83 1.54 26451 L1cam 24.80 16.07 1.54 16728 Gpr27 12.12 7.86 1.54 14761 Rhbdd2 30.94 20.07 1.54 215160 Ogfod1 61.37 39.83 1.54 270086 Rps20 917.55 595.57 1.54 67427 Dclk3 8.19 5.32 1.54 245038 Unc119 90.25 58.62 1.54 22248 Rps26 563.00 365.76 1.54 27370 Rpl13a 1333.13 866.23 1.54 22121 Lmo3 38.04 24.72 1.54 109593 Nme5 14.17 9.21 1.54 75533 Mtch1 538.78 350.27 1.54 56462 Gstm6 10.27 6.68 1.54 14867 Rhov 29.29 19.07 1.54 228543 Sec24d 6.47 4.21 1.54 69608 Tram1l1 24.30 15.83 1.54 229801 Rasgrf2 31.02 20.20 1.54 19418 Slc39a10 17.98 11.71 1.53 227059 Calb2 496.19 323.47 1.53 12308 Stk32c 67.00 43.70 1.53 57740 Slc2a13 12.17 7.94 1.53 239606 Tap2 3.82 2.50 1.53 21355 Tgm2 2.54 1.66 1.53 21817 2900026A02Rik 5.67 3.70 1.53 243219 Ilvbl 16.30 10.64 1.53 216136 Grem2 13.17 8.60 1.53 23893 Mrps34 120.09 78.45 1.53 79044

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Bex4 75.50 49.34 1.53 406217 Cthrc1 8.87 5.80 1.53 68588 Syt4 68.09 44.54 1.53 20983 Tmem9 67.76 44.32 1.53 66241 Shisa9 13.60 8.90 1.53 72555 C85492 24.40 15.97 1.53 215494 Grin1 90.62 59.32 1.53 14810 Spcs1 152.42 99.77 1.53 69019 Itga6 1.71 1.12 1.53 16403 Mchr1 5.26 3.45 1.52 207911 Pcsk4 3.27 2.15 1.52 18551 Tmem50a 82.77 54.33 1.52 71817 Hspa5 66.20 43.48 1.52 14828 Cpt1c 24.41 16.03 1.52 78070 Rps14 1113.37 731.30 1.52 20044 Rpl10 827.88 543.92 1.52 110954 Cdkl4 3.76 2.47 1.52 381113 Tmed3 27.62 18.15 1.52 66111 4930539E08Rik 2.59 1.71 1.52 207819 Agrn 15.22 10.01 1.52 11603 Pcsk2 47.58 31.30 1.52 18549 Tmem59l 262.16 172.52 1.52 67937 Cnih2 136.76 90.03 1.52 12794 Rplp2 505.69 333.21 1.52 67186 Atp9a 134.57 88.70 1.52 11981 Npdc1 189.52 125.01 1.52 18146 Elof1 109.36 72.14 1.52 66126 Cd151 14.51 9.57 1.52 12476 Stat3 18.44 12.17 1.52 20848 Nell2 62.55 41.29 1.51 54003 Tcerg1l 13.88 9.16 1.51 70571 Bcan 39.94 26.38 1.51 12032 BC005764 29.72 19.63 1.51 216152 Mcfd2 51.79 34.24 1.51 193813 Psd 62.79 41.56 1.51 73728 Cdc25b 4.09 2.71 1.51 12531 Scap 18.93 12.54 1.51 235623 Slc27a4 30.53 20.22 1.51 26569 Gfra4 50.32 33.33 1.51 14588

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LepRb Expression Non-LepRb Fold Gene (FPKM) Expression (FPKM) Enrichment NCBI Gene ID Plk5 27.03 17.91 1.51 216166 Traip 2.84 1.88 1.51 22036 Rpl31 502.87 333.36 1.51 114641 9430020K01Rik 4.41 2.93 1.51 240185 Scand3 4.09 2.72 1.51 71970 Usp19 26.75 17.76 1.51 71472 Doc2b 33.58 22.29 1.51 13447 Ap3b2 47.33 31.43 1.51 11775 Fam179b 10.82 7.18 1.51 328108 Hectd1 16.54 10.99 1.50 207304 2610029G23Rik 12.12 8.06 1.50 67683 Tapbp 12.02 8.00 1.50 21356 Fam114a1 4.54 3.02 1.50 68303 Tubb5 332.72 221.70 1.50 22154 1700003E16Rik 17.29 11.52 1.50 71837

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APPENDIX 3

Fold change in gene expression in ob/ob, DIO, STAT3LepRKO and leptin treated mice

Adult LepRbeGFP-L10a mice and LepRbeGFP-L10a;ob/ob mice were treated with leptin (5mg/kg i.p.) or vehicle for 10 hours. Adult LepRbeGFP-L10a mice were also weaned onto a high fat diet for 8 weeks (DIO). Finally, adult STAT3LepRKO and STAT3LepRWT mice were generated as described in Chapter 4. All animals underwent hypothalamic dissection and TRAP-Seq analysis as described. N=3 TRAP-Seq samples per group, each composed of pooled hypothalamic from 4-6 animals. Transcript levels were compared between leptin and vehicle treated mice, between LepRbeGFP-L10a;ob/ob and vehicle treated LepRbeGFP-L10a controls, between DIO LepRbeGFP-L10a and vehicle treated LepRbeGFP-L10a controls, and between STAT3LepRKO and STAT3LepRWT controls. The genes included in this list are all those that were enriched in multiple control groups or that became enriched following a treatment which also caused their upregulation. Enrichment values shown are from 3 hour vehicle treatment (Chapter 3). Fold change values are shown. p<0.05 for fold change in bold and italics vs. control group.

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3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Agrp 43.06 0.69 1.19 5.55 0.94 0.57 3.73 Prlh 24.66 1.18 1.49 0.89 1.02 1.06 1.80 Npy 21.97 0.80 1.29 4.17 0.89 0.60 3.39 Pomc 19.30 0.96 1.56 0.16 1.35 2.17 0.28 Xist 15.31 0.68 1.48 2.47 0.74 1.68 1.48 Ucn 11.66 1.16 0.28 0.35 0.50 0.55 0.31 Gsx1 10.53 1.02 1.41 0.64 1.02 1.28 0.98 Serpina3i 10.12 1.91 2.48 0.13 10.46 1.48 1.37 Cartpt 9.58 0.93 1.12 0.28 0.81 1.53 0.35 Ghrh 9.19 0.65 0.94 2.66 0.96 1.13 1.08 Npw 9.03 1.02 1.26 0.80 1.08 2.67 0.90 Tbx19 8.12 1.33 1.89 0.73 1.84 1.47 1.07 Serpina3n 7.42 0.96 2.06 0.26 5.06 1.89 0.51 Cited1 7.27 1.09 1.97 1.22 1.16 1.41 1.59 Serpina3c 7.19 0.82 2.77 0.20 9.92 1.94 0.36 Serpina3m 6.94 1.05 3.92 0.52 2.79 2.14 0.62 Asb4 6.64 1.24 1.74 0.61 1.64 1.62 0.76 H2-Q8 6.26 0.71 0.99 0.65 1.00 1.04 6.64 Serpina3h 6.07 2.19 6.15 0.32 7.33 2.32 1.54 Nts 6.03 0.88 1.36 0.41 1.10 1.57 0.37 Sprr1a 5.81 0.94 9.22 0.83 0.40 4.31 1.13 Nr5a2 5.61 1.11 2.26 1.00 0.74 1.51 1.14 Bahcc1 5.42 0.98 1.48 1.86 0.72 1.45 1.02 Gch1 5.24 1.43 0.94 0.51 1.34 0.73 0.81 Slc6a3 5.23 1.13 0.42 0.82 1.15 0.74 0.70 Nr0b1 5.18 1.32 1.34 0.49 1.21 1.04 0.82 En1 4.99 1.89 0.68 0.79 0.90 0.68 1.06 Tac1 4.71 0.86 1.34 0.57 1.05 1.26 0.83 Gucy2c 4.69 1.26 1.49 0.63 1.50 1.21 1.05 Irs4 4.65 1.20 1.47 0.60 1.50 1.18 0.82 Fn1 4.56 0.49 0.61 1.24 0.75 1.25 1.09 Atg7 4.55 0.90 0.64 0.42 1.29 0.81 0.68 Atf3 4.45 4.97 4.58 1.02 2.39 6.26 0.91 Abcb1a 4.40 0.43 0.43 1.35 0.99 0.93 1.02 Flt1 4.37 0.54 0.41 1.23 0.92 0.94 1.02 Tnrc18 4.30 0.98 1.29 1.59 0.73 1.42 0.92 Procr 4.21 1.30 2.00 2.45 0.60 3.16 0.42 Mmrn2 4.08 0.38 0.12 1.00 0.59 0.80 1.27 Chrna6 4.05 1.20 0.48 0.83 1.05 0.70 0.73

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3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Rrad 4.02 1.04 1.59 1.24 0.75 1.84 0.94 Prr19 3.95 0.94 1.81 0.92 0.87 1.63 1.07 Ddc 3.87 1.51 0.65 0.79 1.08 0.71 1.12 Slc10a4 3.81 1.11 0.54 0.84 1.14 0.84 0.86 Pitx3 3.73 1.62 0.60 0.77 1.13 0.80 0.85 Traf3ip3 3.61 1.69 2.36 0.53 1.91 1.75 1.05 Serpina3g 3.61 1.19 2.26 1.02 1.47 1.45 1.68 Th 3.61 1.46 0.59 0.78 1.10 0.66 1.09 Serpina3f 3.61 1.19 3.19 0.27 7.83 1.62 29.80 Scpppq1 3.56 0.83 2.58 1.58 0.71 1.49 1.52 Sult5a1 3.52 1.02 0.42 1.06 1.01 0.50 1.42 Otp 3.49 0.94 1.76 1.19 0.83 0.98 1.13 Prdm12 3.49 1.09 1.58 0.68 0.87 1.13 0.65 Tmem176a 3.47 0.82 1.48 0.40 1.40 1.29 0.57 Gal 3.43 0.82 1.60 1.01 0.98 1.77 0.89 Npy2r 3.35 0.85 1.12 0.44 1.32 0.95 0.61 Yeats2 3.33 1.04 0.57 0.62 0.98 0.86 0.78 Socs3 3.14 4.78 3.14 0.25 10.00 2.56 1.05 Crh 3.10 0.81 1.37 1.27 1.00 1.96 1.38 Usp51 3.07 0.87 1.51 1.30 0.76 1.02 1.13 Nkx2-4 3.05 0.96 2.36 0.66 1.23 1.30 1.21 Slc18a2 3.03 1.04 0.54 0.90 1.01 0.81 0.73 Nms 3.03 0.98 1.47 0.67 0.95 1.97 0.90 Fam159a 2.97 0.67 1.25 2.94 0.69 1.28 2.25 Eltd1 2.93 0.45 0.45 1.30 0.81 0.71 0.84 Tie1 2.86 0.47 0.28 1.29 0.98 1.19 0.83 Cdh5 2.86 0.45 0.29 1.00 0.84 0.80 1.01 Bcl3 2.85 1.33 1.75 0.43 1.54 1.92 0.90 Gna14 2.80 1.53 2.03 0.72 1.23 1.69 0.84 Bcor 2.79 0.88 1.53 1.35 0.80 1.34 1.06 Ghsr 2.78 0.87 1.01 1.74 0.82 0.78 1.24 Cpa4 2.78 0.71 1.10 1.28 0.49 1.02 0.92 Acvrl1 2.75 0.54 0.31 1.27 0.87 0.82 1.02 Rbp4 2.74 0.80 0.81 1.92 0.90 0.92 1.74 Sema3g 2.71 0.67 0.27 1.42 0.98 0.91 1.10 Vwf 2.66 0.51 0.23 1.69 0.76 0.63 1.42 Six6 2.61 0.98 1.51 0.87 0.81 1.12 0.91 Tmem176b 2.59 0.78 1.25 0.55 1.14 1.20 0.71 Ret 2.58 1.01 0.51 0.83 1.10 0.88 0.72

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3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Plagl1 2.55 1.14 1.45 0.55 1.22 1.22 0.85 Vwa5a 2.54 1.19 2.01 0.52 1.63 1.42 0.99 Stat3 2.52 1.36 1.62 0.52 2.33 1.47 0.87 Gstm6 2.48 0.95 1.22 0.62 0.98 0.85 0.83 Slco1a4 2.43 0.52 0.40 1.10 0.95 0.83 1.06 Tnfrsf11b 2.43 1.19 1.21 0.42 1.33 1.45 0.58 Crabp1 2.43 1.03 1.30 0.84 0.89 1.57 0.93 Uba7 2.40 0.34 0.66 0.55 1.61 1.07 5.31 Prokr1 2.39 0.91 0.72 0.46 0.96 0.67 0.54 Cd274 2.34 0.56 1.18 0.84 0.89 1.11 3.87 Vgf 2.31 0.90 1.19 1.43 1.13 1.72 0.85 Rgs2 2.30 1.20 1.51 0.85 1.48 1.18 0.89 H2-Q6 2.28 0.39 1.60 0.34 0.94 2.85 19.77 Lck 2.27 1.21 1.58 0.86 1.70 1.12 1.31 Cd24a 2.27 1.17 1.94 0.77 2.30 1.14 0.69 H2-Q7 2.27 0.52 0.86 0.41 0.63 1.61 16.87 Kiss1 2.27 0.84 3.29 0.46 0.97 2.75 0.25 Rplp2 2.26 1.06 0.80 1.51 1.04 1.03 1.24 Podxl 2.22 0.53 0.57 1.12 0.78 1.02 0.92 Camk1g 2.22 0.92 1.15 1.53 0.97 1.00 1.15 Chrna5 2.17 1.31 0.36 0.86 0.77 0.67 0.51 Nup98 2.14 1.32 1.93 1.32 0.85 1.39 0.98 Rpl30 2.14 0.60 1.16 1.02 1.10 1.12 1.17 Maff 2.10 1.42 1.07 1.88 0.91 1.41 2.18 Lgals3bp 2.05 0.74 1.11 0.90 1.32 1.06 1.89 Cldn5 2.05 0.58 0.46 1.48 0.93 0.89 0.79 Ass1 2.04 1.00 0.65 1.24 1.04 0.81 1.09 Junb 2.04 2.01 1.91 0.97 1.67 1.87 0.95 Rpl22l1 2.03 1.11 0.79 1.51 0.87 0.87 1.16 Gem 2.03 1.02 1.03 1.67 0.55 1.63 1.12 Nptx2 2.01 1.03 1.20 0.85 1.73 1.05 1.05 Sox3 2.01 0.97 1.78 0.79 0.85 1.25 0.69 Irf9 2.00 1.37 1.83 0.38 3.01 1.36 2.20 Ptprb 1.97 0.48 0.34 1.24 0.71 0.90 0.94 Psme2 1.97 1.02 1.26 0.87 1.02 1.04 1.59 Ap1s2 1.96 1.04 1.60 0.94 0.98 1.13 0.84 Rnase6 1.96 0.08 0.60 1.24 0.66 0.25 0.67 Chodl 1.95 0.93 1.29 0.62 1.01 1.37 0.79 H2-Gs10 1.95 0.54 0.99 0.98 0.91 1.52 9.50

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3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Fgl2 1.92 1.01 1.31 0.82 2.19 1.69 0.98 Rsad2 1.92 0.69 1.02 1.22 0.71 0.69 4.59 Npy5r 1.90 0.77 1.18 0.71 0.77 1.26 0.54 Ly6c1 1.87 0.37 0.42 1.42 0.89 0.84 1.15 Psmb8 1.86 0.77 1.45 0.80 0.87 1.52 13.81 Gbp9 1.86 0.76 1.04 0.51 1.36 1.39 5.45 Apoa1 1.85 0.95 1.22 2.25 1.00 1.40 1.99 Crem 1.85 1.22 1.53 1.10 1.01 1.78 1.05 Ctla2a 1.85 0.59 1.01 2.97 0.76 1.20 1.19 Bnc2 1.84 1.44 0.59 0.93 1.15 0.62 1.31 St8sia4 1.84 0.80 0.96 0.78 0.97 0.85 0.64 H2-Bl 1.84 0.80 1.14 1.27 0.77 1.02 3.23 H2-D1 1.83 0.67 0.91 0.91 1.10 1.39 2.77 Aldh1a1 1.80 1.53 0.60 0.67 0.98 0.73 0.87 Irx5 1.80 0.91 1.58 0.87 1.02 1.20 1.01 Fosb 1.79 1.04 1.17 3.20 0.53 2.55 1.31 Npy1r 1.79 1.27 1.31 0.72 1.03 1.82 0.57 1500012F01Rik 1.77 1.09 0.98 1.34 0.88 1.20 1.69 N4bp2l1 1.77 1.02 1.22 1.50 0.80 0.90 1.37 B2m 1.76 0.56 1.17 0.82 0.81 1.45 6.09 Ntn1 1.75 0.97 0.50 0.87 0.93 0.63 0.90 Gm885 1.74 0.55 2.60 7.88 1.34 2.14 3.01 Irf1 1.73 1.23 1.38 0.71 1.49 1.17 2.91 Prr12 1.72 0.89 1.72 1.14 0.93 1.06 1.05 Krtap17-1 1.71 0.76 1.07 2.07 0.75 0.94 1.56 Rpl3 1.70 2.74 1.09 1.01 1.01 0.99 1.23 Fam179a 1.70 1.35 1.50 0.66 1.41 1.15 0.80 Ezr 1.69 0.92 1.71 1.51 0.93 1.38 1.45 Btg1 1.69 1.23 1.33 1.55 0.87 1.13 1.29 Tnfaip8l3 1.65 0.99 1.49 0.53 0.96 1.26 0.77 Tap1 1.65 0.57 1.19 0.91 1.08 1.31 7.28 Pcsk1n 1.64 0.71 1.10 0.98 1.25 1.18 0.62 Drd2 1.63 1.18 0.59 0.77 1.07 0.78 0.88 Cd44 1.62 0.96 1.98 0.61 1.35 1.74 0.74 Cebpb 1.62 1.44 2.35 1.29 1.11 1.27 1.58 H2-T23 1.62 0.61 0.91 1.31 1.02 1.05 3.65 Ube2l6 1.61 0.95 1.28 1.05 1.09 1.03 2.96 Gkn3 1.60 0.34 0.12 0.43 0.83 0.34 1.21 BC002163 1.58 0.91 0.23 1.32 0.81 0.11 1.14

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3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Cdhr3 1.58 0.53 0.80 1.84 0.68 0.78 1.89 Tagln2 1.57 0.90 1.50 0.69 0.97 1.19 0.83 Tac2 1.57 0.76 2.25 0.99 1.02 1.65 0.84 Gcnt2 1.56 1.47 1.10 1.12 1.80 0.98 1.00 Mpa2l 1.56 0.86 1.36 0.61 1.01 1.65 16.03 Tapbp 1.56 0.82 0.96 0.95 1.19 1.01 1.63 Notch4 1.54 0.70 1.02 1.86 0.75 0.96 1.00 Tnfsf10 1.53 0.61 0.73 1.01 0.95 0.96 2.17 Gm6548 1.52 1.03 1.07 1.13 1.21 0.95 2.06 Camk1 1.50 1.16 1.51 0.91 1.36 1.10 1.09 Ifi35 1.47 0.78 1.25 0.79 0.79 1.17 5.23 Trim25 1.47 1.02 1.31 1.00 0.95 1.07 1.82 Trim21 1.47 0.79 1.09 0.68 1.20 0.97 4.08 Ndufs5 1.45 1.52 0.68 1.24 1.03 0.99 1.06 Gadl1 1.44 1.31 1.04 2.47 0.64 0.90 1.04 Anxa2 1.44 0.96 1.72 0.63 0.93 1.28 0.89 Myh11 1.44 0.57 0.55 2.31 0.68 1.42 1.14 Snhg8 1.44 1.02 1.08 1.65 0.97 1.20 1.28 Socs1 1.43 1.90 1.29 0.68 1.68 1.10 1.93 Apol10b 1.43 0.66 3.97 0.87 2.10 2.28 11.94 Prokr2 1.42 1.79 2.29 0.61 1.51 2.04 0.58 H2-K1 1.41 0.42 0.87 0.81 0.92 2.10 10.96 Gadd45a 1.41 1.92 1.48 1.04 1.19 1.29 1.17 Gm12250 1.41 0.11 2.72 1.23 0.84 0.95 130.58 Lyve1 1.41 0.43 0.76 6.09 0.72 0.35 2.44 Gbp4 1.41 0.53 0.65 0.38 0.99 0.89 14.57 Bst1 1.41 1.09 1.32 2.56 1.11 0.68 2.00 Stat1 1.40 1.11 1.22 0.56 1.73 1.17 3.49 En2 1.40 1.67 0.45 0.78 1.13 0.55 1.04 Ccl17 1.39 0.91 1.05 2.62 1.60 0.69 4.32 Rpain 1.38 1.14 1.62 0.96 1.10 1.46 1.04 Jun 1.37 1.94 1.21 0.80 1.24 1.42 0.86 Vwce 1.35 1.02 0.59 2.53 0.61 0.89 1.10 H1fx 1.34 1.03 1.55 1.01 1.06 1.16 0.95 Fam46a 1.34 1.05 1.10 1.73 0.67 1.21 1.11 Scand1 1.33 1.01 1.26 0.85 0.87 1.12 0.52 Tubb6 1.33 1.35 0.98 0.75 2.04 1.23 1.06 Psmb10 1.32 0.96 1.09 1.02 1.17 1.07 1.93 Akr1b7 1.31 1.08 2.98 0.55 1.32 0.25 0.63

210

3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Rbm3 1.31 0.94 1.06 1.38 0.88 0.66 1.71 Rdh10 1.30 1.50 1.43 1.04 2.01 1.12 0.97 Oasl2 1.29 0.47 1.29 1.04 1.10 1.36 23.57 Igtp 1.28 0.78 1.00 0.64 1.36 1.06 15.40 Oas1b 1.28 0.70 1.57 1.06 0.94 1.30 4.66 Eif2ak2 1.27 0.91 1.30 1.15 1.02 1.06 3.52 Bst2 1.26 0.31 1.10 1.30 0.70 1.24 49.14 Dtx3l 1.26 0.98 1.29 0.85 1.22 1.08 3.33 Apol9a 1.25 0.59 0.76 1.75 0.70 1.18 8.08 Usp18 1.25 0.34 0.67 0.57 0.82 1.02 13.53 Cyp4v3 1.25 0.62 0.73 2.20 0.64 0.81 1.32 Fos 1.24 1.69 1.45 1.45 0.77 2.46 1.16 Rprm 1.23 1.19 1.57 0.90 1.10 1.28 0.95 Fosl2 1.22 1.76 1.04 3.26 0.68 2.41 1.50 Gm1082 1.22 0.80 2.04 0.41 1.10 1.89 0.71 Slc39a4 1.22 1.05 0.28 0.77 0.73 0.63 0.81 Gdpd3 1.22 0.48 1.22 1.54 0.73 0.90 2.06 Samhd1 1.18 1.00 1.12 1.06 1.05 0.99 1.78 Rtp4 1.18 0.76 1.86 1.02 1.27 1.08 25.99 Lix1 1.17 1.35 0.67 0.76 1.05 0.60 1.00 Ifi47 1.17 1.19 1.06 0.48 2.00 1.18 49.06 4931414P19Rik 1.17 1.28 1.53 0.83 1.25 1.30 1.02 Plscr3 1.16 0.92 1.65 0.82 1.03 1.36 0.92 Cish 1.14 1.79 1.14 0.67 1.65 1.24 1.78 Gadd45g 1.14 2.33 1.49 1.31 1.33 1.60 1.41 Lamc2 1.13 1.13 1.78 1.07 2.86 1.53 1.06 Irgm1 1.12 0.89 1.14 1.05 0.85 1.00 7.24 Irf7 1.12 0.69 1.06 1.09 0.96 1.41 5.68 Ifit1 1.11 0.45 1.06 0.92 0.78 1.20 29.69 Parp10 1.11 0.86 0.92 0.95 1.08 0.97 9.23 Brca2 1.09 0.93 1.05 1.56 0.86 0.93 1.27 Sbno2 1.07 1.70 1.46 0.74 1.78 1.69 0.77 Ier3 1.06 1.63 1.35 0.99 1.51 1.21 1.17 Cd38 1.05 1.30 2.97 1.15 1.63 5.72 0.84 Ddx58 1.04 0.75 1.10 1.25 0.84 1.03 2.81 Gbp2 1.03 0.37 0.50 1.00 0.56 0.78 5.41 Rhoc 1.03 1.05 1.56 0.75 1.11 1.26 0.98 Psmb9 1.02 0.56 1.25 0.61 0.72 1.22 11.95 Irgm2 1.01 1.19 1.77 0.59 1.71 1.45 22.91

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3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Drd1a 1.01 1.80 0.72 0.66 1.66 0.84 0.77 Nmi 1.01 0.63 0.94 0.76 1.07 0.97 2.02 Socs2 0.99 1.92 1.19 0.68 1.68 1.17 1.36 Myof 0.98 0.86 0.57 1.83 0.67 0.92 2.48 Vdr 0.97 1.03 1.40 2.88 0.85 1.20 1.88 Trh 0.97 0.69 1.26 0.95 0.95 2.10 0.92 Rasl11a 0.97 1.68 1.57 0.74 1.83 1.44 1.15 Zc3hav1 0.97 0.93 0.85 0.81 1.01 1.08 2.42 Trim62 0.96 1.68 1.05 1.03 1.16 1.09 1.10 Nlrc5 0.95 1.12 6.98 1.68 3.09 3.72 8.19 Ifih1 0.94 0.84 1.15 0.83 0.81 0.79 1.99 Nmb 0.93 0.85 0.99 4.14 0.77 1.08 1.73 Cdkn1a 0.93 1.08 1.18 4.00 1.24 1.54 2.40 Ada 0.90 0.97 1.85 1.02 1.12 1.41 1.40 Stat2 0.90 1.19 1.04 0.91 1.19 1.20 1.67 Trim56 0.88 0.83 1.12 1.47 0.56 0.71 2.44 Gbp3 0.88 0.37 0.69 0.54 1.08 1.40 27.82 Adrb2 0.88 0.52 0.72 2.07 0.50 0.63 3.08 Hs3st1 0.87 0.68 0.78 2.06 0.71 0.98 1.33 Gbp6 0.79 0.89 0.85 0.69 1.10 0.92 3.99 Ddx60 0.77 0.84 0.76 0.71 0.73 0.54 3.06 Dhx58 0.76 0.90 0.79 0.83 0.85 0.95 3.26 1700024P16Rik 0.75 0.49 1.44 8.42 0.43 0.84 3.47 Etv6 0.73 2.65 1.23 0.96 1.59 1.14 1.26 Ifi203 0.69 0.42 0.81 0.60 1.51 1.45 4.48 Apol6 0.69 0.81 0.88 1.12 1.48 0.93 4.82 Gpr151 0.68 1.36 2.30 0.77 2.33 2.15 0.92 Ifi44 0.68 0.11 1.07 0.91 0.22 1.47 15.12 Ifit2 0.68 0.98 1.11 1.14 0.87 1.06 2.93 Spink8 0.68 0.46 0.85 3.50 1.04 1.87 5.07 Parp14 0.63 0.72 0.40 0.83 0.81 0.99 6.88 Trim34a 0.59 0.71 0.63 0.87 0.86 0.78 2.71 Iigp1 0.57 0.26 0.72 0.60 1.01 1.39 33.53 Casp1 0.56 0.91 1.01 0.90 0.98 0.72 4.39 Arid5a 0.54 4.52 1.30 1.00 2.50 1.50 1.19 Samd9l 0.53 0.78 0.77 1.51 0.56 1.24 4.44 Gm4951 0.51 0.48 0.69 1.26 0.55 1.13 39.21 Isg15 0.48 0.76 1.03 0.53 1.51 1.15 31.03 9230105E10Rik 0.47 0.70 0.65 1.61 0.60 1.24 4.28

212

3hr 10hr ob/ob + Gene Enrichment Leptin Leptin ob/ob Leptin DIO STAT3LepRKO Il18bp 0.46 0.64 1.37 2.28 0.66 1.07 5.62 Isg20 0.46 1.20 1.03 2.64 0.83 1.37 7.83 Zbp1 0.44 0.15 1.51 0.66 0.62 0.99 127.49 I830012O16Rik 0.44 0.62 0.74 0.87 0.64 0.76 3.42 Ifit3 0.43 0.65 0.84 1.07 0.77 1.06 6.37 Gm4841 0.41 0.00 1.43 1.03 1.47 1.64 11.99 H2-Aa 0.36 0.21 0.60 0.95 0.55 0.65 3.32 Xaf1 0.33 0.74 0.64 0.81 1.35 0.97 4.96 Irf8 0.32 1.06 0.68 0.97 0.81 0.82 4.21 Gm12185 0.28 0.91 1.21 1.10 0.68 1.08 4.58 Tuba1c 0.05 1.32 1.52 0.37 2.48 1.35 0.15

213